pyrimidinones and forodesine

Nucleoside analogues have proven to be highly successful chemotherapeutic agents in the treatment of a wide variety of cancers. Several such compounds, including gemcitabine and cytarabine, are the go-to option in first-line treatments. However, these materials do have limitations and the development of next generation compounds remains a topic of significant interest and necessity. Herein, we discuss recent advances in the chemical synthesis and biological evaluation of nucleoside analogues as potential anticancer agents. Focus is paid to 4'-heteroatom substitution of the furanose oxygen, 2'-, 3'-, 4'- and 5'-position ring modifications and the development of new prodrug strategies for these materials.

Topics: Adenosine; Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Design; Drug Screening Assays, Antitumor; Furans; Humans; K562 Cells; Mice; Molecular Structure; Nucleosides; Oxygen; Prodrugs; Purine Nucleosides; Pyrimidinones; Thionucleosides; Vitamin E

Cutaneous T-cell lymphoma (CTCL) is characterized by the accumulation of neoplastic CD4+ T lymphocytes in the skin. Given the lack of curative treatments for CTCL, there is a significant need for new, superior therapies. Forodesine is a transition-state analogue that inhibits purine nucleoside phosphorylase. Because it selectively targets T lymphocytes, it represents a drug of interest for the treatment of CTCL. Areas covered: Phase I/II dose-ranging studies of intravenous (IV) and oral forodesine demonstrated its activity, safety, and tolerability for refractory CTCL. Response rates were 31% and 27%, respectively. No dose-limiting toxicities were observed. These studies were followed by a phase II trial of oral forodesine 200 mg daily. This oral formulation showed only partial activity, with a response rate of 11%, likely attributable to underdosing. Common adverse events in these trials included infection, fatigue, peripheral edema, nausea, pruritus, headache, and insomnia. Expert opinion: IV and oral formulations of forodesine have demonstrated partial activity and an acceptable safety profile in patients with refractory CTCL. A higher oral dose, or sequential therapy consisting of IV forodesine followed by maintenance oral forodesine, may be more effective. With proper dosing, forodesine may emerge as a safe and effective treatment for refractory CTCL.

Topics: Administration, Intravenous; Administration, Oral; Animals; Antineoplastic Agents; Dose-Response Relationship, Drug; Humans; Lymphoma, T-Cell, Cutaneous; Purine Nucleosides; Pyrimidinones; Skin Neoplasms

Advances in acute lymphocytic leukemia (ALL) therapy has led to long-term survival rates in children. However, only 30%-40% of adults achieve long-term disease-free survival. After relapse, the outcome of salvage chemotherapy is very disappointing with less than 10% of long survival. Novel agents are therefore desperately required to improve response rates and survival, but also the quality of life of patients. Areas covered: The following review is a comprehensive summary of various novel options reported over the past few years in the therapeutic area of adult ALL. Expert opinion: Identifying key components involved in disease pathogenesis may lead to new approaches. In a near future, the incorporation of monoclonal antibodies and T-cell directed approaches including blinatumomab and chimeric antigen receptor T cells may increase the cure rates and may reduce the need for intensive therapy.

Topics: Adenine Nucleotides; Adult; Antibodies, Monoclonal; Antineoplastic Agents; Arabinonucleosides; Clofarabine; Disease-Free Survival; Drug Discovery; Humans; Molecular Targeted Therapy; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Pyrimidinones; Quality of Life; Recurrence; Salvage Therapy

Malaria is a leading cause of morbidity and mortality in the tropics. Chemotherapeutic and vector control strategies have been applied for more than a century but have not been efficient in disease eradication. Increased resistance of malaria parasites to drug treatment and of mosquito vectors to insecticides requires the development of novel chemotherapeutic agents. Malaria parasites exhibit rapid nucleic acid synthesis during their intraerythrocytic growth phase. Plasmodium purine and pyrimidine metabolic pathways are distinct from those of their human hosts. Thus, targeting purine and pyrimidine metabolic pathways provides a promising route for novel drug development. Recent developments in enzymatic transition state analysis have provided an improved route to inhibitor design targeted to specific enzymes, including those of purine and pyrimidine metabolism. Modern transition state analogue drug discovery has resulted in transition state analogues capable of binding to target enzymes with unprecedented affinity and specificity. These agents can provide specific blocks in essential pathways. The combination of tight binding with the high specificity of these logically designed inhibitors, results in low toxicity and minor side effects. These features reduce two of the major problems with the current antimalarials. Transition state analogue design is being applied to generate new lead compounds to treat malaria by targeting purine and pyrimidine pathways.

Topics: Antimalarials; Binding Sites; Drug Design; Enzyme Inhibitors; Humans; Malaria, Falciparum; Models, Molecular; Plasmodium falciparum; Protein Binding; Protozoan Proteins; Purine Nucleosides; Purines; Pyrimidines; Pyrimidinones; Pyrroles; Substrate Specificity

Cytogenetic, molecular and phenotyping features of malignant hematologic diseases succeeded in improving their management by a more accurate stratification of patients according to several groups of risk and by providing a rational for targeted therapy. Three major types of treatment (excluding cellular therapy) are currently available in onco-hematology: conventional chemotherapy, small molecules for targeted therapy and monoclonal antibodies. Conventional chemotherapy with optimization of doses and multidrug-based regimens allowed to substantially improve survival of patients and keeps a place of choice in treatment of these diseases. Targeted treatments came from the cytogenetic and molecular characterization of hemopathies. Thus, the kinase Bcr-Abl, as a result of the translocation t(9;22)(q34;q11), has been successfully targeted by tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. Molecular abnormalities like internal-tandem duplication/point activating mutations in FLT3 in some acute myeloblastic leukemia or epigenetic dysregulations in some blood malignancies can also be targeted by small molecules. Hematopoietic malignant cells are phenotypically characterized by expression of cluster of differentiation (CD) on their surface. These CD are detected by flow cytometry using specific antibodies. Monoclonal antibodies targeting different CD have been developed for treatment. Rituximab, an anti-CD20 antibody, was the first monoclonal antibody successfully developed for treatment of malignant hematologic diseases. Since rituximab, many other monoclonal antibodies are being developed. Trends in malignant hematologic diseases presented here will include treatments, which have at least entered phase I/II clinical trials in adult or childhood leukemia. They include some novel drugs of conventional chemotherapy like second-generation nucleoside analogues. We will give an overview of the small molecules targeting the different cellular pathways and we will highlight those appearing as the most promising like novel TKIs. The large field of monoclonal antibodies will be also approached focusing on antibodies developed in leukemias.

Topics: Adenine Nucleotides; Antibodies, Monoclonal; Antineoplastic Agents; Arabinonucleosides; Chemistry, Pharmaceutical; Clofarabine; Hematologic Neoplasms; Humans; Molecular Targeted Therapy; Purine Nucleosides; Pyrimidinones

Though peripheral T-cell lymphoma (PTCL) is an area of significant unmet therapeutic need, a number of new treatment options are available for patients, especially those with relapsed or refractory disease. A plethora of drugs are now in development for PTCL, but drugs that truly target novel disease biology are noticeably absent. Combinations of T-cell centric agents could produce novel platforms of therapy to replace the relatively ineffective CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-based regimens. Among agents with T-cell activity are the folate analog pralatrexate, histone deacetylase inhibitors (HDACi) like romidepsin, the proteasome inhibitor bortezomib, the immunomodulatory agent lenalidomide, the purine nucleoside phosphorylase (PNP) inhibitor forodesine, the nucleoside analog gemcitabine, and BH3-only mimetics like ABT-263 and ABT-737.

Topics: Aminopterin; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Clinical Trials as Topic; Depsipeptides; Drug Design; Drugs, Investigational; Histone Deacetylase Inhibitors; Humans; Immunologic Factors; Lenalidomide; Lymphoma, T-Cell, Peripheral; Purine Nucleosides; Pyrazines; Pyrimidinones; Salvage Therapy; Thalidomide

The most common subtype of T-/natural killer (NK) cell lymphoma in Japan is adult T-cell leukemia-lymphoma (ATL), which is associated with the human T-cell lymphotropic virus type I (HTLV-1). The investigators in Japan have conducted several clinical trials on multi-agent chemotherapy and stem cell transplantation for patients with ATL. They have also initiated several new clinical trials with a number of agents: an anti-CCR4 antibody, KW-0761; forodesine, a purine nucleoside phosphorylase inhibitor; and lenalidomide, an immunomodulatory agent. Clinical trials with pralatrexate, a folate analog, and denileukin diftitox, an immunoconjugate, are under discussion for patients with ATL and peripheral T-cell lymphoma (PTCL).

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Combined Modality Therapy; Drugs, Investigational; Hematopoietic Stem Cell Transplantation; Humans; Immunologic Factors; Japan; Lenalidomide; Leukemia-Lymphoma, Adult T-Cell; Multicenter Studies as Topic; Purine Nucleosides; Pyrimidinones; Thalidomide; Treatment Outcome

Purine nucleoside phosphorylase (PNP) is an important catalytic enzyme in the purine salvage pathway; its deficiency is associated with T-cell lymphopenia and with humoral deficiency. This clinical observation led to the investigation of PNP inhibitors and their possible clinical application in the management of hematologic malignancies, notably those of T-cell lineage. Forodesine is the most potent of the PNP inhibitors. Its effect appears to be linked to increased 2 -deoxyguanosine levels in plasma, which in turn is converted to 2 -deoxyguanosine triphosphate in target cells and disrupts DNA synthesis. Several preclinical studies have shown forodesine's effect against lymphocytes in vitro and in vivo, and these findings have led to several Phase I/II studies in patients with lymphoid neoplasms. Early clinical trials show that forodesine has promise as a single agent for the treatment of relapsed/refractory hematologic malignancies, and combination therapies might be warranted to improve clinical results.

Topics: Animals; Clinical Trials as Topic; Drug Evaluation, Preclinical; Humans; Neoplasms; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; T-Lymphocytes

Recently, the search for more effective and safer antineoplastic agents has led to synthesis and introduction into preclinical and clinical studies of a few new purine nucleoside analogues (PNA). Three of them: clofarabine (CAFdA), nelarabine, and forodesine (immucillin H, BCX-1777), despite belonging to the same group of drugs such as PNA, have shown some differences concerning their active forms, metabolic properties and mechanism of action. However, all these drugs have demonstrated promising activity in patients with relapsed and refractory acute lymphoblastic leukemia (ALL). CAFdA was approved for the therapy of relapsed or refractory ALL in the third line of treatment. It has proved promising in pediatric patients as well as in some patients who are able to proceed to allogenic hematopietic stem cell transplantation (HSCT). Moreover, the drug exhibits an efficacy in acute myeloid leukemia (AML), blast crisis of chronic myelogenous leukemia (CML-BP) and myelodysplastic syndrome (MDS). Nelarabine is recommended for T-ALL and T-cell lymphoblastic lymphoma (T-LBL) with the overall response rates ranging from 11 to 60%. However, the use of the drug is limited by potentially severe neurotoxicity. Forodesine is a purine nucleoside phosphorylase (PNP) inhibitor and it has shown activity in relapsed and refractory T- and B-cells leukemias as well as in cutaneous T-cell lymphoma (CTCL). Recently patented, a few of inventions in the field of pharmaceutical preparation of new PNA have also been published. Great hopes are currently set on the use of these drugs in the treatment of lymphoid and myeloid malignancies in adult and in pediatric patients, however ongoing studies will help to define their role in the standard therapy.

Topics: Adenine Nucleotides; Antineoplastic Agents; Arabinonucleosides; Clinical Trials as Topic; Clofarabine; Hematologic Neoplasms; Humans; Purine Nucleosides; Pyrimidinones

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Bendamustine Hydrochloride; Clinical Trials as Topic; Cyclophosphamide; Doxorubicin; Gene Targeting; Hematopoietic Stem Cell Transplantation; Humans; Indoles; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Nitrogen Mustard Compounds; Prednisone; Prognosis; Purine Nucleosides; Pyrimidinones; Risk; Rituximab; Salvage Therapy; Sirolimus; Transplantation, Autologous; Vincristine

This review will describe the recent advances in the field of aza-C-nucleosides with a particular emphasis on immucillins and related compounds. The review will cover both chemical and biological aspects concerning their preparation and/or occurrence in Nature as well as their biological properties which include glycosidase, glycosyl transferase, and nucleoside hydrolase and phosphorylase inhibition, among others. These enzymatic inhibitory properties are the basis for the potential use of the title compounds in viral and parasitic infections, cancer and genetic disorders.

Topics: Adenine; Adenosine; Aza Compounds; Glycoside Hydrolases; N-Glycosyl Hydrolases; Nucleosides; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Pyrrolidines

There are few approved therapies for cutaneous T-cell lymphoma (CTCL). The retinoids are the major biologic response modifiers used in CTCL, producing good response rates but few complete responses. For patients with early-stage disease, the oral retinoids can be combined with other therapies, such as psoralen plus ultraviolet A or interferon alpha, to improve response rates. Combined-modality therapy with oral retinoids, combined chemotherapy, electron-beam therapy, and topical mustargen has also proved effective. For the treatment of advanced-stage disease, the targeted therapy denileukin diftitox (Ontak) provides a nonimmunosuppressive alternative to conventional chemotherapy or radiation therapy. Of the conventional chemotherapies that have been tested in CTCL, gemcitabine (Gemzar) has demonstrated good efficacy in producing responses, particularly in patients with tumors. This agent can be used in combination with a maintenance therapy of bexarotene (Targretin) to manage the plaques and patches of mycosis fungoides. Several other targeted therapies are now also in testing, for example, alemtuzumab (CamPath), HuMax-CD4, several histone deacetylase inhibitors, and the transition-state inhibitor forodesine. These drugs, in combination with currently used therapies, may increase the number and combinations of therapies available for the treatment of this chronic condition to optimize long-lasting responses in CTCL.

Topics: Alemtuzumab; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Neoplasm; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Chemotherapy, Adjuvant; Diphtheria Toxin; Dose-Response Relationship, Drug; Humans; Hydroxamic Acids; Immunologic Factors; Interferon-alpha; Interleukin-2; Lymphoma, T-Cell, Cutaneous; Mechlorethamine; Purine Nucleosides; Pyrimidinones; Radiotherapy, Adjuvant; Recombinant Fusion Proteins; Retinoids; Sezary Syndrome; Skin Neoplasms; Vorinostat

Recently a few new purine nucleoside analogues (PNA) have been synthesized and introduced into preclinical and clinical trials. The transition-state theory has led to the design of 9-deazanucleotide analogues that are purine nucleoside phosphorylase (PNP) inhibitors, termed immucillins. Among them the most promising results have been obtained with forodesine. Forodesine (BCX-1777, Immucillin H, 1-(9-deazahypoxanthin)-1,4-dideoxy-1,4-imino-D-ribitol) has carbon-carbon linkage between a cyclic amine moiety that replaces ribose and 9-deaza-hypixanthine. The drug is a novel T-cell selective immunosuppressive agent which in the presence of 2'-deoxyguanosine (dGuo) inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction and phytohemagglutinin. In the mechanism of forodesine action two enzymes are involved: PNP and deoxycytidine kinase (dCK). PNP catalyzes the phosphorolysis of dGuo to guanine (Gu) and 2'-deoxyribose-1-phosphate, whereas dCK converts dGuo to deoxyguanosino-5'-monophosphate (dGMP) and finally to deoxyguanosino-5'-triphosphate (dGTP). The affinity of dGuo is higher for PNP than for dCK. Nevertheless, if PNP is blocked by forodesine, plasma dGuo is not cleaved to Gu, but instead it is intracellularly converted to dGTP by high dCK activity, which leads to inhibition of ribonucleotide reductase (RR), an enzyme required for DNA synthesis and cell replication, which eventually results in apoptosis. Forodesine is active in some experimental tumors in mice, however it could be used for the treatment of human T-cell proliferative disorders and it is undergoing phase II clinical trials for the treatment of T-cell non-Hodgkin's lymphoma, which includes cutaneous T-cell lymphoma (CTCL). Moreover, recent preclinical and clinical data showed activity of forodesine in B-cell acute lympholastic leukemia (ALL).

Topics: Animals; Enzyme Inhibitors; Humans; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

The search for more effective and safer anti-leukemia therapies has led to the identification of several new agents that show activity against specific types of acute lymphoblastic leukemia (ALL). Recently, three novel purine nucleoside analogues (nelarabine, clofarabine, and forodesine) have shown promising activity in patients with relapsed or refractory ALL. Of these, nelarabine has shown clinically meaningful benefit in patients with T-cell ALL, with overall response rates ranging from 33% to 60%, the induction of durable complete remissions, and an overall 1-year survival rate of 28% in adults. Clofarabine has also shown promising clinical activity in pediatric patients, with an overall response rate of 30%, and some patients are able to proceed to allogeneic hematopoietic cell transplantation. Forodesine is the most recent novel agent, with a unique mechanism that has shown single-agent activity in relapsed and refractory T- and B-cell leukemias and cutaneous lymphomas. Although clinical experience is limited, treatment-related toxicities appear to be mild. The rationale, pharmacology, and clinical experience to date with these agents in the treatment of patients with refractory acute leukemia are reviewed, with a highlight on ALL.

Topics: Adenine Nucleotides; Antineoplastic Agents; Apoptosis; Arabinonucleosides; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clofarabine; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of non-Hodgkin's lymphomas characterized by skin infiltration of neoplastic T lymphocytes. Mycosis fungoides and its leukemic variant Sézary syndrome represent the most common CTCL subtypes. Current treatment for patients with mycosis fungoides involves topical and systemic therapies for the cutaneous manifestations. However, no therapy is curative and patients often progress to advanced extracutaneous CTCL with visceral organ complications or relapsed disease that is frequently refractory to most topical and aggressive systemic regimens. The emergence of novel targeted therapies such as biologic agents, histone deacetylase inhibitors, and purine nucleoside phosphorylase inhibitors offers promise for more effective and safer treatment strategies for refractory CTCLs.

Topics: Clinical Trials as Topic; Histone Deacetylases; Humans; Immunologic Factors; Mycosis Fungoides; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Sezary Syndrome; Skin Neoplasms

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of ribonucleosides and 2'-deoxyribonucleosides to their respective bases. Endogenous PNP deficiency leads to specific T-cell immunodeficiency, a genetic disease that has prompted the development of PNP inhibitors as potential therapies for T-cell-mediated diseases. PNP inhibition leads to the elevation of 2'-deoxyguanosine levels and accumulation of intracellular deoxyguanosine 5'-triphosphate, inducing cellular apoptosis. Forodesine is a highly potent, orally active, rationally designed PNP inhibitor that has shown activity in preclinical studies with malignant cells and clinical utility against T-cell acute lymphoblastic leukemia and cutaneous T-cell lymphoma. Additional preliminary findings support its use for the management of some B-cell malignancies.

Topics: Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Deoxyguanine Nucleotides; Humans; Leukemia, B-Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

Purine nucleoside phosphorylase (PNP) was recognized more than 30 years ago as a potential target for the treatment of patients with T-cell malignancies when an inherited deficiency of PNP was reported to be associated with a profound T-cell lymphopenia. The biochemical basis for this T-cell deficiency was subsequently shown to be related to the accumulation of plasma 2'-deoxyguanosine (dGuo) and intracellular dGuo triphosphate (dGTP). These observations have led to a search for PNP inhibitors that would be useful clinically in the management of T cell-derived malignancies. The most potent inhibitor of PNP described to date is forodesine, a rationally designed, transition-state analogue inhibitor. The preclinical and clinical pharmacology of forodesine showed its effectiveness in inhibiting PNP and augmenting dGuo levels in plasma. Increased dGTP concentrations in leukemia cells of different lineages provides strong support for the potential use of this agent in the treatment of patients with hematologic malignancies of both T- and B-cell origin.

Topics: Biosynthetic Pathways; Clinical Trials as Topic; Deoxyguanine Nucleotides; Deoxyguanosine; Humans; Leukemia, T-Cell; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; T-Lymphocytes

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage metabolic pathway. In humans, the loss of functional PNP results in significant T-cell-mediated immunodeficiency (and may also affect B-cell function). Forodesine is a potent PNP inhibitor that acts by elevating plasma 2'-deoxyguanosine (dGuo) and intracellular deoxyguanosine triphosphate, which in turn affects deoxynucleotide-triphosphate pools and induces cell death by apoptosis. BioCryst Pharmaceuticals Inc, under license from the Albert Einstein College of Medicine, is developing intravenous and oral formulations of forodesine for the potential treatment of various T-cell and B-cell lymphomas and leukemias, as well as for solid tumors; MundiPharma AG is also investigating the drug for leukemia. Forodesine effectively inhibits T-cell proliferation in vitro in the presence of dGuo. In early clinical trials, forodesine has demonstrated an acceptable safety profile and indications of biological activity. Few drug-related serious adverse events have been reported, and generally only mild-to-moderate nonhematological toxicity has been observed. Forodesine has the potential to lead the development of other novel therapies with broad-based activity for hematological malignancies; the drug may also be useful for the treatment of a wide variety of other T-cell-mediated disorders, as well as for the potential treatment for other B-cell lymphomas/leukemias.

Topics: Animals; Enzyme Inhibitors; Humans; Leukemia; Lymphoma; Neoplasms; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Structure-Activity Relationship

Purine nucleoside phosphorylase (PNP) deficiency is a rare, inherited immunodeficiency disorder in which the specific molecular defect was identified. Clinically, a lack of PNP manifests as profound T-cell deficiency with minor or variable changes in the humoral system. Biochemically, the absence of PNP results in an increase in plasma deoxyguanosine (dGuo) and a T-cell-specific increase in intracellular deoxyguanosine triphosphate (dGTP). This observation has been the impetus for the search for either inhibitors of the enzyme or PNP-resistant dGuo analogues as potential anti-T-cell-lineage agents over the past 30 years. Forodesine (an inhibitor of PNP) and nelarabine (a PNP-resistant dGuo analogue) proved to be T-cell selective when tested in clinic. This review summarises the preclinical, clinical and pharmacokinetic investigations with these novel agents.

Topics: Adult; Animals; Antimetabolites, Antineoplastic; Arabinonucleosides; Child; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Deoxyguanosine; Drug Design; Drug Screening Assays, Antitumor; Drugs, Investigational; Humans; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Experimental; Lymphoma, T-Cell; Mice; Neoplasm Proteins; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; T-Lymphocytes

Kinetic isotope effects and computational chemistry have defined the transition state structures for several members of the N-ribosyltransferase family. Transition state analogues designed to mimic their cognate transition state structures are among the most powerful enzyme inhibitors. In complexes of N-ribosyltransferases with their transition state analogues, the dynamic nature of the transition state is converted to an ordered, thermodynamic structure closely related to the transition state. This phenomenon is documented by peptide bond H/D exchange, crystallography and computational chemistry. Complexes with substrate, transition state and product analogues reveal reaction coordinate motion and catalytic interactions. Isotope-edited spectroscopic analysis and binding specificity of these complexes provides information about specific enzyme-transition state contacts. In combination with protein dynamic QM/MM models, it is proposed that the transition state is reached by stochastic dynamic excursions of the protein groups near the substrates in the closed conformation. Examples from fully dissociated (D(N) *A(N)), hybrid (D(N)A(N)) and symmetric nucleophilic displacement (A(N)D(N)) transition states are found in the N-ribosyltransferases. The success of transition state analogue inhibitor design based on kinetic isotope effects validates this approach to understanding enzymatic transition states.

Topics: Animals; Catalysis; Crystallography, X-Ray; Deuterium; Drug Design; Enzyme Inhibitors; Enzymes; Humans; Hydrogen; Hydrogen Bonding; Inosine; Kinetics; Models, Molecular; Motion; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Static Electricity; Substrate Specificity; Thermodynamics

The genetic deficiency of human PNP causes a specific immunodeficiency by inducing apoptosis in dividing T-cells. Powerful inhibitors of PNP have been designed from the experimental determination of the transition state structure of PNPs. The Immucillins are transition state analogue inhibitors with Kd values as low as 7 pM. In the presence of deoxyguanosine the Immucillins kill activated human T-cells but not other cell types. The Immucillins are orally available and of low toxicity to mice. Immucillins also inhibit PNP from Plasmodium falciparum. Parasites cultured in human erythrocytes are killed by purine starvation in the presence of Immucillins and can be rescued by hypoxanthine.

Topics: Animals; Anti-Bacterial Agents; Cell Proliferation; Deoxyguanosine; Erythrocytes; Humans; Hypoxanthine; Kinetics; Lymphocyte Activation; Malaria; Models, Chemical; Phenotype; Plasmodium falciparum; Purine Nucleosides; Purines; Pyrimidinones; Pyrroles; T-Lymphocytes

Topics: Animals; Enzyme Inhibitors; Humans; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Structure-Activity Relationship

Newborns with a genetic deficiency of purine nucleoside phosphorylase (PNP) are normal, but exhibit a specific T-cell immunodeficiency during the first years of development. All other cell and organ systems remain functional. The biological significance of human PNP is degradation of deoxyguanosine, and apoptosis of T-cells occurs as a consequence of the accumulation of deoxyguanosine in the circulation, and dGTP in the cells. Control of T-cell proliferation is desirable in T-cell cancers, autoimmune diseases, and tissue transplant rejection. The search for powerful inhibitors of PNP as anti-T-cell agents has culminated in the immucillins. These inhibitors have been developed from knowledge of the transition state structure for the reactions catalyzed by PNP, and inhibit with picomolar dissociation constants. Immucillin-H (Imm-H) causes deoxyguanosine-dependent apoptosis of rapidly dividing human T-cells, but not other cell types. Human T-cell leukemia cells, and stimulated normal T-cells are both highly sensitive to the combination of Imm-H to block PNP and deoxyguanosine. Deoxyguanosine is the cytotoxin, and Imm-H alone has low toxicity. Single doses of Imm-H to mice cause accumulation of deoxyguanosine in the blood, and its administration prolongs the life of immunodeficient mice in a human T-cell tissue xenograft model. Immucillins are capable of providing complete control of in vivo PNP levels and hold promise for treatment of proliferative T-cell disorders.

Topics: Animals; Antineoplastic Agents; Drug Design; Enzyme Inhibitors; Humans; Leukemia, T-Cell; Mice; Nucleosides; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Static Electricity; T-Lymphocytes; Transplantation, Heterologous

Peripheral T cell lymphomas are an aggressive group of non-Hodgkin lymphomas with poor outcomes for most subtypes and no accepted standard of care for relapsed patients. This study evaluated the efficacy and safety of forodesine, a novel purine nucleoside phosphorylase inhibitor, in patients with relapsed peripheral T cell lymphomas. Patients with histologically confirmed disease, progression after ≥ 1 prior treatment, and an objective response to last treatment received oral forodesine 300 mg twice-daily. The primary endpoint was objective response rate (ORR). Secondary endpoints included duration of response, progression-free survival (PFS), overall survival (OS), and safety. Forty-eight patients (median age, 69.5 years; median of 2 prior treatments) received forodesine. In phase 1 (n = 3 evaluable), no dose-limiting toxicity was observed during the first 28 days of forodesine treatment. In phase 2 (n = 41 evaluable), the ORR for the primary and final analyses was 22% (90% CI 12-35%) and 25% (90% CI 14-38%), respectively, including four complete responses (10%). Median PFS and OS were 1.9 and 15.6 months, respectively. The most common grade 3/4 adverse events were lymphopenia (96%), leukopenia (42%), and neutropenia (35%). Dose reduction and discontinuation due to adverse events were uncommon. Secondary B cell lymphoma developed in five patients, of whom four were positive for Epstein-Barr virus. In conclusion, forodesine has single-agent activity within the range of approved therapies in relapsed peripheral T cell lymphomas, with a manageable safety profile, and may represent a viable treatment option for this difficult-to-treat population.

Topics: Administration, Oral; Adult; Aged; Female; Humans; Lymphoma, T-Cell, Peripheral; Male; Middle Aged; Purine Nucleosides; Pyrimidinones; Recurrence

Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP) that leads to intracellular accumulation of deoxyguanosine triphosphate (dGTP) in T and B cells, resulting in apoptosis. Forodesine has demonstrated impressive antitumor activity in early phase clinical trials in cutaneous T-cell lymphoma (CTCL).. In this phase II study, patients with CTCL who had already failed three or more systemic therapies were recruited. We investigated the response rate, safety and tolerability of oral forodesine treatment in subjects with cutaneous manifestations of CTCL, stages IB, IIA, IIB, III and IVA. The safety population encompassing all stages was used for analysis of accountability, demographics and safety. The efficacy population differed from the safety population by exclusion of stage IB and IIA patients.. All 144 patients had performance status 0-2. The median duration of CTCL from diagnosis was 53 months (5-516 months). The median number of pretreatments was 4 (range: 3-15). No complete remissions were observed. In the efficacy group of patients, 11% achieved partial remission and 50% had stable disease. The median time to response was 56 days and the median duration of response was 191 days. A total of 96% of all treated patients reported one or more adverse events (AEs) and 33% reported a serious AE. The majority of AEs were classified as mild or moderate in severity. The most commonly reported AEs (>10%) were peripheral edema, fatigue, insomnia, pruritus, diarrhea, headache and nausea. Overall eight patients died during the study: five due to sepsis and infections, one due to a second malignancy (esophageal cancer), one due to disease progression and one due to liver failure.. Oral forodesine at a dose of 200 mg daily is feasible and shows partial efficacy in this highly selected CTCL population and some durable responses.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Female; Humans; Male; Middle Aged; Mycosis Fungoides; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Sezary Syndrome; Skin Neoplasms; Treatment Failure

The discovery that purine nucleoside phosphorylase (PNP) deficiency leads to T-cell lymphopenia was the basis for introducing PNP inhibitors for T-cell leukemias. Forodesine is an orally bioavailable PNP inhibitor with picomolar potency. Because T lymphoblasts and indolent chronic lymphocytic leukemia (CLL) B cells inherently elicit favorable pharmacokinetics to accumulate deoxyguanosine triphosphate (dGTP), forodesine demonstrated promising activity in preclinical and clinical settings for patients with T-cell acute lymphoblastic leukemia (T-ALL) and B-cell CLL (B-CLL). However, the use of forodesine in B-cell ALL (B-ALL) is unknown.. Leukemic blasts obtained from pediatric patients with de novo B-ALL (n = 10) were incubated with forodesine and deoxyguanosine (dGuo), and the biological end points of apoptosis, intracellular dGTP accumulation, and inhibition of RNA and DNA synthesis were measured. Additionally, adult patients with B-ALL (n = 2) were intravenously infused with 80 mg/m(2)/d daily for 5 days. After therapy, clinical response, toxicity, laboratory biomarkers including PNP enzyme inhibition, and plasma forodesine, dGuo, and intracellular dGTP levels were analyzed.. Our in vitro investigations demonstrated that forodesine treatment inhibited proliferation and induced modest apoptosis in de novo B-ALL lymphoblasts. There was time-dependent accumulation of dGTP and inhibition of RNA and DNA synthesis. During therapy, neither patient achieved a complete response (CR), but there was disease stabilization for several weeks in both patients. There was significant maintained inhibition of PNP enzyme in red blood cells, accumulation of forodesine and dGuo in plasma, and intracellular dGTP accumulation in both patients.. Our preclinical and clinical investigations suggest that forodesine has activity in B-ALL. However, it needs to be either infused with dGuo or combined with established chemotherapeutic agents based on mechanistic rationale.

Topics: Acute Disease; Adolescent; Adult; Apoptosis; Child; Child, Preschool; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Female; Humans; Infant; Male; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Pyrimidinones; Young Adult

BCX1777 (forodesine), a novel purine nucleoside phosphorylase inhibitor, induces apoptosis, mainly in T cells. To evaluate the safety, tolerability, and pharmacokinetics of BCX1777, we conducted a phase I study in patients with relapsed or refractory peripheral T/natural killer-cell malignancies. Eligible patients had relapsed or refractory peripheral T/natural killer-cell malignancies without any major organ dysfunction. BCX1777 was administered orally once daily (dose escalation: 100, 200, and 300 mg) until disease progression requiring new therapy or unacceptable adverse events occurred. A total of 13 patients were enrolled and treated in three dose cohorts (100 mg/day, five patients; 200 mg/day, three patients; 300 mg/day, five patients). Although none of the patients developed dose-limiting toxicities, further dose escalation was not performed based on data from overseas. Therefore, the maximum tolerated dose was not determined. Adverse events of grade 3 or greater (≥2 patients) included lymphopenia (62%), anemia (15%), leukopenia (8%), and pyrexia (8%). Plasma pharmacokinetics parameter of BCX1777 (area under the plasma concentration-time curve) at day 1 in each cohort was 1948 ± 884, 4608 ± 1030, and 4596 ± 939 ng•h/mL, respectively. Disease control was achieved in approximately half of patients. One patient with anaplastic large cell lymphoma, which was negative for anaplastic lymphoma kinase, achieved a complete response, and two patients with cutaneous T-cell lymphoma achieved partial responses. BCX1777 was well tolerated at doses up to 300 mg once daily and showed preliminary evidence of activity in relapsed or refractory peripheral T/natural killer-cell malignancies, warranting further investigation.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Area Under Curve; Cohort Studies; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Resistance, Neoplasm; Exanthema; Female; Humans; Lymphoma, Large-Cell, Anaplastic; Lymphoma, T-Cell; Lymphoma, T-Cell, Peripheral; Lymphopenia; Male; Metabolic Clearance Rate; Middle Aged; Mycosis Fungoides; Natural Killer T-Cells; Purine Nucleosides; Pyrimidinones; Recurrence; Skin Neoplasms; Treatment Outcome

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bendamustine Hydrochloride; Boronic Acids; Bortezomib; Brentuximab Vedotin; Diphtheria Toxin; Drug Discovery; Everolimus; Humans; Hydroxamic Acids; Immunoconjugates; Indoles; Inotuzumab Ozogamicin; Interleukin-2; Lenalidomide; Lymphoma; Lymphoma, B-Cell; Nitrogen Mustard Compounds; Purine Nucleosides; Pyrazines; Pyrimidinones; Recombinant Fusion Proteins; Sirolimus; Thalidomide; Vorinostat

Forodesine is a new and potent purine nucleoside phosphorylase (PNP) inhibitor. Patients with chronic lymphocytic leukemia (CLL) with primary resistance to fludarabine-based therapy or with progressive disease were eligible for oral forodesine (200 mg/d) for up to 24 weeks. Eight patients with median lymphocyte count of 35.9 x 10(9)/L and median serum beta2 microglobulin level of 6.45 mg/L were treated. Six had Rai stage III to IV and were previously heavily treated (median prior therapy = 5). Two had transient decrease in lymphocyte count to normal, whereas in 5, disease progressed. Adverse events were mild. Steady-state level of forodesine ranged from 200 to 1300 nM and did not reach desired 2 microM level. PNP inhibition ranged from 57% to 89% and steady-state 2'-deoxyguanosine (dGuo) concentration median was 1.8 microM. Intracellular deoxyguanosine triphosphate (dGTP) increase was very modest, from median of 6 microM to 10 microM. Compared with in vivo, in vitro incubations of CLL lymphocytes with 10 or 20 microM dGuo and forodesine (2 microM) resulted in accumulation of higher levels of dGTP (40-250 microM) which resulted in increase in apoptosis. Forodesine has biologic activity in CLL; pharmacodynamic parameters suggest that an alternate dosing schedule and/or higher doses to achieve greater intracellular dGTP may be beneficial in this patient population.

Topics: Administration, Oral; Aged; Antineoplastic Agents; Apoptosis; Drug Administration Schedule; Drug Therapy, Combination; Enzyme Inhibitors; Female; Follow-Up Studies; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Count; Lymphocytes; Male; Middle Aged; Phosphoric Monoester Hydrolases; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Severity of Illness Index; Vidarabine

The discovery of purine nucleoside phosphorylase (PNP) deficiency and T lymphocytopenia suggested that inhibition of this enzyme could serve as a therapeutic target. Inhibitors of PNP failed until structure-based synthesis of immucillin-H (BCX-1777, forodesine), a transition-state analog of PNP. The picomolar potency for PNP, T cell-selective cytotoxicity, and animal studies provided the rationale for use of forodesine in T-cell malignancies. Five patients were treated with an intravenous infusion of forodesine (40 mg/m2) on day 1; treatment continued on day 2; forodesine was administered every 12 hours for an additional 8 doses. Plasma and cellular pharmacokinetics and pharmaco-dynamics were investigated. Median peak level of forodesine (5.4 microM) was achieved at the end of infusion. This level was sufficient to increase plasma 2'-deoxyguanosine (dGuo) concentrations in all patients. Intracellular deoxyguanosine triphosphate (dGTP) increased by 2- to 40-fold in 4 of 5 patients (8 of 9 courses) and correlated with antileukemia activity in 4 patients. However, objective responses were not observed. This was the first clinical study in humans to demonstrate the plasma pharmacokinetics and the pharmacodynamic effectiveness of the PNP inhibitor, forodesine; however, regrowth of leukemia cells in the blood and marrow after course 1 suggested that a different therapeutic schedule should be considered for future studies.

Topics: Adult; Aged; Deoxyguanine Nucleotides; Enzyme Inhibitors; Female; Humans; Leukemia, Lymphoid; Male; Middle Aged; Molecular Structure; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Treatment Outcome

Topics: Central Nervous System; Humans; Lymphoma, T-Cell, Peripheral; Purine Nucleosides; Pyrimidinones

Topics: Humans; Lymphoma, T-Cell, Peripheral; Purine Nucleosides; Pyrimidinones; Retrospective Studies

The de novo synthesis of piperidine nucleosides from our homologating agent 5,6-dihydro-1,4-dithiin is herein reported. The structure and conformation of nucleosides were conceived to faithfully resemble the well-known nucleoside drugs Immucillins H and A in their bioactive conformation. NMR analysis of the synthesized compounds confirmed that they adopt an iminosugar conformation bearing the nucleobases and the hydroxyl groups in the appropriate orientation.

Topics: Adenine; Adenosine; Magnetic Resonance Spectroscopy; Molecular Conformation; Nucleosides; Piperidines; Purine Nucleosides; Pyrimidinones; Pyrrolidines; Structure-Activity Relationship

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

Topics: Animals; Deoxyguanine Nucleotides; Drug Resistance, Neoplasm; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Mice; Mice, Inbred C57BL; Purine Nucleosides; Pyrimidinones; SAM Domain and HD Domain-Containing Protein 1

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 μM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.

Topics: Adenine; Adenosine; Animals; Antiprotozoal Agents; Cell Proliferation; Enzyme Inhibitors; Female; Humans; In Vitro Techniques; Kinetics; Leishmania infantum; Leishmania mexicana; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; N-Glycosyl Hydrolases; Purine Nucleosides; Pyrimidinones; Pyrroles; Pyrrolidines

Immucillins ImmA (IA), ImmH (IH) and SerMe-ImmH (SMIH) are synthetic deazapurine nucleoside analogues that inhibit Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro without macrophage toxicity. Immucillins are compared to the Glucantime standard drug in the chemotherapy of Leishmania (L.) infantum chagasi infection in mice and hamsters. These agents are tested for toxicity and immune system response.. BALB/c mice were infected with 107 amastigotes, treated with IA, IH, SMIH or Glucantime (2.5mg/kg/day) and monitored for clinical variables, parasite load, antibody levels and splenocyte IFN-γ, TNF-α, and IL-10 expression. Cytokines and CD4+, CD8+ and CD19+ lymphocyte frequencies were assessed in uninfected controls and in response to immucillins. Urea, creatinine, GOT and GPT levels were monitored in sera. Anti-Leishmania-specific IgG1 antibodies (anti-NH36) increased in untreated animals. IgG2a response, high levels of IFN-γ, TNF-α and lower levels of IL-10 were detected in mice treated with the immucillins and Glucantime. Immucillins permitted normal weight gain, prevented hepato-splenomegaly and cleared the parasite infection (85-89%) without renal and hepatic toxicity. Immucillins promoted 35% lower secretion of IFN-γ in uninfected controls than in infected mice. IA and IH increased the CD4+ T and CD19+ B cell frequencies. SMIH increased only the proportion of CD-19 B cells. IA and IH also cured infected hamsters with lower toxicity than Glucantime.. Immucillins IA, IH and SMIH were effective in treating leishmaniasis in mice. In hamsters, IA and IH were also effective. The highest therapeutic efficacy was obtained with IA, possibly due to its induction of a TH1 immune response. Low immucillin doses were required and showed no toxicity. Our results disclose the potential use of IA and IH in the therapy of visceral leishmaniasis.

Topics: Adenine; Adenosine; Animals; Antibodies, Protozoan; Antiprotozoal Agents; Blood Chemical Analysis; Disease Models, Animal; Drug-Related Side Effects and Adverse Reactions; Female; Gene Expression; Immunophenotyping; Interferon-gamma; Interleukin-10; Leishmania; Leishmaniasis, Visceral; Leukocytes, Mononuclear; Mesocricetus; Mice, Inbred BALB C; Parasite Load; Purine Nucleosides; Pyrimidinones; Pyrrolidines; Spleen; T-Lymphocyte Subsets; Treatment Outcome; Tumor Necrosis Factor-alpha

The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5'-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5' groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5'-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2'-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5'-methylthioinosine and similarly 5'-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.

Topics: Catalysis; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Kinetics; Protozoan Proteins; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Substrate Specificity; Toxoplasma

Forodesine inhibits purine nucleoside phosphorylase, resulting in an accumulation of intracellular dGTP and consequently cell death. 9-β-D-Arabinofuranosylguanine (ara-G) is an active compound of nelarabine that is intracellularly phosphorylated to a triphosphate form, which inhibits DNA synthesis. Both agents show cytotoxicity toward T-cell malignancies. In the present study, we investigated the cytotoxicity of forodesine in vitro using ara-G-resistant leukemia cells.. T-Lymphoblastic leukemia cell line CCRF-CEM and ara-G-resistant CEM variant cell line CEM/ara-G that we had previously established were used.. A growth-inhibition assay demonstrated that CEM cells were insensitive to single-agent forodesine treatment. The cells were also insensitive to deoxyguanosine at a maximal concentration of 10 μM. CEM/ara-G cells were 80-fold more resistant to ara-G than were CEM cells, and the mode of sensitivity to forodesine and deoxyguanosine was similar to that of CEM cells. In the presence of 10 μM deoxyguanosine, forodesine effectively inhibited the growth of CEM cells but not that of CEM/ara-G cells. Flow cytometric analyses showed that combination of forodesine and deoxyguanosine induced apoptosis of CEM cells but not of CEM/ara-G cells. The addition of ara-G did not augment the cytotoxicity of the forodesine/deoxyguanosine combination towards CEM cells or CEM/ara-G cells. The combination index revealed antagonism between forodesine and ara-G. The intracellular production of ara-G triphosphate was reduced in the presence of forodesine.. Nelarabine-resistant CEM/ara-G cells are insensitive to forodesine.

Topics: Antineoplastic Agents; Arabinonucleosides; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; HL-60 Cells; Humans; Lymphoma, B-Cell; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme that catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to their respective bases and (deoxy)ribose-1-phosphate. It is a key enzyme in the purine salvage pathway of mammalian cells. The present investigation sought to determine whether the PNP transition state analog inhibitor (Immucillin-H) arrests bone loss in two models of induced periodontal disease in rats. Periodontal disease was induced in rats using ligature or LPS injection followed by administration of Immucillin-H for direct analysis of bone loss, histology and TRAP staining. In vitro osteoclast differentiation and activation of T CD4+ cells in the presence of Immucillin-H were carried out for assessment of RANKL expression, PNP and Cathepsin K activity. Immucillin-H inhibited bone loss induced by ligatures and LPS, leading to a reduced number of infiltrating osteoclasts and inflammatory cells. In vitro assays revealed that Immucillin-H could not directly abrogate differentiation of osteoclast precursor cells, but affected lymphocyte-mediated osteoclastogenesis. On the other hand, incubation of pre-activated T CD4+ with Immucillin-H decreased RANKL secretion with no compromise of cell viability. The PNP transition state analog Immucillin-H arrests bone loss mediated by T CD4+ cells with no direct effect on osteoclasts. PNP inhibitor may have an impact in the treatment of diseases characterized by the presence of pathogens and imbalances of bone metabolism.

Topics: Animals; CD4-Positive T-Lymphocytes; Coculture Techniques; Enzyme Inhibitors; Lymphocyte Activation; Mice; Periodontal Diseases; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Rats; Rats, Wistar

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.

Topics: Animals; Binding Sites; Calorimetry; Catalytic Domain; Cattle; Fluorometry; Hypoxanthine; Ligands; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Recombinant Proteins; Thermodynamics

A simple, efficient, and highly sensitive in-line CE method was developed for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) present in membrane preparations of human 1539 melanoma cells. After filling the running buffer (50 mM borate buffer, 100 mM SDS, pH 9.10) into a fused-silica capillary (50 cm effective length × 75 μm), a large sample volume was loaded by hydrodynamic injection (5 psi, 36 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-20 kV). The current was monitored and the polarity was reversed when 95% of the current had been recovered. The separation of the neutral analytes (nucleosides and nucleobases) was performed by applying a voltage of 15 kV. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. For inosine and adenine detection limits as low as 60 nM were achieved. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analysis using CE with UV detection reported so far. The Michaelis-Menten constants (K(m)) for PNP and ADA and the inhibition constants (K(i)) for standard inhibitors determined with the new method were consistent with literature data.

Topics: Adenosine Deaminase; Borates; Cell Line, Tumor; Electrodes; Electrophoresis, Capillary; Enzyme Inhibitors; Humans; Kinetics; Linear Models; Melanoma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Sensitivity and Specificity

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Topics: Antineoplastic Agents; Arabinonucleosides; Cell Line, Tumor; Child; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Leukemia, Prolymphocytic, B-Cell; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Purine Nucleosides; Purines; Pyrimidinones; RNA, Messenger; RNA, Neoplasm

Purine nucleoside phosphorylase (PNP) (EC.2.4.2.1) is an enzyme that catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is involved in purine-salvage pathway and has been proposed as a promising target for design and development of antimalarial and antibacterial drugs. Recent elucidation of the three-dimensional structure of PNP by X-ray protein crystallography left open the possibility of structure-based virtual screening initiatives in combination with molecular dynamics simulations focused on identification of potential new antimalarial drugs. Most of the previously published molecular dynamics simulations of PNP were carried out on human PNP, a trimeric PNP. The present article describes for the first time molecular dynamics simulations of hexameric PNP from Plasmodium falciparum (PfPNP). Two systems were simulated in the present work, PfPNP in ligand free form, and in complex with immucillin and sulfate. Based on the dynamical behavior of both systems the main results related to structural stability and protein-drug interactions are discussed.

Topics: Binding Sites; Biocatalysis; Enzyme Stability; Molecular Dynamics Simulation; Phosphates; Plasmodium falciparum; Pliability; Protein Multimerization; Protein Structure, Tertiary; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Sulfates; Time Factors

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the K(m) and/or K(i) for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the K(i) for PfENT1 transport of hypoxanthine and adenosine is in the 300-700microM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport K(m). PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [(3)H]hypoxanthine or [(3)H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Topics: Animals; Biological Transport; Enzyme Inhibitors; Kinetics; Metabolic Networks and Pathways; Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins; Oocytes; Plasmodium falciparum; Protozoan Proteins; Purine Nucleosides; Purines; Pyrimidinones; Tubercidin

Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

Topics: Amides; Amino Acid Sequence; Binding Sites; Catalysis; Deuterium Exchange Measurement; Entropy; Humans; Molecular Sequence Data; Peptides; Protein Binding; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Thermodynamics; Water

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*i(1/4) 58 pM, first generation)contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*i(1/4) 9 pM, second-generation),uses a methylene-bridged dihydroxypyrrolidine cation with twoasymmetric centers.DATMe-Immucillin-H (K*i(1/4)9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*i(1/4) 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5'-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Topics: Animals; Catalytic Domain; Cattle; Enzyme Inhibitors; Humans; Models, Molecular; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines; Thermodynamics

We present a technique that uses (13)C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (k(cat)/K(m)) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase-catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.

Topics: Bacterial Proteins; Carbon Isotopes; Catalysis; Enzymes; Hydrolysis; Isotope Labeling; Isotopes; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; N-Acetylneuraminic Acid; Neuraminidase; Purine Nucleosides; Pyrimidinones; Vibrio cholerae

Forodesine, a purine nucleoside phosphorylase inhibitor, displays in vitro activity in chronic lymphocytic leukemia (CLL) cells in presence of dGuo, which is the basis for an ongoing clinical trial in patients with fludarabine-refractory CLL. Initial clinical data indicate forodesine has significant activity on circulating CLL cells, but less activity in clearing CLL cells from tissues such as marrow. In tissue microenvironments, lymphocytes interact with accessory stromal cells that provide survival and drug-resistance signals, which may account for residual disease. Therefore, we investigated the impact of marrow stromal cells (MSCs) on forodesine-induced response in CLL lymphocytes. We demonstrate that spontaneous and forodesine-induced apoptosis of CLL cells was significantly inhibited by human and murine MSCs. Forodesine-promoted dGuo triphosphate (dGTP) accumulation and GTP and ATP depletion in CLL cells was inhibited by MSCs, providing a mechanism for resistance. Also, MSCs rescued CLL cells from forodesine-induced RNA- and protein-synthesis inhibition and stabilized and increased Mcl-1 transcript and protein levels. Conversely, MSC viability was not affected by forodesine and dGuo. Collectively, MSC-induced biochemical changes antagonized forodesine-induced CLL cell apoptosis. This provides a biochemical mechanism for MSC-derived resistance to forodesine and emphasizes the need to move toward combinations with agents that interfere with the microenvironment's protective role for improving current therapeutic efforts.

Topics: Animals; Apoptosis; Blotting, Western; Bone Marrow; Cell Proliferation; Cells, Cultured; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Mesenchymal Stem Cells; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stromal Cells

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit-subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top (beta) surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solvent-exposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an on-enzyme thermodynamic equilibrium constant (Keq) near unity in the temperature range between 20 and 30 degrees C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano- to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (approximately 200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Topics: Catalytic Domain; Humans; Kinetics; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Protein Binding; Protein Conformation; Protein Subunits; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Thermodynamics; Tryptophan

We investigate the kinetic effects of antibody variable domain fragments derived from heavy chain antibodies (VHH domains) that behave as allosteric effectors of the nucleoside hydrolase from Trypanosoma vivax (TvNH). Strikingly, these antibodies can stimulate or inhibit TvNH steady-state activity, depending on the substrate used. This effect was investigated in greater detail using steady-state and pre-steady-state kinetic experiments. The most potent allosteric effector, VHH 1589, inhibits certain steps on the TvNH catalytic pathway (e.g. N-glycosidic bond cleavage) but increases the rates of others (e.g. substrate and product release). For the natural nucleoside 7-methyl guanosine, where product ribose release is rate determining, the net effect of VHH 1589 binding is to increase k(cat). For the poor substrate pNPR, VHH 1589 causes chemistry (O-glycosidic bond cleavage) to become rate determining and both k(cat)/K(m) and k(cat) to decrease. Thus, the substrate-dependent effects of VHH 1589 binding are caused by differences in the relative rates of chemistry with respect to subsequent steps on the catalytic pathway for these two substrates. We discuss possible mechanisms for these kinetic effects and the implications for allosteric effector drug development.

Topics: Allosteric Regulation; Animals; Catalysis; Immunoglobulin Variable Region; Kinetics; N-Glycosyl Hydrolases; Peptide Fragments; Protein Structure, Tertiary; Purine Nucleosides; Pyrimidinones; Trypanosoma vivax

Forodesine HCl is being investigated as a potential therapeutic target for the control of T-cell proliferation. During the filing process for a New Drug Application (NDA) it became evident that there was a need to synthesize some stereo-isomers of forodesine HCl. Herein we present the synthesis of these three novel compounds (2-4).

Topics: Cell Proliferation; Enzyme Inhibitors; Humans; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; T-Lymphocytes

Forodesine HCl is being investigated as a potential therapeutic target for the control of T-cell proliferation. During our ongoing process development work on forodesine HCl several novel compounds were identified as possible impurities in the process. Herein we present the synthesis of three novel compounds (2-4).

Topics: Cell Proliferation; Enzyme Inhibitors; Humans; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; T-Lymphocytes

Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiotreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase-a (GPa), and phosphotransacetylase (PTA). Using a multidisciplinary approach, we explored the mechanism whereby these enzymes mediate AsV reduction. It is known that PNP cleaves inosine with AsV into hypoxanthine and ribose-1-arsenate. In presence of inosine, AsV and DTT, PNP mediates AsIII formation. In this study, we incubated PNP first with inosine and AsV, allowing the arsenolytic reaction to run, then blocked this reaction with the PNP inhibitor BCX-1777, added DTT and continued the incubation. Despite inhibition of PNP, large amount of AsIII was formed in these incubations, indicating that PNP does not reduce AsV directly but forms a product (i.e., ribose-1-arsenate) that is reduced to AsIII by DTT. Similar studies with the other arsenolytic enzymes (GPa, GAPDH, and PTA) yielded similar results. Various thiols that differentially supported AsV reduction when present during PNP-catalyzed arsenolysis (DTT approximately dimercaptopropane-1-sulfonic acid > mercaptoethanol > DMSA > GSH) similarly supported AsV reduction when added only after a transient PNP-catalyzed arsenolysis, which preformed ribose-1-arsenate. Experiments with progressively delayed addition of DTT after BCX-1777 indicated that ribose-1-arsenate is short-lived with a half-life of 4 min. In conclusion, phosphorolytic enzymes, such as PNP, GAPDH, GPa, and PTA, promote thiol-dependent AsV reduction because they convert AsV into arsenylated products reducible by thiols more readily than AsV. In support of this view, reactivity studies using conceptual density functional theory reactivity descriptors (local softness, nucleofugality) indicate that reduction by thiols of the arsenylated metabolites is favored over AsV.

Topics: Acetyl Coenzyme A; Animals; Arsenates; Arsenites; Bacterial Proteins; Cattle; Dithiothreitol; Enzyme Inhibitors; Glutathione; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycogen Phosphorylase; Half-Life; Inosine; Kinetics; Mercaptoethanol; Models, Chemical; Oxidation-Reduction; Phosphate Acetyltransferase; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Rabbits; Sodium Compounds; Succimer; Sulfhydryl Compounds; Unithiol

Chronic lymphocytic leukemia (CLL) is an incurable disease derived from the monoclonal expansion of CD5(+) B lymphocytes. High expression levels of ZAP-70 or CD38 and deletions of 17p13 (TP53) and 11q22-q23 (ATM) are associated with poorer overall survival and shorter time to disease progression. DNA damage and p53 play a pivotal role in apoptosis induction in response to conventional chemotherapy, because deletions of ATM or p53 identify CLL patients with resistance to treatment. Forodesine is a transition-state inhibitor of the purine nucleoside phosphorylase with antileukemic activity. We show that forodesine is highly cytotoxic as single agent or in combination with bendamustine and rituximab in primary leukemic cells from CLL patients regardless of CD38/ZAP-70 expression and p53 or ATM deletion. Forodesine activates the mitochondrial apoptotic pathway by decreasing the levels of antiapoptotic MCL-1 protein and induction of proapoptotic BIM protein. Forodesine induces transcriptional up-regulation of p73, a p53-related protein able to overcome the resistance to apoptosis of CLL cells lacking functional p53. Remarkably, no differences in these apoptotic markers were observed based on p53 or ATM status. In conclusion, forodesine induces apoptosis of CLL cells bypassing the DNA-damage/ATM/p53 pathway and might represent a novel chemotherapeutic approach that deserves clinical investigation.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Bendamustine Hydrochloride; Cyclophosphamide; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Gene Expression Regulation, Leukemic; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Membrane Proteins; Mitochondria; Nitrogen Mustard Compounds; Nuclear Proteins; Proto-Oncogene Proteins; Purine Nucleosides; Pyrimidinones; Rituximab; Tumor Cells, Cultured; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Vidarabine

Neighboring-group participation in the reaction catalyzed by purine nucleoside phosphorylase involves a compression mode between the 5'- and 4'-ribosyl oxygens, facilitated by His257. The His257Gly mutant opens a space in the catalytic site. Hydrophobic 5'-substituted Immucillins are transition-state analogue inhibitors of this mutant enzyme. Dissociation constants as low as 2pM are achieved, with K(m)/K(d) as high as 400,000,000.

Topics: Catalytic Domain; Crystallography, X-Ray; Glycine; Histidine; Humans; Imidazoles; Kinetics; Mutation; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Structure-Activity Relationship

Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.

Topics: Crystallography, X-Ray; Enzyme Inhibitors; Humans; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines; Stereoisomerism; Substrate Specificity

Topics: Binding, Competitive; Humans; Inosine; Kinetics; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Thermodynamics; Tritium

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. (3)H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas (3)H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Deoxycytidine Kinase; Deoxyguanosine; Drug Screening Assays, Antitumor; Humans; Hypoxanthine Phosphoribosyltransferase; Leukemia, T-Cell; Models, Biological; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

Peripheral T-cell lymphomas (PTCLs) are a rare and diverse group of neoplasms with a poor prognosis. Management of these disorders has been largely extrapolated from the treatment of aggressive B-cell lymphomas; however, therapeutic responses to this approach are neither adequate nor durable for most patients with PTCL. Given the rarity of PTCL, much of the literature consists of studies with small sample size and anecdotal case reports. Therefore, no consensus exists on the best therapeutic strategy for either newly diagnosed or relapsed/refractory PTCL. This article reviews promising novel approaches in the treatment of PTCL and its subtypes. Investigation into the pathogenesis of PTCL has also identified new targets for treatment. These emerging therapies include new uses of existing agents and the development of novel agents specifically targeted against T-cell lymphoma. Results using antimetabolites, immunotherapies, and histone deacetylase inhibitors have been particularly encouraging. These novel therapies are being tested as single agents and in combination with conventional lymphoma regimens in the frontline and salvage settings. Because of the rarity and heterogeneity of PTCL, national and international cooperation is needed to conduct the clinical studies required for the development of more effective treatment paradigms. These efforts are ongoing and will hopefully guide new strategies to improve the historically poor outcome of PTCL.

Topics: Alemtuzumab; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Neoplasm; Antineoplastic Agents; Arabinonucleosides; Deoxycytidine; Diphtheria Toxin; Gemcitabine; Histone Deacetylase Inhibitors; Humans; Immunosuppressive Agents; Interleukin-2; Lymphoma, T-Cell, Peripheral; Practice Guidelines as Topic; Protease Inhibitors; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Recombinant Fusion Proteins; Vascular Endothelial Growth Factor A

This article presents two case studies of patients diagnosed with T-cell acute lymphoblastic leukemia who relapsed following allogeneic hematopoietic stem cell transplantation and were subsequently enrolled in a clinical trial in which they received forodesine hydrochloride, a rationally designed, potent, transition-state inhibitor of purine nucleoside phosphorylase. Forodesine induced complete remission in both patients. Graft-versus-host disease developed subsequently but was treated successfully with conventional immunosuppressive therapy. Both patients remain in complete remission at the most recent follow-up. We hypothesize that forodesine contributed to a primary anti-leukemic cytotoxic effect as well as a secondary immunologic effect by allowing the development of an ongoing graft-versus-leukemia effect in these patients.

Topics: Adult; Antineoplastic Agents; Child, Preschool; Disease-Free Survival; Female; Graft vs Leukemia Effect; Hematopoietic Stem Cell Transplantation; Humans; Male; Neoplasm Recurrence, Local; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

(1R)-1-(9-Deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-L-ribitol [(+)-5] and (3S,4S)-1-[(9-deazahypoxanthin-9-yl)methyl]-4-(hydroxymethyl)pyrrolidin-3-ol [(-)-6] are the L-enantiomers of immucillin-H (D-ImmH) and DADMe-immucillin-H (D-DADMe-ImmH), respectively, these D-isomers being high affinity transition state analogue inhibitors of purine nucleoside phosphorylases (PNPases) developed as potential pharmaceuticals against diseases involving irregular activation of T-cells. The C-nucleoside hydrochloride D-ImmH [(-)-5) x HCl], now "Fodosine" is in phase II clinical trials as an anti-T-cell leukaemia agent, while D-DADMe-ImmH is a second generation inhibitor with extreme binding to the target enzyme and has entered the clinic for phase I testing as an anti-psoriasis drug. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the L-nucleoside analogues (+)-5 x HCl and (-)-6, respectively, of D-ImmH and D-DADMe-ImmH, were prepared and their PNPase binding properties were studied. For the synthesis of compound (-)-6 suitable enzyme-based routes to the enantiomerically pure starting material (3S,4S)-4-(hydroxymethyl)pyrrolidin-3-ol [(-)-6] and its enantiomer were developed. The L-enantiomers (+)-5 x HCl and (-)-6 bind to the PNPases approximately 5- to 600-times less well than do the D-compounds, but nevertheless remain powerful inhibitors with nanomolar dissociation constants.

Topics: Enzyme Inhibitors; Indicators and Reagents; Kinetics; Models, Molecular; Molecular Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines; Stereoisomerism

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase with an "open" active site with a highly flexible loop (loop 2) in its vicinity. Here, we present the crystal structures of the T. vivax nucleoside hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co)) transition-state inhibitor immucillin H (ImmH or (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and 2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is ordered and folds over the active site, establishing previously unobserved enzyme-inhibitor interactions. As such this structure presents the first complete picture of a purine-specific NH active site, including leaving group interactions. In the closed active site, a water channel of highly ordered water molecules leads out from the N7 of the nucleoside toward bulk solvent, while Trp260 approaches the nucleobase in a tight parallel stacking interaction. Together with mutagenesis results, this structure rules out a mechanism of leaving group activation by general acid catalysis, as proposed for base-aspecific nucleoside hydrolases. Instead, the structure is consistent with the previously proposed mechanism of leaving group protonation in the T. vivax nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow protonation by solvent. A mechanism that couples loop closure to the positioning of active site residues is proposed based on a comparison of the soaked structure with the co-crystallized structure. Interestingly, the dimer interface area increases by 40% upon closure of loop 2, with loop 1 of one subunit interacting with loop 2 of the other subunit, suggesting a relationship between the dimeric form of the enzyme and its catalytic activity.

Topics: Animals; Binding Sites; Crystallography, X-Ray; Ligands; Models, Molecular; Molecular Structure; N-Glycosyl Hydrolases; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Purine Nucleosides; Pyrimidinones; Pyrroles; Trypanosoma vivax

Transition state analogues of PNP, the Immucillins and DADMe-Immucillins, were designed to match transition state features of bovine and human PNPs, respectively. The inhibitors with or without the hydroxyl and hydroxymethyl groups of the substrate demonstrate that inhibitor geometry mimicking that of the transition state confers binding affinity discrimination. This finding is remarkable since crystallographic analysis indicates complete conservation of active site residues and contacts to ligands in human and bovine PNPs.

Topics: Animals; Arsenates; Cattle; Enzyme Inhibitors; Erythrocytes; Humans; Mice; Molecular Structure; Protein Binding; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients.

Topics: Antineoplastic Agents; Apoptosis; B-Lymphocytes; Caspases; DNA Damage; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Mitochondria; Phosphorylation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; T-Lymphocytes; Tumor Suppressor Protein p53

Human purine nucleoside phosphorylase has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Recently, several structures of human PNP have been reported, which allowed redefinition of the active site and understanding of the structural basis for inhibition of PNP by acyclovir and immucillin-H. Based on previously solved human PNP structures, we proposed here a new catalytic mechanism for human PNP, which is supported by crystallographic studies and explains previously determined kinetic data.

Topics: Acyclovir; Binding Sites; Catalysis; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Models, Molecular; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

Immucillin and DADMe-Immucillin inhibitors are tight binding transition state mimics of purine nucleoside phosphorylases (PNP). 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is proposed to form a similar transition state structure as PNP. The companion paper describes modifications of the Immucillin and DADMe-Immucillin inhibitors to better match transition state features of MTAN and have led to 5'-thio aromatic substitutions that extend the inhibition constants to the femtomolar range (Singh, V., Evans, G. B., Lenz, D. H., Mason, J., Clinch, K., Mee, S., Painter, G. F., Tyler, P. C., Furneaux, R. H., Lee, J. E., Howell, P. L., and Schramm, V. L. (2005) J. Biol. Chem. 280, 18265-18273). 5'-Methylthio-Immucillin A (MT-ImmA) and 5'-methylthio-DADMe-Immucillin A (MT-DADMe-ImmA) exhibit slow-onset inhibition with K(i)(*) of 77 and 2 pm, respectively, and were selected for structural analysis as the parent compounds of each class of transition state analogue. The crystal structures of Escherichia coli MTAN complexed with MT-ImmA and MT-DADMe-ImmA were determined to 2.2 A resolution and compared with the existing MTAN inhibitor complexes. These MTAN-transition state complexes are among the tightest binding enzyme-ligand complexes ever described and analysis of their mode of binding provides extraordinary insight into the structural basis for their affinity. The MTAN-MT-ImmA complex reveals the presence of a new ion pair between the 4'-iminoribitol atom and the nucleophilic water (WAT3) that captures key features of the transition state. Similarly, in the MTAN-MT-DADMe-ImmA complex a favorable hydrogen bond or ion pair interaction between the cationic 1'-pyrrolidine atom and WAT3 is crucial for tight affinity. Distance analysis of the nucleophile and leaving group show that MT-ImmA is a mimic of an early transition state, while MT-DADMe-ImmA is a better mimic of the highly dissociated transition state of E. coli MTAN.

Topics: Deoxyadenosines; Enzyme Inhibitors; Escherichia coli; Models, Molecular; N-Glycosyl Hydrolases; Purine Nucleosides; Pyrimidinones; Pyrroles; Thionucleosides

Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5'-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k(chem)/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5'-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5'-OH neighboring group participation. When PfPNP acts on 5'-methylthioinosine, this interaction is not possible. Compensation for the 5'-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.

Topics: Animals; Aspartic Acid; Cattle; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Plasmodium falciparum; Polyamines; Protein Binding; Protein Structure, Tertiary; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines; Pyrimidinones; Pyrroles; Static Electricity; Substrate Specificity; T-Lymphocytes

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the beta-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 A resolution), 3'-deoxyguanosine (at 2.86 A resolution) and 8-azaguanine (at 2.85 A resolution). These structures were compared with the PNP-guanine, PNP-inosine and PNP-immucillin-H complexes solved previously.

Topics: Azaguanine; Binding Sites; Crystallography, X-Ray; Guanine; Guanosine; Humans; Inosine; Ligands; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

Methythioadenosine phosphorylase (MTAP) functions solely in the polyamine pathway of mammals to remove the methylthioadenosine (MTA) product from both spermidine synthase (2.5.1.16) and spermine synthase (2.5.1.22). Inhibition of polyamine synthesis is a validated anticancer target. We designed and synthesized chemically stable analogues for the proposed transition state of human MTAP on the basis of the known ribooxacarbenium character at all reported N-ribosyltransferase transition states [Schramm, V. L. (2003) Acc. Chem. Res. 36, 588-596]. Methylthio-immucillin-A (MT-ImmA) is an iminoribitol tight-binding transition state analogue inhibitor with an equilibrium dissociation constant of 1.0 nM. The immucillins resemble the ribooxacarbenium ion transition states of N-ribosyltransferases and are tightly bound as the N4' cations. An ion pair formed between the iminoribitol cation and phosphate anion mimics the ribooxacarbenium cation-phosphate anion pair formed at the transition state and is confirmed in the crystal structure. The X-ray crystal structure of human MTAP with bound MT-Imm-A also reveals that the 5'-methylthio group lies in a flexible hydrophobic pocket. Substitution of the 5'-methylthio group with a 5'-phenylthio group gives an equilibrium binding constant of 1.0 nM. Methylthio-DADMe-immucillin-A is a pyrrolidine analogue of the transition state with a methylene bridge between the 9-deazaadenine group and the pyrrolidine ribooxacarbenium mimic. It is a slow-onset inhibitor with a dissociation constant of 86 pM. Improved binding energy with DADMe-immucillin-A suggests that the transition state is more closely matched by increasing the distance between leaving group and ribooxacarbenium mimics, consistent with a more dissociative transition state. Increasing the hydrophobic volume near the 5'-position at the catalytic site with 5'-phenylthio-DADMe-immucillin-A gave a dissociation constant of 172 pM, slightly weaker than the 5'-methylthio group. p-Cl-phenylthio-DADMe-immucillin-A binds with a dissociation constant of 10 pM (K(m)/K(i) value of 500000), the tightest binding inhibitor reported for MTAP. These slow-onset, tight-binding transition state analogue inhibitors are the most powerful reported for MTAP and have sufficient affinity to be useful in inhibiting the polyamine pathway.

Topics: Binding Sites; Catalysis; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Phosphates; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Recombinant Proteins; Ribitol

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and alpha-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1', ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4' is protonated to the cation. It binds tightly to the PNPs with a K(d) value 56 pM for human PNP. Previous NMR studies of PNP. ImmH.PO(4) have shown that the N-4' of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP. ImmH.PO(4) complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using (15)N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only approximately 1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP. ImmH.PO(4) complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP. ImmH complex. The approximately 4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to O-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.

Topics: Binding, Competitive; Catalysis; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen Bonding; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protons; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Recombinant Proteins; Solutions; Thermodynamics

Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of PfPNP.Immucillin-H (ImmH).SO(4) reveals a homohexamer with ImmH and SO(4) bound at each catalytic site. A solvent-filled cavity close to the 5'-hydroxyl group of ImmH suggested that PfPNP can accept additional functional groups at the 5'-carbon. Assays established 5'-methylthioinosine (MTI) as a substrate for PfPNP. MTI is not found in human metabolism. These properties of PfPNP suggest unusual purine pathways in P. falciparum and provide structural and mechanistic foundations for the design of malaria-specific transition state analogue inhibitors. 5'-Methylthio-Immucillin-H (MT-ImmH) was designed to resemble the transition state of PfPNP and binds to PfPNP and human-PNP with K(d) values of 2.7 and 303 nm, respectively, to give a discrimination factor of 112. MT-ImmH is the first inhibitor that favors PfPNP inhibition. The structure of PfPNP.MT-ImmH.SO(4) shows that the hydrophobic methylthio group inserts into a hydrophobic region adjacent to the more hydrophilic 5'-hydroxyl binding site of ImmH. The catalytic features of PfPNP indicate a dual cellular function in purine salvage and polyamine metabolism. Combined metabolic functions in a single enzyme strengthen the rationale for targeting PfPNP in anti-malarial action.

Topics: Animals; Catalysis; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Hydrophobic and Hydrophilic Interactions; Methylthioinosine; Molecular Structure; Plasmodium falciparum; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the latter stages of polyamine biosynthesis (the synthesis of spermidine and spermine) is 5'-methylthioadenosine (MTA). In humans, MTA is processed by 5'-methylthioadenosine phosphorylase (MTAP) so that significant amounts of MTA do not accumulate. Potent inhibitors of MTAP might allow the buildup of sufficient levels of MTA to generate feedback inhibition of polyamine biosynthesis. We have designed and synthesized a family of potential transition-state analogue inhibitors of MTAP on the basis of our knowledge of the transition-state structure of purine nucleoside phosphorylase and the assumption that it is likely the two enzymes share a common catalytic mechanism. Several of the inhibitors display slow-onset tight-binding properties, consistent with them being transition-state analogues, with the most potent having a dissociation constant of 166 pM.

Topics: Adenine; Humans; Polyamines; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Pyrrolidines; Stereoisomerism; Structure-Activity Relationship

Difference Raman and FTIR studies complemented by vibrational analysis based on ab initio calculations show that the dianionic phosphate in the PNP.ImmH.PO4 complex is forced into a unique bonding arrangement in which one of the PO bonds is greatly polarized by enzyme active site interactions, such that it resembles a PO bond that is about one-quarter of the way toward forming a bridging P-O-C single P-O bond.

Topics: Animals; Binding Sites; Catalysis; Cattle; Humans; Hydrogen Bonding; Kinetics; Organophosphates; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Thermodynamics

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that, unlike the upper part, remains fed with substrates originating from the degradation of 2,3-bisphosphoglycerate in RBC depleted of glucose by pyruvate. (6) Fluoride, which arrests glycolysis at enolase and thus prevents NAD formation, inhibited AsV reduction in glucose-sufficient RBC, but increased it in glucose-deficient (NAD-enriched) cells, suggesting that the section of glycolysis coupled to AsV reduction lies between GAPDH and enolase. In conclusion, besid

Topics: Arsenates; Arsenite Transporting ATPases; Blood Glucose; Dithiothreitol; Erythrocytes; Ferricyanides; Glucose Oxidase; Glutathione; Humans; In Vitro Techniques; Inosine; Ion Pumps; Multienzyme Complexes; NADP; Oxidation-Reduction; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Pyruvic Acid; Spectrophotometry, Atomic

We have previously reported that compounds dimethyl-substituted on the phenyl ring of N-n-propyl-3-phenylpiperidines (PPEs) have a high (nM) affinity and selectivity toward the D(4) dopamine receptor (D(4) DAR) with m,p-dimethyl PPE (1) having the highest affinity and selectivity. In the present paper we have investigated the role of the methyl substitution by the synthesis of monomethylated (2a-c) and nonmethylated (2d) PPEs followed by the characterization of their biological properties using receptor binding assays. Our findings reveal that the methyl substitution of the phenyl ring is not necessary for a high and selective binding affinity to the D(4) DAR. Moreover, we have also synthesized cyclohexylpiperidines (CHPEs, 3a-d), which all showed higher binding affinities for the D(4) DAR than their aromatic counterparts. These results indicate that a pi-pi type interaction of the phenyl ring of PPEs with the D(4) DAR might not be essential, whereas a simple hydrophobic attraction between the cyclohexyl substituent of CHPEs and a hypothesized lipophilic pocket of the receptor might be crucial. Furthermore, functional assays indicate that 3d, as well as 1, are partial agonist at the D(4) DAR and therefore might represent new pharmacological tools to investigate the role of D(4) DAR activation in the control of cognitive functions and emotional states in health and disease.

Topics: Animals; Binding, Competitive; Corpus Striatum; Crystallography, X-Ray; Dopamine Agonists; Guanine Nucleotides; Guinea Pigs; In Vitro Techniques; Male; Melatonin; Piperidines; Purine Nucleosides; Pyrimidinones; Pyrroles; Radioligand Assay; Receptors, Dopamine D2; Receptors, Dopamine D4; Retina; Structure-Activity Relationship

Administration of BCX-1777 to primates results in a rapid elevation of plasma 2'-deoxyguanosine (up to 0.4 microg/ml, 1.5 microM). Maximum 2'-deoxyguanosine C(max), 0.4 microg/ml, was achieved with the lowest IV dose of BCX-1777 and increasing the IV dose of BCX-1777 did not increase the 2'-deoxyguanosine C(max). However, plasma 2'-deoxyguanosine remained elevated longer as the dose of BCX-1777 increased. In contrast, increases in the oral dose of BCX-1777 did increase the plasma C(max) of 2'-deoxyguanosine. This was in spite of the observation that overall oral bioavailability of BCX-1777 was only 8.2%. This suggests that the BCX-1777 was absorbed slowly producing a sustained low concentration of BCX-1777, resulting in prolonged plasma concentrations of 2'-deoxyguanosine. After IV dosing, the BCX-1777 was cleared relatively quickly and the plasma 2'-deoxyguanosine tracked slightly behind the BCX-1777. IV administration of 5 mg/kg of BCX-1777 twice daily maintains the plasma 2'-deoxyguanosine concentrations at around 0.3 microg/ml (1.1 microM). These data indicate that oral and IV administration of BCX-1777 induce a rapid rise in 2'-deoxyguanosine and that oral dosing at 8.8 and 17.6 mg/kg are at least equivalent to 4.4 mg/kg IV in effecting the accumulation of 2'-deoxyguanosine. Finally, 2'-deoxyguanosine plasma concentration was maintained longer in the three highest oral doses in comparison to all IV doses.

Topics: Administration, Oral; Animals; Area Under Curve; Biological Availability; Deoxyguanosine; Enzyme Inhibitors; Erythrocytes; Injections, Intravenous; Inosine; Macaca fascicularis; Male; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated alpha-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1'-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5'-PO(4) (ImmHP) resemble the transition state charge when protonated at 4'-N and bind tightly to these enzymes with K(d) values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4'-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP*ImmH*PO(4) and HGPRTase*ImmHP*MgPP(i) indicate protonation at N-4' for the tightly bound inhibitors. The 1'-(13)C and 1'-(1)H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1'-C toward sp(2) geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4'-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.

Topics: Catalytic Domain; Guanosine; Guanosine Monophosphate; Humans; Hydrogen-Ion Concentration; Hypoxanthine Phosphoribosyltransferase; In Vitro Techniques; Ions; Kinetics; Molecular Structure; Mycobacterium tuberculosis; Nuclear Magnetic Resonance, Biomolecular; Phosphorylation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Recombinant Proteins; Substrate Specificity

Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 A) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.

Topics: Catalytic Domain; Crystallography, X-Ray; Inosine; Kinetics; Models, Molecular; Molecular Mimicry; Mycobacterium tuberculosis; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Static Electricity; Substrate Specificity

In previous studies, treatment with 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) enhanced malignant transformation of immortal human epidermal (RHEK-1) keratinocytes. In contrast, arsenic (As) alone or in a mixture of As, cadmium (Cd), chromium (Cr), and lead (Pb) inhibited this process. Microarray analysis showed unique gene expression patterns in RHEK-1 exposed to MNNG, As, or the metal mixture. From this analysis, we have selected 16 genes potentially involved in the enhancement or inhibition of transformation. These 16 genes, nine (IFN inducible protein 9-27, MAA A32, CCLB protein, integrin beta4, XRCC1, K8, K18, MT3, MAPKK6) of which were altered in a chemical-specific manner and seven (MIC1, bikunin, MTS1, BMP4, RAD23A, DOC2, vimentin) of which were commonly affected by the MNNG and As or mixture treatments, were examined for expression in detail by real-time RT-PCR. Qualitatively, both microarray and real-time RT-PCR analyses gave comparable results for 15 of 16 genes, i.e., genes were consistently induced or suppressed under the different treatment regimens when measured by either technique. Of the seven genes altered in their expression by multiple chemical treatments, five showed patterns consistent with a role in the transformation process, i.e., they were oppositely regulated in MNNG-transformed RHEK-1 cells (designated as OM3) as compared to the nonmalignant As- and mixture-exposed cells. Through time-course studies, we also identified markers whose expression correlates with acquisition of transformation-associated characteristics in OM3. Identification of a battery of genes altered during progressive transformation of RHEK-1 should aid in developing a mechanistic understanding of this process, as well as strengthening the utility of these genes as biomarkers.

Topics: Animals; Arsenates; Bile; Biotransformation; Chromatography, High Pressure Liquid; Dithiothreitol; Enzyme Inhibitors; Erythrocytes; Humans; In Vitro Techniques; Inosine; Male; Oxidation-Reduction; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Rats; Rats, Wistar; Spectrophotometry, Atomic; Sulfhydryl Reagents

Purine nucleoside phosphorylase (PNP) deficiency in humans produces a relatively selective depletion of T cells. BCX-1777 is a potent inhibitor of PNP. BCX-1777 in the presence of deoxyguanosine (dGuo) inhibits the proliferation of CEM-SS [T-acute lymphoblastic leukemia (T-ALL)] cells with an IC(50)=0.015 microM. This inhibition by BCX-1777 and dGuo is accompanied by elevation of dGTP (154-fold) and dATP (8-fold). Deoxycytidine (dCyt) completely and lamivudine (3TC) partially reverse this inhibition caused by BCX-1777 and dGuo. dNTP analysis of these samples indicates that, in the presence of dCyt, where complete reversal of inhibition is observed, dGTP and dATP pools revert back to the control levels. In samples containing 3TC, where partial reversal of inhibition was observed, dGTP decreased from 154-fold to 38-fold and dATP levels further increased from 8-fold to 30-fold compared to the control sample. In CEM-SS cells, inhibition of proliferation by BCX-1777 and dGuo is not due to accumulation of dATP because in the presence of 3TC, where reversal of inhibition is observed, dATP levels are further increased. These studies clearly indicate that inhibition of T cells is due to accumulation of dGTP resulting in cell death with characteristics of apoptosis. The half-life of dGTP in CEM-SS cells is 18 h, which is longer than that observed in human lymphocytes (4 h), suggesting that the nucleotidase level in CEM-SS cells is lower than in human lymphocytes. A 154-fold accumulation of dGTP in CEM-SS cells in the presence of BCX-1777 and dGuo compared to a 15-fold accumulation of dGTP in human lymphocytes suggests that kinase level is higher in CEM-SS cells compared to human lymphocytes. High kinase and low nucleotidase levels make CEM-SS cells more sensitive to inhibition by BCX-1777 and dGuo than human lymphocytes. Currently, BCX-1777 is in phase I/II clinical trial for the treatment of T cell malignancies.

Topics: Apoptosis; Cell Division; Cell Line; Culture Media; Half-Life; HIV Reverse Transcriptase; Humans; Lamivudine; Leukemia-Lymphoma, Adult T-Cell; Lymphocytes; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Reverse Transcriptase Inhibitors; Stavudine

The aza-C-nucleosides, Immucillin-H and Immucillin-G, are transition state analogue inhibitors of purine nucleoside phosphorylase, a therapeutic target for the control of T-cell proliferation. Immucillin analogues modified at the 2'-, 3'-, or 5'-positions of the azasugar moiety or at the 6-, 7-, or 8-positions of the deazapurine, as well as methylene-bridged analogues, have been synthesized and tested for their inhibition of human purine nucleoside phosphorylase. All analogues were poorer inhibitors, which reflects the superior capture of transition state features in the parent immucillins.

Topics: Animals; Cattle; Enzyme Inhibitors; Humans; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Structure-Activity Relationship

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6A resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP.

Topics: Crystallography, X-Ray; Enzyme Inhibitors; Humans; Ligands; Models, Molecular; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

Knowledge of enzymatic transition states permits the logical design of transition state analogues. Kinetic isotope effects have been used to solve transition state structures for several N-ribosyltransferases. Nucleoside hydrolases from protozoan parasites show ribooxacarbenium ion character at their transition states, but with different extents of activation at the leaving group and the oxacarbenium ion. Transition state analogues are designed to capture these interactions and provide isozyme-specific inhibitors. Ricin A-chain is an RNA N-ribohydrolase for a single site on 28S rRNA. Its transition state resembles a fully dissociated ribooxacarbenium ion. A transition state analogue for ricin A-chain mimics the fully dissociated purine and cationic ribosyl transition state. The transition state for human purine nucleoside phosphorylase (PNP) is more dissociated than for the bovine enzyme. Immucillin-H, a powerful transition state inhibitor for human PNP, has entered clinical trials as an anti T-cell agent.

Topics: Animals; Cattle; Enzyme Inhibitors; Enzymes; Humans; Kinetics; Nucleic Acids; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Ricin

Sodium arsenite is much more potent than sodium arsenate in producing adverse effects in animals and in cultured cells. Although arsenate may exhibit toxicity as a phosphate analogue, its potency in vivo appears to be enhanced by reduction to arsenite. To understand the relative importance of this reduction, which is critical in evaluating the responsiveness of cell culture models to the different oxidation states and thus to elucidating the mechanism of arsenic action, present work has correlated the extent of reduction with biological activity in human keratinocytes. The results show that at biologically relevant concentrations, arsenate reduction to appreciable levels required several days, helping rationalize a previous empirical observation that it was approximately one-third as potent as arsenite. The relatively low conversion rate also emphasizes a limitation of culture; arsenate was nearly as efficacious as arsenite, but the time required for it to reach maximal effect exceeded ordinary medium change intervals. In keratinocytes, an important role for purine nucleoside phosphorylase in the reduction could not be demonstrated, indicating that another pathway is dominant in this cell type. Methylation of inorganic arsenic, uptake of methylated forms, and their reduction were all very slow. These findings suggest that the reduced methylated forms have only a small contribution to skin carcinogenesis unless they are supplied through the circulation. In parallel experiments, trivalent antimony was similar to arsenite in potency and efficacy, whereas pentavalent antimony was virtually without biological effect. Conversion of antimony in the pentavalent to the trivalent oxidation state was not detectable in keratinocytes. These findings emphasize the importance of intracellular reduction of the metalloids for biological effects.

Topics: Animals; Antimony; Arsenic; Biotransformation; Cattle; Cell Line; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme Inhibitors; Fibroblasts; Guanine; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Keratinocytes; Membrane Proteins; Oxidation-Reduction; Protein Precursors; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Spleen

Purine nucleoside phosphorylase (PNP) deficiency in humans produces a relatively selective depletion of T-cells. Inhibitors of PNP are therefore of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP and in vitro T-cell proliferation. Inhibition of human T-cells by BCX-1777 and deoxyguanosine (dGuo) is accompanied by deoxyguanosine triphosphate (dGTP) accumulation. Unlike human T-cells, mouse, rat, dog and monkey T-cells are neither inhibited (IC50>100 microM) nor accumulate dGTP in the presence of BCX-1777 and dGuo. Cells pretreated with BCX-1777 and dGuo for 24 h (to elevate dGTP levels) prior to stimulation demonstrated 80% inhibition similar to the inhibition observed with cells treated with BCX-1777 and dGuo during the stimulation and proliferation process. This further confirms that inhibition of T-cells is due to the accumulation of dGTP in these cells. Deoxynucleotide (dNTP) analysis of the cells treated with BCX-1777 and dGuo for 48 h showed no significant change in deoxycytidine triphosphate (dCTP) and deoxyadenosine triphosphate (dATP) pools. However, a decrease (2-fold) in thymidine triphosphate (dTTP) pools, and a large increase in dGTP pools (15-fold) were observed. Results from various groups have shown that alteration in the dNTP supply results in DNA fragmentation and cell death with characteristics of apoptosis. Indeed, apoptosis is observed in human T-lymphocytes treated with BCX-1777 and dGuo. To compare the in vivo efficacy of BCX-1777 with another potent T-cell inhibitor, cyclosporin, these drugs were tested in a xenogeneic graft-vs.-host disease model (XGVHD). In this model, human lymphocytes are engrafted into severe combined immunodeficient mice (SCID) mice inducing severe XGVHD. The efficacy of BCX-1777 in the XGVHD model was comparable to cyclosporin and a combination of BCX-1777 and cyclosporin treatment showed a trend towards increased efficacy compared to cyclosporin alone. These results suggest that BCX-1777 may be useful for the treatment of disease characterized by activated T-cell responses.

Topics: Animals; Cyclosporine; Deoxyguanine Nucleotides; Dogs; Drug Therapy, Combination; Graft vs Host Disease; Growth Inhibitors; Humans; Immunosuppressive Agents; Lymphocytes; Macaca fascicularis; Mice; Mice, Inbred BALB C; Mice, SCID; Purine Nucleosides; Pyrimidinones; Pyrroles; Rats; Rats, Sprague-Dawley; Survival Rate; Transplantation, Heterologous

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.

Topics: Animals; Asparagine; Binding Sites; Catalysis; Cattle; Crystallography, X-Ray; Enzyme Inhibitors; Glutamic Acid; Hydrogen Bonding; Hydrogen-Ion Concentration; Hypoxanthine; Imines; Nuclear Magnetic Resonance, Biomolecular; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Ribonucleosides; Static Electricity; Substrate Specificity; Thermodynamics

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP). Immucillins are powerful transition state analogue inhibitors of mammalian PNP and also inhibit P. falciparum PNP as illustrated in the accompanying article (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219-3225). This work tests the hypothesis that erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes with an IC(50) of 35 nm. Growth inhibition by Imm-H is reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. The concentration of Imm-H required for inhibition of parasite growth varies as a function of culture hematocrit, reflecting stoichiometric titration of human erythrocyte PNP by the inhibitor. Human and P. falciparum PNPs demonstrate different specificity for inhibition by immucillins, with the 2'-deoxy analogues showing marked preference for the human enzyme. The IC(50) values for immucillin analogue toxicity to P. falciparum cultures indicate that inhibition of PNP in both the erythrocytes and the parasite is necessary to induce a purine-less death.

Topics: Animals; Biological Transport; Cell Death; Enzyme Inhibitors; Erythrocytes; Humans; Hypoxanthine; Kinetics; Models, Biological; Plasmodium falciparum; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines; Pyrimidinones; Pyrroles; Time Factors

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.

Topics: Animals; Binding Sites; Catalysis; Cattle; Crystallography, X-Ray; Deuterium; Electron Transport; Enzyme Inhibitors; Hydrolysis; Inosine; Macromolecular Substances; Motion; Phosphates; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Topics: Apoptosis; Cell Division; Deoxyguanine Nucleotides; Enzyme Inhibitors; Humans; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; T-Lymphocytes; Tumor Cells, Cultured

Patients with purine nucleoside phosphorylase (PNP) deficiency present a selective T-cell immunodeficiency. Inhibitors of PNP are, therefore, of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP from various species including human, mouse, rat, monkey and dog, with IC50 values ranging from 0.48 to 1.57 nM. BCX-1777, in the presence of 2'-deoxyguanosine (dGuo, 3-10 microM), inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction (MLR) and phytohemagglutinin (PHA) (IC50 values < 0.1-0.38 microM). BCX-1777 is a 10-100-fold more potent inhibitor of human lymphocyte proliferation than other known PNP inhibitors like PD141955 and BCX-34. Nucleotide analysis of human lymphocytes indicate that inhibition of proliferation by BCX-1777 correlates with dGTP levels in the cells. BCX-1777 has excellent oral bioavailability (63%) in mice. At a single dose of 10 mg/kg in mice, BCX-1777 elevates dGuo to approximately 5 microM. BCX-1777 was not effective in mouse T-cell models such as delayed type hypersensitivity (DTH) and splenomegaly because mouse T-cells do not accumulate dGTP as do human T-cells. However, in the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mouse model, BCX-1777 was effective in prolonging the life span 2-fold or more. This is the first known example of a PNP inhibitor that elevates dGuo in mice similar to the levels observed in PNP-deficient patients. Furthermore, these dGuo levels are also required for in vitro T-cell inhibition by BCX-1777. Thus, BCX-1777 represents a novel class of selective immunosuppressive agents that could have therapeutic utility in various T-cell disorders.

Topics: Administration, Oral; Animals; Biological Availability; Enzyme Inhibitors; Graft vs Host Reaction; Guanosine Triphosphate; Immunosuppressive Agents; Indicators and Reagents; Injections, Intravenous; Lymphocyte Culture Test, Mixed; Mice; Mice, Inbred BALB C; Mice, SCID; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Survival Analysis; T-Lymphocytes

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].

Topics: Binding, Competitive; Catalysis; Cloning, Molecular; Enzyme Inhibitors; Gene Expression Regulation, Bacterial; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Mycobacterium tuberculosis; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Recombinant Proteins

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.

Topics: Actinomycetales; Animals; Binding Sites; Catalysis; Cattle; Enzyme Inhibitors; Enzyme Stability; Escherichia coli; Macromolecular Substances; Models, Molecular; Mycobacterium tuberculosis; Phosphates; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.

Topics: Amino Acid Sequence; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Protein Binding; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Solvents

The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.

Topics: Animals; Binding Sites; Catalysis; Diphosphates; Drug Design; Enzyme Inhibitors; Guanosine Monophosphate; Humans; Hydrogen Bonding; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Inosine Monophosphate; Kinetics; Magnesium Compounds; Nuclear Magnetic Resonance, Biomolecular; Phosphoribosyl Pyrophosphate; Phosphorylation; Plasmodium falciparum; Protein Binding; Protons; Purine Nucleosides; Pyrimidinones; Pyrroles

Malaria is a leading cause of worldwide mortality from infectious disease. Plasmodium falciparum proliferation in human erythrocytes requires purine salvage by hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase). The enzyme is a target for the development of novel antimalarials. Design and synthesis of transition-state analogue inhibitors permitted cocrystallization with the malarial enzyme and refinement of the complex to 2.0 A resolution. Catalytic site contacts in the malarial enzyme are similar to those of human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) despite distinct substrate specificity. The crystal structure of malarial HGXPRTase with bound inhibitor, pyrophosphate, and two Mg(2+) ions reveals features unique to the transition-state analogue complex. Substrate-assisted catalysis occurs by ribooxocarbenium stabilization from the O5' lone pair and a pyrophosphate oxygen. A dissociative reaction coordinate path is implicated in which the primary reaction coordinate motion is the ribosyl C1' in motion between relatively immobile purine base and (Mg)(2)-pyrophosphate. Several short hydrogen bonds form in the complex of the enzyme and inhibitor. The proton NMR spectrum of the transition-state analogue complex of malarial HGXPRTase contains two downfield signals at 14.3 and 15.3 ppm. Despite the structural similarity to the human enzyme, the NMR spectra of the complexes reveal differences in hydrogen bonding between the transition-state analogue complexes of the human and malarial HG(X)PRTases. The X-ray crystal structures and NMR spectra reveal chemical and structural features that suggest a strategy for the design of malaria-specific transition-state inhibitors.

Topics: Animals; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Macromolecular Substances; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Pentosyltransferases; Plasmodium falciparum; Protein Conformation; Protein Folding; Protein Structure, Secondary; Protons; Purine Nucleosides; Pyrimidinones; Pyrroles

Genetic defects in human purine nucleoside phosphorylase cause T-cell deficiency as the major phenotype. It has been proposed that efficient inhibitors of the enzyme might intervene in disorders of T-cell function. Compounds with features of the transition-state structure of purine nucleoside phosphorylase were synthesized and tested as inhibitors. The transition-state structure for purine nucleoside phosphorylase is characterized by (1) an elevated pKa at N7 of the purine ring for protonation or favorable H-bond interaction with the enzyme and (2) oxocarbenium ion formation in the ribosyl ring (Kline, P. C., and Schramm, V. L. (1995) Biochemistry 34, 1153-1162). Both features have been incorporated into the stable transition-state analogues, (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (immucillin-H) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1, 4-imino-D-ribitol (immucillin-G). Both inhibitors exhibit slow-onset tight-binding inhibition of calf spleen and human erythrocyte purine nucleoside phosphorylase. The inhibitors exhibit equilibrium dissociation constants (Ki) from 23 to 72 pM and are the most powerful inhibitors reported for the enzyme. Complete inhibition of the homotrimeric enzyme occurs at one mole of inhibitor per mole of enzymic trimer. Binding of the transition-state inhibitor at one site per trimer prevents inhibitor binding at the remaining two sites of the homotrimer. A mechanism of sequential catalysis at each subunit, similar to that of F1 ATPase, is supported by these results. Slow inhibitor dissociation (e.g., t1/2 of 4.8 h) suggests that these compounds will have favorable pharmacologic properties. Interaction of transition-state inhibitors with purine nucleoside phosphorylase is different from reactant-state (substrate and product analogue) inhibitors of the enzyme which bind equally to all subunits of the homotrimer.

Topics: Animals; Cattle; Enzyme Inhibitors; Humans; Kinetics; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Structure-Activity Relationship; Substrate Specificity; T-Lymphocytes