immucillin-g and forodesine

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

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

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

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

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

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

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

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

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