thioinosine and 6-benzylthioinosine

Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10-25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60-70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Death; Cell Line, Tumor; Drug Synergism; Fetal Blood; fms-Like Tyrosine Kinase 3; Glycolysis; Humans; Inverted Repeat Sequences; Leukemia, Myeloid, Acute; Metformin; STAT5 Transcription Factor; Thioinosine

We previously demonstrated that 6-benzylthioinosine (6-BT) could induce the differentiation of a subset of acute myeloid leukemia (AML) cell lines and primary AML cells regardless of their cytogenetics. In this study we investigated whether Wnt signaling pathways played roles in 6-BT-induced differentiation of AML cells.. We induced differentiation of HL-60 leukemic cells and primary AML cells in vitro using 6-BT. Real-time PCR (qPCR), western blot, and luciferase assays were used to examine the molecules' expression and biological activity in canonical and noncanonical Wnt signaling pathways. AML cell differentiation was measured by the Nitroblue tetrozolium (NBT) reduction assay.. 6-BT regulated the expression of both canonical and non-canonical Wnt signaling molecules in HL-60 cells. Both 6-BT and all-trans-retinoic-acid (ATRA) reduced canonical Wnt signaling and activated noncanonical Wnt/Ca2+ signaling in HL-60 cells. Pre-treatment of HL-60 cells with an inhibitor of glycogen synthase kinase-3β (GSK-3β), which activated canonical Wnt signaling, partly abolished the differentiation of HL-60 cells induced by 6-BT. Pre-treatment of HL-60 cells with an inhibitor of protein kinase C (PKC), resulting in inactivation of non-canonical Wnt/Ca2+ signaling, abolished 6-BT-induced differentiation of HL-60 cells. Several molecules in the non-canonical Wnt/Ca2+ pathway were detected in bone marrow samples from AML patients, and the expression of FZD4, FZD5, Wnt5a and RHOU were significantly reduced in newly diagnosed AML samples compared with normal controls.. Both canonical and non-canonical Wnt signaling were involved in 6-BT-induced differentiation of HL-60 cells, and played opposite roles in this process. Wnt signaling could be involved in the pathogenesis of AML not only by regulating self-renewal of hematopoietic stem cells, but also by playing a role in the differentiation of AML cells.

Topics: Calcium Signaling; Cell Differentiation; Cells, Cultured; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Thioinosine; Tretinoin; Wnt Signaling Pathway

Despite advances in oncology drug development, most commonly used cancer therapeutics exhibit serious adverse effects. Often the toxicities of chemotherapeutics are due to the induction of significant DNA damage that is necessary for their ability to kill cancer cells. In some clinical situations, the direct induction of significant cytotoxicity is not a requirement to meet clinical goals. For example, differentiation, growth arrest, and/or senescence is a valuable outcome in some cases. In fact, in the case of acute myeloid leukemia (AML), the use of the differentiation agent all-trans-retinoic acid (ATRA) has revolutionized the therapy for a subset of leukemia patients and led to a dramatic survival improvement. Remarkably, this therapeutic approach is possible even in many elderly patients, who would not be able to tolerate therapy with traditional cytotoxic chemotherapy. Because of the success of ATRA, there is widespread interest in identifying differentiation strategies that may be effective for the 90-95% of AML patients who do not clinically respond to ATRA. Utilizing an AML differentiation agent that is in development, we found that AML differentiation can be induced through ATP depletion and the subsequent activation of DNA damage signaling through an ATR/Chk1-dependent and p53-independent pathway. This study not only reveals mechanisms of AML differentiation but also suggests that further investigation is warranted to investigate the potential clinical use of low dose chemotherapeutics to induce differentiation instead of cytotoxicity. This therapeutic approach may be of particular benefit to patients, such as elderly AML patients, who often cannot tolerate traditional AML chemotherapy.

Topics: Acute Disease; Adenosine; Adenosine Triphosphate; Antibiotics, Antineoplastic; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Caffeine; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Comet Assay; DNA Damage; Doxorubicin; HEK293 Cells; Humans; Leukemia, Myeloid; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Signal Transduction; Staurosporine; Thioinosine; Tumor Suppressor Protein p53

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity.

Topics: Adenosine Kinase; Animals; Drug Design; Inosine; Structure-Activity Relationship; Substrate Specificity; Thioinosine; Toxoplasma

Toxoplasma gondii adenosine kinase (EC 2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Several 6-benzylthioinosines have already been identified as subversive substrates of the T. gondii but not human adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not its host. In the present study, we report the testing of the metabolism of several carbocyclic 6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the carbocyclic 6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the carbocyclic 6-benzylthioinosine analogues showed a selective anti-toxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. These results indicate that the oxygen atom of the sugar is not critical for substrate binding. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were carbocyclic 6-(p-methylbenzylthio)inosine (IC(50)=11.9 microM), carbocyclic 6-(p-methoxybenzylthio)inosine (IC(50)=12.1 microM), and carbocyclic 6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=12.8 microM). These compounds have about a 1.5-fold better efficacy relative to their corresponding 6-benzylthioinosine analogues (Rais et al., Biochem Pharmacol 2005;69:1409-19 [29]). The results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that carbocyclic 6-benzylthioinosines are potential anti-toxoplasmic agents.

Topics: Adenosine Kinase; Animals; Female; Inorganic Chemicals; Inosine; Ligands; Mice; Nucleotides; Organic Chemicals; Thioinosine; Toxoplasma; Toxoplasmosis

This paper described the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 6-benzylthioinosine (6BT), a novel myeloid leukemia differentiation-inducing agent, in mouse and human plasma. In this method, 2-amino-6-benzylthioinosine (2A6BT) was used as internal standard and ethyl acetate was used as organic solvent for the extraction of 6BT and internal standard from plasma samples. The extracted samples were separated on YMC ODS-AQ((R)) column (2.0mmx50mm), and the eluates from the column were monitored by the positive-electrospray-ionization tandem mass spectrometer (ESI(+)-MS/MS). Quantification of 6BT by internal calibration with 2A6BT was carried out using multiple-reaction-monitoring (MRM) mode. This method had a linear calibration range of 3.00-1.00x10(3)ng/mL with correlation coefficients of 1.00 and 0.999 in mouse and human plasma. The lower limit of quantification (LLOQ) was 3.00ng/mL in either mouse or human plasma. The method had recovery of 6BT of 82-87% from mouse plasma and 90-98% from human plasma. Both accuracy (as percent error) and precision (as coefficient of variation) of the method were

Topics: Animals; Chromatography, Liquid; Humans; Male; Mice; Mice, Inbred C57BL; Spectrometry, Mass, Electrospray Ionization; Thioinosine

As the pathophysiology of acute myelogenous leukemia (AML) involves a block of myeloid maturation, a desirable therapeutic strategy is to induce leukemic cell maturation to increase the efficacy and to avoid the side effects of traditional chemotherapeutics. Through a compound library screen, 6-benzylthioinosine (6BT) was identified as a promising differentiation-inducing agent. 6BT induces monocytic differentiation of myeloid leukemia cell lines such as HL-60 and OCI-AML3, as well as primary patient samples as evidenced by morphology, immunophenotyping, and nitroblue tetrazolium reduction. Not only can 6BT induce differentiation but a subset of AML cell lines such as MV4-11 and HNT34 instead undergo 6BT-mediated cell death. Despite inducing cell death in some leukemic cells, 6BT exhibits extremely low toxicity on several nonmalignant cells such as fibroblasts, normal bone marrow, and endothelial cells. This toxicity profile may relate to the function of 6BT as an inhibitor of the nucleoside transporter, ent1, which is thought to prevent it from entering many cell types. In contrast, 6BT likely enters at least some leukemic cell lines as shown by its requirement for phosphorylation for its differentiation activity. 6BT is also able to synergize with currently used myeloid differentiation agents such as ATRA and decitabine. Early studies indicate that the mechanism of action of this compound may involve ATP depletion that leads to growth inhibition and subsequent differentiation. Besides in vitro activity, 6BT also shows the ability to impair HL-60 and MV4-11 tumor growth in nude mice. 6BT is a promising new monocytic differentiation agent with apparent leukemic cell-specific activity.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line, Tumor; DNA Primers; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Nude; Phosphorylation; Polymerase Chain Reaction; Thioinosine; Transplantation, Heterologous

Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-substituted purine nucleosides act as subversive substrates of T. gondii, but not the human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Herein, we report the testing of newly synthesized 6-benzylthioinosine analogues with various substituents on the phenyl ring of their benzyl group as subversive substrates of T. gondii adenosine kinases. The binding affinity of these compounds to T. gondii adenosine kinase and their efficacy as antitoxoplasmic agents varied depending on the nature and position of the various substituents on the phenyl ring of their benzyl group. p-Cyano-6-benzylthioinosine and 2,4-dichloro-6-benzylthioinosine were the best ligands. In general, analogues with substitution at the para position of the phenyl ring were better ligands than those with the same substitutions at the meta or ortho position. The better binding of the para-substituted analogues is attributed to the combined effect of hydrophobic as well as van der Waals interactions. The 6-benzylthioinosine analogues were devoid of host-toxicity but all showed selective anti-toxoplasmic effect in cell culture and animal models. These results further confirm that toxoplasma adenosine kinase is an excellent target for chemotherapy and that 6-substituted purine nucleosides are potential selective antitoxoplasmic agents.

Topics: Adenosine Kinase; Animals; Antiprotozoal Agents; Female; Mice; Thioinosine; Toxoplasma

Toxoplasma gondii is the most common cause of secondary CNS infections in immunocompromised persons such as AIDS patients. The major route of adenosine metabolism in T. gondii is direct phosphorylation to adenosine 5'-monophosphate (AMP) catalyzed by the enzyme adenosine kinase (EC 2.7.1.20). Adenosine kinase in T. gondii is significantly more active than any other purine salvage enzyme in this parasite and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Subversive substrates of T. gondii,but not the human, adenosine kinase are preferentially metabolized to their monophosphorylated forms and become selectively toxic to the parasites but not their host. 6-Benzylthioinosine (BTI) was identified as an excellent subversive substrate of T. gondii adenosine kinase. Herein, we report the synthesis of new analogues of BTI as subversive substrates for T. gondii adenosine kinase. These new subversive substrates were synthesized starting from tribenzoyl protected d-ribose. To accomplish the lead optimization process, a divergent and focused combinatorial library was synthesized using a polymer-supported trityl group at the 5'-position. The combinatorial library of 20 compounds gave several compounds more active than BTI. Structure-activity relationship studies showed that substitution at the para position plays a crucial role. To investigate the reasons for this discrimination, substrates with different substituents at the para position were studied by molecular modeling using Monte Carlo Conformational Search followed by energy minimization of the enzyme-ligand complex.

Topics: Adenosine Kinase; Animals; Cells, Cultured; Coccidiostats; Combinatorial Chemistry Techniques; Humans; Models, Molecular; Structure-Activity Relationship; Thioinosine; Toxoplasma