The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Aleem Gangjee - One of the best experts on this subject based on the ideXlab platform.
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Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.
Biochemistry, 2016Co-Authors: Siobhan M. Deis, Aleem Gangjee, Larry H Matherly, Zhanjun Hou, Arpit Doshi, Charles E. DannAbstract:Pemetrexed and methotrexate are Antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine Antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted Antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine Antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl Antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel Antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used Antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.
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Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates
Molecular Pharmacology, 2016Co-Authors: M. Roy Wilson, Juiwanna Kushner, Manohar Ratnam, Aleem Gangjee, Si Yang, Kathryn White, Jenny Huang, Lisa Polin, Larry H MatherlyAbstract:Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3- d ]pyrimidine antifolate used for therapy of nonsquamous nonsmall cell lung cancer (NS-NSCLC). PMX is transported by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Unlike RFC, PCFT is active at acidic pH levels characterizing the tumor microenvironment. By real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, PCFT transcripts and proteins were detected in primary NS-NSCLC specimens. In six NS-NSCLC cell lines (A549, H1437, H460, H1299, H1650, and H2030), PCFT transcripts and proteins were detected by real-time RT-PCR and western blots, respectively. 6-Substituted pyrrolo[2,3- d ]pyrimidine thienoyl Antifolates related to PMX [compound 1 (C1) and compound 2 (C2), respectively] are selective substrates for PCFT over RFC. In the NS-NSCLC cell lines, both [3H]PMX and [3H]C2 were transported by PCFT. C1 and C2 inhibited proliferation of the NS-NSCLC cell lines; A549, H460, and H2030 cells were more sensitive to C1 than to PMX. C1 and C2 inhibited glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. When treated at pH 6.8, which favors PCFT uptake, C1 and C2 inhibited clonogenicity of H460 cells greater than PMX; PMX inhibited clonogenicity more than C1 or C2 at pH 7.2, which favors RFC transport over PCFT. Knockdown of PCFT in H460 cells resulted in decreased [3H]PMX and [3H]C2 transport and decreased growth inhibition by C1 and C2, and to a lesser extent by PMX. In vivo efficacy of C1 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Our results suggest that 6-substituted pyrrolo[2,3- d ]pyrimidine thienoyl Antifolates offer significant promise for treating NS-NSCLC by selective uptake by PCFT.
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Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase
2016Co-Authors: Siobhan M. Deis, Aleem Gangjee, Larry H Matherly, Zhanjun Hou, Arpit Doshi, Charles E. DannAbstract:Pemetrexed and methotrexate are Antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine Antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted Antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine Antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl Antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel Antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used Antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition
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Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine Antifolates as dual-acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: i
Journal of medicinal chemistry, 2013Co-Authors: Shermaine Mitchell-ryan, Christina Cherian, Yiqiang Wang, Zhanjun Hou, Sudhir Raghavan, Steven Orr, Manasa P. Ravindra, Eric C. Hales, Aleem GangjeeAbstract:We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine Antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.
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Discovery of 5‑Substituted Pyrrolo[2,3‑d]pyrimidine Antifolates as Dual-Acting Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5‑Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase in De Novo Purine Nucleotide Biosynthesis: I
2013Co-Authors: Shermaine Mitchell-ryan, Christina Cherian, Yiqiang Wang, Zhanjun Hou, Sudhir Raghavan, Steven Orr, Manasa Punaha Ravindra, Eric Hales, Aleem GangjeeAbstract:We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine Antifolates (compounds 5–10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action
John J. Mcguire - One of the best experts on this subject based on the ideXlab platform.
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synthesis and evaluation of a classical 2 4 diamino 5 substituted furo 2 3 d pyrimidine and a 2 amino 4 oxo 6 substituted pyrrolo 2 3 d pyrimidine as Antifolates
Bioorganic & Medicinal Chemistry, 2006Co-Authors: Aleem Gangjee, John J. Mcguire, Jie Yang, Roy L KisliukAbstract:Two classical Antifolates, a 2,4-diamino-5-substituted furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine, were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The syntheses were accomplished by condensation of 2,6-diamino-3(H)-4-oxo-pyrimidine with alpha-chloro-ketone 21 to afford two key intermediates 23 and 24, followed by hydrolysis, coupling with l-glutamate diethyl ester and saponification of the diethyl ester to afford the classical Antifolates 13 and 14. Compounds 13 and 14 with a single carbon atom bridge are both substrates for folylpoly-gamma-glutamate synthetase (FPGS), the enzyme responsible for forming critical poly-gamma-glutamate antifolate metabolites with increased potency and/or increased cell retention. Compound 14 is a highly efficient FPGS substrate demonstrating that 2,4-diamino-5-substituted furo[2,3-d]pyrimidines are important lead structures for the design of Antifolates with FPGS substrate activity. It retains inhibitory potency for DHFR and TS compared to the two atom bridged analog 5. Compound 13 is a poor inhibitor of purified DHFR and TS, and both 13 and 14 are poor inhibitors of the growth of CCRF-CEM human leukemia cells in culture, indicating that single carbon bridged compounds in these series though conducive to FPGS substrate activity were not potent inhibitors.
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5 amino 4 imidazolecarboxamide riboside potentiates both transport of reduced folates and Antifolates by the human reduced folate carrier and their subsequent metabolism
Cancer Research, 2006Co-Authors: John J. Mcguire, William H Haile, Chenchen YehAbstract:Transport is required before reduced folates and anticancer Antifolates [e.g., methotrexate (MTX)] exert their physiologic functions or cytotoxic effects. The folate/antifolate transporter with the widest tissue distribution and greatest activity is the reduced folate carrier (RFC). There is little evidence that RFC-mediated influx is posttranscriptionally regulated. We show that [3H]MTX influx in CCRF-CEM human childhood T-leukemia cells is potentiated up to 6-fold by exogenous 5-amino-4-imidazolecarboxamide riboside (AICAr) in a AICAr and MTX concentration–dependent manner. Metabolism to more biologically active polyglutamate forms is also potentiated for MTX and other Antifolates. That potentiation of influx by AICAr is mediated by effects on the RFC is supported by analyses ±AICAr showing ( a ) similarity and magnitude of kinetic constants for [3H]MTX influx; ( b ) similarity of inhibitory potency of known RFC substrates; ( c ) lack of potentiation in a CCRF-CEM subline that does not express the RFC; and ( d ) similarity of time and temperature dependence. Potentiation occurs rapidly and does not require new protein synthesis. Effects of specific inhibitors of folate metabolism and the time and sequence of AICAr incubation with cells suggest that both dihydrofolate reductase inhibition and metabolism of AICAr are essential for potentiation. Acute folate deficiency or incubation of CCRF-CEM with AICAr-related metabolites (e.g., adenosine) does not initiate potentiation. AICAr increases growth inhibitory potency of MTX and aminopterin against CCRF-CEM cells when both AICAr and antifolate are present for the first 24 hours of a 120-hour growth period. AICAr is the first small molecule that regulates RFC activity. (Cancer Res 2006; 66(7): 3836-44)
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synthesis of n 4 2 4 diamino 5 methyl 4 7 dihydro 3h pyrrolo 2 3 d pyrimidin 6 yl thio benzoyl l glutamic acid and n 4 2 amino 4 oxo 5 methyl 4 7 dihydro 3h pyrrolo 2 3 d pyrimidin 6 yl thio benzoyl l glutamic acid as dual inhibitors of dihydrofolate
Journal of Medicinal Chemistry, 2005Co-Authors: Aleem Gangjee, Roy L Kisliuk, Xin Lin, John J. McguireAbstract:Two novel classical Antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-l-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-l-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR−TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the gr...
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anticancer Antifolates current status and future directions
Current Pharmaceutical Design, 2003Co-Authors: John J. McguireAbstract:Abstract Antifolates are the oldest of the antimetabolite class of anticancer agents and were one of the first modern anticancer drugs. The first clinically useful antifolate, described in 1947, was 2,4-diamino-pteroylglutamate (4-amino-folic acid; aminopterin; AMT) which yielded the first-ever remissions in childhood leukemia. AMT was soon superseded by its 10-methyl congener, methotrexate (MTX), based on toxicity considerations; MTX remains, with one limited exception, the only antifolate anticancer agent in clinical use to this date. Because of the safety and utility of MTX, considerable effort has been invested in attempting to design more therapeutically selective Antifolates or Antifolates with a wider tumor spectrum. Initially, the design was based on the burgeoning knowledge of folate-dependent pathways and the determinants of the mechanism of action of MTX. These determinants include transport, the tight-binding inhibition of its target (the folate-dependent enzyme dihydrofolate reductase (DHFR)), and metabolism of MTX to poly-gamma-glutamate (Glu(n)) metabolites. These early studies led to the development of other antifolate DHFR inhibitors of two types: (1). "classical" analogs that use the same cellular transport systems as MTX and are also metabolized to Glu(n); and (2). "nonclassical" (i.e., lipophilic) analogs that do not require transport systems and that are not metabolized to Glu(n). Although several of these analogs have undergone clinical trial, none is proved superior to MTX. Detailed examination of the mechanisms of cytotoxicity and selectivity of MTX showed that inhibition of both dTMP synthesis and de novo purine synthesis, secondary to DHFR inhibition, led to DNA synthesis inhibition and subsequent cell death; inhibition of other folate-dependent pathways did not appear necessary for cell death. Further studies showed that the contribution of inhibition of dTMP or purine synthesis to cell death varied in different cell types. These data suggested that inhibition of one of these pathways individually might (at least in some cases) be therapeutically superior to the dual inhibition induced by MTX. Thus in rational design and in structure-based design studies, two new classes of antifolate enzyme inhibitors were elaborated-direct inhibitors of thymidylate synthase (TMPS) and direct inhibitors of one or both of the two folate-dependent enzymes of de novo purine synthesis. Members of each class included both classical and nonclassical types. After preclinical evaluation, several of these have moved into clinical trials. To date only one new TMPS inhibitor has successfully completed clinical trials and been approved for routine use; this drug, Tomudex (D1694, raltitrexed) is currently approved only in Europe and only for the treatment of colon cancer. This still represents a step forward for Antifolates, however, since MTX is well-known to be ineffective in colon cancer; thus Tomudex extends the tumor range of Antifolates. Antifolate development continues. Based on the immense body of knowledge now extant on Antifolates, specific aspects of the mechanism of action have been the focus. Newer Antifolates have been described that inhibit more than one pathway in folate metabolism, that have improved delivery, or that inhibit other targets in folate metabolism. These new analogs are in various stages of preclinical and clinical development.
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Exploitation of folate and antifolate polyglutamylation to achieve selective anticancer chemotherapy
Investigational New Drugs, 1996Co-Authors: John J. Mcguire, Takashi Tsukamoto, Barry P. Hart, James K. Coward, Thomas I. Kalman, John GalivanAbstract:Synthesis of poly(γ-glutamate) metabolites of natural folates and Antifolates is a critical process. Folylpolyglutamates are essential for cell proliferation. Polyglutamates of glutamate (Glu)-containing Antifolates are often critical for their cytotoxic action and are relevant to antifolate resistance. However, the role of polyglutamate synthesis in selectivity is less clear. We have undertaken a research program to further define the significance of polyglutamate metabolism and to devise ways to exploit this metabolism to achieve greater therapeutic selectivity in cancer chemotherapy. This article briefly reviews several approaches tested thus far. Inhibition of folylpolyglutamate synthesis should lead to cell death. Current ornithine (Orn)-containing folate-based inhibitors of the enzyme responsible for their synthesis, folylpolyglutamate synthetase (FPGS), are poorly transported, apparently because of interference by the protonated δ-amine. Replacement of Orn with 4,4-difluoroOrn, the δ-amine of which has a much lower pK a and is thus less protonated at physiological pH, was explored. Since it is unclear how polyglutamylation contributes to selectivity, we explored generic means either to eliminate or to enhance polyglutamylation. The data indicate that substitution for Glu in an antifolate by some Glu analogs in which the γ-COOH is either altered or replaced (e.g., γ-tetrazole-Glu) leads to loss of both FPGS substrate activity and binding; antifolate target specificity is unchanged, while uptake is actually enhanced. Substitution of 3,3-difluoroGlu for Glu leads to enhanced polyglutamylation (although probably only to the diglutamate), retention of target specificity, and at least equal uptake. Comparative studies of the same antifolate containing different replacements for Glu, such as γ-tetrazole-Glu (no polyglutamylation) or 3,3-difluoroGlu (enhanced polyglutamylation), will be useful in exploring the role and significance of polyglutamylation.
Roy L Kisliuk - One of the best experts on this subject based on the ideXlab platform.
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2 4 diamino 5 methyl 6 substituted arylthio furo 2 3 d pyrimidines as novel classical and nonclassical Antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors
Bioorganic & Medicinal Chemistry, 2010Co-Authors: Aleem Gangjee, Hiteshkumar D Jain, Jaclyn Phan, Xin Guo, Sherry F Queener, Roy L KisliukAbstract:Abstract A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}- l -glutamic acid 5 and 11 nonclassical Antifolates 6–16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6–16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6–16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.
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synthesis and evaluation of a classical 2 4 diamino 5 substituted furo 2 3 d pyrimidine and a 2 amino 4 oxo 6 substituted pyrrolo 2 3 d pyrimidine as Antifolates
Bioorganic & Medicinal Chemistry, 2006Co-Authors: Aleem Gangjee, John J. Mcguire, Jie Yang, Roy L KisliukAbstract:Two classical Antifolates, a 2,4-diamino-5-substituted furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine, were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The syntheses were accomplished by condensation of 2,6-diamino-3(H)-4-oxo-pyrimidine with alpha-chloro-ketone 21 to afford two key intermediates 23 and 24, followed by hydrolysis, coupling with l-glutamate diethyl ester and saponification of the diethyl ester to afford the classical Antifolates 13 and 14. Compounds 13 and 14 with a single carbon atom bridge are both substrates for folylpoly-gamma-glutamate synthetase (FPGS), the enzyme responsible for forming critical poly-gamma-glutamate antifolate metabolites with increased potency and/or increased cell retention. Compound 14 is a highly efficient FPGS substrate demonstrating that 2,4-diamino-5-substituted furo[2,3-d]pyrimidines are important lead structures for the design of Antifolates with FPGS substrate activity. It retains inhibitory potency for DHFR and TS compared to the two atom bridged analog 5. Compound 13 is a poor inhibitor of purified DHFR and TS, and both 13 and 14 are poor inhibitors of the growth of CCRF-CEM human leukemia cells in culture, indicating that single carbon bridged compounds in these series though conducive to FPGS substrate activity were not potent inhibitors.
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synthesis of n 4 2 4 diamino 5 methyl 4 7 dihydro 3h pyrrolo 2 3 d pyrimidin 6 yl thio benzoyl l glutamic acid and n 4 2 amino 4 oxo 5 methyl 4 7 dihydro 3h pyrrolo 2 3 d pyrimidin 6 yl thio benzoyl l glutamic acid as dual inhibitors of dihydrofolate
Journal of Medicinal Chemistry, 2005Co-Authors: Aleem Gangjee, Roy L Kisliuk, Xin Lin, John J. McguireAbstract:Two novel classical Antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-l-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-l-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR−TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the gr...
Yehuda G. Assaraf - One of the best experts on this subject based on the ideXlab platform.
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folylpoly γ glutamate synthetase a key determinant of folate homeostasis and antifolate resistance in cancer
Drug Resistance Updates, 2016Co-Authors: Shachar Raz, Michal Stark, Yehuda G. AssarafAbstract:Mammalians are devoid of autonomous biosynthesis of folates and hence must obtain them from the diet. Reduced folate cofactors are B9-vitamins which play a key role as donors of one-carbon units in the biosynthesis of purine nucleotides, thymidylate and amino acids as well as in a multitude of methylation reactions including DNA, RNA, histone and non-histone proteins, phospholipids, as well as intermediate metabolites. The products of these S-adenosylmethionine (SAM)-dependent methylations are involved in the regulation of key biological processes including transcription, translation and intracellular signaling. Folate-dependent one-carbon metabolism occurs in several subcellular compartments including the cytoplasm, mitochondria, and nucleus. Since folates are essential for DNA replication, intracellular folate cofactors play a central role in cancer biology and inflammatory autoimmune disorders. In this respect, various folate-dependent enzymes catalyzing nucleotide biosynthesis have been targeted by specific folate antagonists known as Antifolates. Currently, Antifolates are used in drug treatment of multiple human cancers, non-malignant chronic inflammatory disorders as well as bacterial and parasitic infections. An obligatory key component of intracellular folate retention and intracellular homeostasis is (anti)folate polyglutamylation, mediated by the unique enzyme folylpoly-γ-glutamate synthetase (FPGS), which resides in both the cytoplasm and mitochondria. Consistently, knockout of the FPGS gene in mice results in embryonic lethality. FPGS catalyzes the addition of a long polyglutamate chain to folates and Antifolates, hence rendering them polyanions which are efficiently retained in the cell and are now bound with enhanced affinity by various folate-dependent enzymes. The current review highlights the crucial role that FPGS plays in maintenance of folate homeostasis under physiological conditions and delineates the plethora of the molecular mechanisms underlying loss of FPGS function and consequent antifolate resistance in cancer.
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binding of a smad4 ets 1 complex to a novel intragenic regulatory element in exon12 of fpgs underlies decreased gene expression and antifolate resistance in leukemia
Oncotarget, 2014Co-Authors: Shachar Raz, Michal Stark, Yehuda G. AssarafAbstract:Polyglutamylation of Antifolates catalyzed by folylpoly-γ-glutamate synthetase (FPGS) is essential for their intracellular retention and cytotoxic activity. Hence, loss of FPGS expression and/or function results in lack of antifolate polyglutamylation and drug resistance. Members of the TGF-β/Smad signaling pathway are negative regulators of hematopoiesis and deregulation of this pathway is considered a major contributor to leukemogenesis. Here we show that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12 of FPGS in both acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. We demonstrate that antifolate resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS which disrupts FPGS activity by abolishing ATP binding, and alters the binding pattern of transcription factors to the genomic region of exon12. This in turn results in the near complete silencing of the wild type allele leading to a 97% loss of FPGS activity. We show that exon12 is a novel intragenic transcriptional regulator, endowed with the ability to drive transcription in vitro, and is occupied by transcription factors and chromatin remodeling agents (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.
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Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest
Cell Death & Disease, 2014Co-Authors: D Sheban, N Gonen, M Stark, B Berman, Yehuda G. AssarafAbstract:Antifolates have a crucial role in the treatment of various cancers by inhibiting key enzymes in purine and thymidylate biosynthesis. However, the frequent emergence of inherent and acquired antifolate resistance in solid tumors calls for the development of novel therapeutic strategies to overcome this chemoresistance. The core of solid tumors is highly hypoxic due to poor blood circulation, and this hypoxia is considered to be a major contributor to drug resistance. However, the cytotoxic activity of Antifolates under hypoxia is poorly characterized. Here we show that under severe hypoxia, gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis is downregulated. We further demonstrate that carcinoma cells become completely refractory, even at sub-millimolar concentrations, to all hydrophilic and lipophilic Antifolates tested. Moreover, tumor cells retained sensitivity to the proteasome inhibitor bortezomib and the topoisomerase II inhibitor doxorubicin, which are independent of cell cycle. We provide evidence that this antifolate resistance, associated with repression of folate metabolism, is a result of the inability of Antifolates to induce DNA damage under hypoxia, and is attributable to a hypoxia-induced cell cycle arrest, rather than a general anti-apoptotic mechanism. Our findings suggest that solid tumors harboring a hypoxic core of cell cycle-arrested cells may display antifolate resistance while retaining sensitivity to the chemotherapeutics bortezomib and doxorubicin. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors.
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Antifolates in cancer therapy structure activity and mechanisms of drug resistance
Drug Resistance Updates, 2012Co-Authors: Nitzan Gonen, Yehuda G. AssarafAbstract:In the past 65 years, Antifolates targeting folate metabolism played a pivotal role in drug treatment of malignant, microbial, parasitic and chronic inflammatory diseases. Drug discovery of novel Antifolates with improved properties and superior activities remains an attractive strategy both in academia and in the pharmaceutical industry. Among novel Antifolates are pemetrexed which primarily targets thymidylate synthase as well as pralatrexate which blocks dihydrofolate reductase, and displays enhanced transport and cellular retention properties. The present review describes the evolution and pharmacological activity of Antifolates and prospects for the development of the next generation Antifolates. Pre-clinical and clinical studies identified a plethora of mechanisms of antifolate resistance that are a primary hindrance to curative cancer chemotherapy; these are frequently associated with qualitative and/or quantitative alterations in influx and/or efflux transporters of Antifolates and in folate-dependent enzymes. Current advances including for example the deciphering of the dominant folate transporter proton-coupled folate transporter (PCFT/SLC46A1) facilitated the synthesis of experimental Antifolates aimed at selectively targeting solid tumor cells, which reside in an acidic microenvironment where PCFT supposedly functions optimally. Moreover, drugs that are structurally and mechanistically distinct from folates were conjugated to folic acid (e.g. Vintafolide/EC145, a folic acid desacetylvinblastine conjugate) to facilitate endocytosis via the folate receptor (FR) which is markedly overexpressed in various solid tumors. In an alternative approach, novel Antifolates selectively targeting the FR but not other folate transporters are being developed (e.g. BGC 945). Hence, targeting mechanisms of antifolate-resistance could facilitate the development of rationally-based novel Antifolates and strategies that overcome chemoresistance.
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Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia
Blood, 2009Co-Authors: Michal Stark, Chen Wichman, Irit Avivi, Yehuda G. AssarafAbstract:Folylpoly-γ-gluatamate synthetase (FPGS) catalyzes the polyglutamylation and thus intracellular retention of folates and Antifolates (eg, methotrexate; MTX) through the addition of multiple glutamate equivalents to their γ-carboxyl residue. Since polyglutamylation of Antifolates is crucial for their pharmacological activity in leukemia, loss of FPGS function results in decreased cellular levels of polyglutamylation-dependent Antifolates and consequent drug resistance. Whereas resistance to pulse exposure to Antifolates is frequently associated with loss of FPGS activity, the underlying molecular mechanism remains elusive. Here we explored the molecular basis of antifolate resistance in human MTX-resistant leukemia cell lines displaying marked loss of FPGS activity. We demonstrate that these MTX-resistant cells exhibit impaired splicing of FPGS mRNA based on intron retention and/or exon skipping, thereby resulting in loss of FPGS function due to premature translation termination. Furthermore, analysis of FPGS transcripts in blood or bone marrow specimens from patients with acute lymphoblastic leukemia revealed exon 12 skipping, both at diagnosis and at relapse, the latter of which occurs after high-dose MTX-containing chemotherapy. These results constitute the first demonstration of the loss of FPGS function via aberrant mRNA splicing, thereby resulting in loss of antifolate retention and drug resistance. The clinical ramifications of these novel findings are discussed.
Zhanjun Hou - One of the best experts on this subject based on the ideXlab platform.
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Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.
Biochemistry, 2016Co-Authors: Siobhan M. Deis, Aleem Gangjee, Larry H Matherly, Zhanjun Hou, Arpit Doshi, Charles E. DannAbstract:Pemetrexed and methotrexate are Antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine Antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted Antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine Antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl Antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel Antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used Antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.
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Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase
2016Co-Authors: Siobhan M. Deis, Aleem Gangjee, Larry H Matherly, Zhanjun Hou, Arpit Doshi, Charles E. DannAbstract:Pemetrexed and methotrexate are Antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine Antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted Antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine Antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl Antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel Antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used Antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition
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Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine Antifolates as dual-acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: i
Journal of medicinal chemistry, 2013Co-Authors: Shermaine Mitchell-ryan, Christina Cherian, Yiqiang Wang, Zhanjun Hou, Sudhir Raghavan, Steven Orr, Manasa P. Ravindra, Eric C. Hales, Aleem GangjeeAbstract:We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine Antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.
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Discovery of 5‑Substituted Pyrrolo[2,3‑d]pyrimidine Antifolates as Dual-Acting Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5‑Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase in De Novo Purine Nucleotide Biosynthesis: I
2013Co-Authors: Shermaine Mitchell-ryan, Christina Cherian, Yiqiang Wang, Zhanjun Hou, Sudhir Raghavan, Steven Orr, Manasa Punaha Ravindra, Eric Hales, Aleem GangjeeAbstract:We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine Antifolates (compounds 5–10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action
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structure and function of the reduced folate carrier a paradigm of a major facilitator superfamily mammalian nutrient transporter
Vitamins and Hormones Series, 2008Co-Authors: Larry H Matherly, Zhanjun HouAbstract:Folates are essential for life and folate deficiency contributes to a host of health problems including cardiovascular disease, fetal abnormalities, neurological disorders, and cancer. Antifolates, represented by methotrexate, continue to occupy a unique niche among the modern day pharmacopoeia for cancer along with other pathological conditions. This article focuses on the biology of the membrane transport system termed the "reduced folate carrier" or RFC with a particular emphasis on RFC structure and function. The ubiquitously expressed RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC expression or function portends potentially profound physiological or developmental consequences. For chemotherapeutic Antifolates used for cancer, loss of RFC expression or synthesis of mutant RFC protein with impaired function results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and low levels of substrate for polyglutamate synthesis. The functional properties for RFC were first documented nearly 40 years ago in murine leukemia cells. Since 1994, when RFC was first cloned, tremendous advances in the molecular biology of RFC and biochemical approaches for studying the structure of polytopic membrane proteins have led to an increasingly detailed picture of the molecular structure of the carrier, including its membrane topology, its N-glycosylation, identification of functionally and structurally important domains and amino acids, and helix packing associations. Although no crystal structure for RFC is yet available, biochemical and molecular studies, combined with homology modeling, based on homologous bacterial major facilitator superfamily transporters such as LacY, now permit the development of experimentally testable hypotheses designed to establish RFC structure and mechanism.