Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors.

We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRß, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.

Discovery of N-(6-Fluoro-1-oxo-1,2-dihydroisoquinolin-7-yl)-5-[(3R)-3-hydroxypyrrolidin-1-yl]thiophene-2-sulfonamide (LSN 3213128), a Potent and Selective Nonclassical Antifolate Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase (AICARFT) Inhibitor Effective at Tumor Suppression in a Cancer Xenograft Model.

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

Novel 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines as potential antitumor agents with multitargeted inhibition of TS, GARFTase and AICARFTase.

A novel series of 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential antitumor agents targeting both thymidylate and purine nucleotide biosynthesis. Starting from the key intermediate 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, target compounds 1-6 were successfully obtained through two sequential condensation and saponification reactions in decent yield. The newly synthesized compounds showed antiproliferative potencies against a panel of tumor cell lines including KB, SW620 and MCF7. In particular, most compounds of this series exhibited nanomolar to subnanomolar inhibitory activities toward KB tumor cells, significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX). Along with the results of nucleoside protection assays, molecular modeling studies suggested that the antitumor activity of compound 6 could be attributed to multitargeted inhibition of folate-dependent enzymes thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). Growth inhibition by compound 6 also induced distinct early apoptosis and cell cycle arrest at S-phase, which resulted in cell death.

Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of TS and AICARFTase and as potential antitumor agents.

A new series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines, with an isosteric replacement of the side chain amide moiety to a sulfur atom, were designed and synthesized as multitargeted antifolates as well as potential antitumor agents. Starting from previously synthesized 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, a reduction by lithium triethylborohydride and successive mesylation afforded the key mesylate. Nucleophilic substitution by mercaptoacetic or mercaptopropionic acid methyl esters, followed by hydrolysis and condensation with pyridinyl-methylamines provided the nonclassical compounds 1-6, whereas condensation with glutamic acid diethyl ester hydrochloride and saponification afforded the classical analogs 7-8. All target compounds exhibited inhibitory activities toward KB, SW620 and A549 tumor cell lines. The most potent compounds of this series, 7 and 8, are better inhibitors against A549 cells than methotrexate (MTX) and pemetrexed (PMX). Nucleoside protection assays establish compound 8 a dual inhibitor of thymidylate synthase (TS) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) targeting both de novo thymidylate and purine nucleotide biosynthesis, which is further verified by the molecular modeling studies. Analogous to PMX, target compound 8 alternates the cell cycle of SW620 cells with S-phase accumulation and induces apoptosis, leading to cell death.

Novel 5-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and as potential antitumor agents.

A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.

Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase.

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.

Fibroblasts from methotrexate-sensitive mice accumulate methotrexate polyglutamates but those from methotrexate-resistant mice do not.

OBJECTIVES: We and others have previously demonstrated that methotrexate (MTX) mediates its anti-inflammatory effects through an increase in cellular release of adenosine. Consistent with this observation, there is no increase in adenosine from exudates of mouse strains resistant to MTX. Because intracellular MTX polyglutamates inhibit AICAR transformylase (ATIC) activity and thereby promote adenosine release we determined whether there is any difference in the capacity of cells from MTX-resistant mice to accumulate MTX polyglutamates. METHODS: Dermal fibroblasts (DF) from BALBc, MTX-sensitive, and DBA/1J, MTX-resistant, mice were cultured in the presence or absence of MTX. Adenosine concentration in the supernatant and intracellular MTX polyglutamate (MTXPG1-5) concentrations were measured by liquid chromatography. ATIC activity in DF was monitored spectrophotometrically by the formation of formytetrahydrofolate. RESULTS: MTX (1 µM) increased adenosine production by DF from BALBc sensitive-mice from 269±40 nM to 446±4 nM. No adenosine production was found in supernates of cultured DF from DBA/1J mice regardless of MTX treatment. Intracellular MTX polyglutamates (MTXPG2-4) were detected only in BALBc DFs, not in DBA/1J DF. Further investigation demonstrated that ATIC activity was inhibited following MTX treatment in DF from BALBc mice. CONCLUSIONS: These data suggest that resistance to the anti-inflammatory effects of MTX could be due to diminished MTX polyglutamate accumulation resulting in diminished ATIC inhibition and adenosine accumulation.

Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization.

Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å(2), is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K(i) of 17 µM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K(i) value of 84 µM. A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K(i) value of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.

Methotrexate and erythro-9-(2-hydroxynon-3-yl) adenine therapy for rat adjuvant arthritis and the effect of methotrexate on in vivo purine metabolism.

The objectives were: (1) to test the association of methotrexate (MTX) efficacy in rat adjuvant arthritis (rat AA) with interference of purine biosynthesis and adenosine metabolism and (2) to test the efficacy of erythro-9-(2-hydroxynon-3-yl) adenine (EHNA), an inhibitor of adenosine deaminase, and the efficacy of aminoimidazolecarboxamide (AICA) riboside plus MTX in rat AA. Radiographic and histologic examinations of the hind limbs were measures of efficacy. Urinary excretions of AICA and adenosine were markers of AICA ribotide transformylase inhibition (i.e., blockage of purine biosynthesis) and interference with adenosine metabolism, respectively. AICA and adenosine excretions increased during the day of MTX dosing (treatment day) compared to the previous baseline day in animals responding well to MTX (i.e., low radiographic and histologic scores). Based on radiographic and histologic scores, adjuvant injected rats were separated into two disease categories (i.e., no/mild and moderate/severe). Only AICA excretion was significantly elevated on the treatment day in rat AA with no/mild disease (i.e., those responding well to MTX therapy). AICA (not adenosine) excretion was significantly correlated with the above scores. EHNA was not efficacious, even at toxic levels, while AICA riboside potentiated the efficacy of MTX. The data suggests that efficacious MTX therapy in rat AA (1) blocks purine biosynthesis; (2) increases in in vivo AICA levels. Also adenosine accumulation and blockage of adenosine deaminase (i.e., by EHNA) appear to be less critical to MTX efficacy. Increased levels of AICA metabolites may suppress the immune response in rat AA.

Structure-based design, synthesis, evaluation, and crystal structures of transition state analogue inhibitors of inosine monophosphate cyclohydrolase.

The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATIC in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.

Methotrexate enhances the antianabolic and antiproliferative effects of 5-aminoimidazole-4-carboxamide riboside.

Because of its ability to mimic a low energy status of the cell, the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside was proposed as an antineoplastic agent switching off major energy-consuming processes associated with the malignant phenotype (lipid production, DNA synthesis, cell proliferation, cell migration, etc.). Key to the antineoplastic action of AICA riboside is its conversion to ZMP, an AMP mimetic that at high concentrations activates the AMP-activated protein kinase (AMPK). Here, in an attempt to increase the efficacy of AICA riboside, we pretreated cancer cells with methotrexate, an antimetabolite blocking the metabolism of ZMP. Methotrexate enhanced the AICA riboside-induced accumulation of ZMP and led to a decrease in the levels of ATP, which functions as an intrasteric inhibitor of AMPK. Consequently, methotrexate markedly sensitized AMPK for activation by AICA riboside and potentiated the inhibitory effects of AICA riboside on tumor-associated processes. As cotreatment elicited antiproliferative effects already at concentrations of compounds that were only marginally effective when used alone, our findings on the cooperation between methotrexate and AICA riboside provide new opportunities both for the application of classic antimetabolic chemotherapeutics, such as methotrexate, and for the exploitation of the energy-sensing machinery as a target for cancer intervention.