A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Methotrexate-based PROTACs as DHFR-specific chemical probes.

Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.

Delving in folate metabolism in the parasite Leishmania major through a chemogenomic screen and methotrexate selection.

Most of our understanding of folate metabolism in the parasite Leishmania is derived from studies of resistance to the antifolate methotrexate (MTX). A chemical mutagenesis screen of L. major Friedlin and selection for resistance to MTX led to twenty mutants with a 2- to 400-fold decrease in MTX susceptibility in comparison to wild-type cells. The genome sequence of the twenty mutants highlighted recurrent mutations (SNPs, gene deletion) in genes known to be involved in folate metabolism but also in novel genes. The most frequent events occurred at the level of the locus coding for the folate transporter FT1 and included gene deletion and gene conversion events, as well as single nucleotide changes. The role of some of these FT1 point mutations in MTX resistance was validated by gene editing. The gene DHFR-TS coding for the dihydrofolate reductase-thymidylate synthase was the second locus with the most mutations and gene editing confirmed a role in resistance for some of these. The pteridine reductase gene PTR1 was mutated in two mutants. The episomal overexpression of the mutated versions of this gene, but also of DHFR-TS, led to parasites several fold more resistant to MTX than those overexpressing the wild-type versions. Genes with no known link with folate metabolism and coding for a L-galactolactone oxidase or for a methyltransferase were mutated in specific mutants. Overexpression of the wild-type versions of these genes in the appropriate mutants reverted their resistance. Our Mut-seq approach provided a holistic view and a long list of candidate genes potentially involved in folate and antifolate metabolism in Leishmania.

Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate.

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

Methotrexate recognition by the human reduced folate carrier SLC19A1.

Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4-6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4-8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment.

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRß is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRß-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens.

Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.

Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection.

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

The evolving biology of the proton-coupled folate transporter: New insights into regulation, structure, and mechanism.

The human proton-coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high-level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo-electron microscopy structures of chicken PCFT in a substrate-free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH-regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo-oligomer, and evidence suggests that homo-oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer.

Structural basis of antifolate recognition and transport by PCFT.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.

Combined gene deletion of dihydrofolate reductase-thymidylate synthase and pteridine reductase in Leishmania infantum.

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously.

In Silico Study Identified Methotrexate Analog as Potential Inhibitor of Drug Resistant Human Dihydrofolate Reductase for Cancer Therapeutics.

Drug resistance is a core issue in cancer chemotherapy. A known folate antagonist, methotrexate (MTX) inhibits human dihydrofolate reductase (hDHFR), the enzyme responsible for the catalysis of 7,8-dihydrofolate reduction to 5,6,7,8-tetrahydrofolate, in biosynthesis and cell proliferation. Structural change in the DHFR enzyme is a significant cause of resistance and the subsequent loss of MTX. In the current study, wild type hDHFR and double mutant (engineered variant) F31R/Q35E (PDB ID: 3EIG) were subject to computational study. Structure-based pharmacophore modeling was carried out for wild type (WT) and mutant (MT) (variant F31R/Q35E) hDHFR structures by generating ten models for each. Two pharmacophore models, WT-pharma and MT-pharma, were selected for further computations, and showed excellent ROC curve quality. Additionally, the selected pharmacophore models were validated by the Guner-Henry decoy test method, which yielded high goodness of fit for WT-hDHFR and MT-hDHFR. Using a SMILES string of MTX in ZINC15 with the selections of 'clean', in vitro and in vivo options, 32 MTX-analogs were obtained. Eight analogs were filtered out due to their drug-like properties by applying absorption, distribution, metabolism, excretion, and toxicity (ADMET) assessment tests and Lipinski's Rule of five. WT-pharma and MT-pharma were further employed as a 3D query in virtual screening with drug-like MTX analogs. Subsequently, seven screening hits along with a reference compound (MTX) were subjected to molecular docking in the active site of WT- and MT-hDHFR. Through a clustering analysis and examination of protein-ligand interactions, one compound was found with a ChemPLP fitness score greater than that of MTX (reference compound). Finally, a simulation of molecular dynamics (MD) identified an MTX analog which exhibited strong affinity for WT- and MT-hDHFR, with stable RMSD, hydrogen bonds (H-bonds) in the binding site and the lowest MM/PBSA binding free energy. In conclusion, we report on an MTX analog which is capable of inhibiting hDHFR in wild type form, as well as in cases where the enzyme acquires resistance to drugs during chemotherapy treatment.

Design, synthesis and biological activity of novel substituted 3-benzoic acid derivatives as MtDHFR inhibitors.

The enzyme dihydrofolate reductase from M.tuberculosis (MtDHFR) has a high unexploited potential to be a target for new drugs against tuberculosis (TB), due to its importance for pathogen survival. Preliminary studies have obtained fragment-like molecules with low affinity to MtDHFR which can potentially become lead compounds. Taking this into account, the fragment MB872 was used as a prototype for analogue development by bioisosterism/retro-bioisosterism, which resulted in 20 new substituted 3-benzoic acid derivatives. Compounds were active against MtDHFR, with IC50 values ranging from 7 to 40 µM, where compound 4e not only had the best inhibitory activity (IC50 = 7 µM), but also was 71-fold more active than the original fragment MB872. The 4e inhibition kinetics indicated an uncompetitive mechanism, which was supported by molecular modeling which suggested that the compounds can access an independent backpocket from the substrate and competitive inhibitors. Thus, based on these results, substituted 3-benzoic acid derivatives have strong potential to be developed as novel MtDHFR inhibitors and also anti-TB agents.

Design, synthesis and biological evaluation of novel pyrrolo[2,3-d]pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier.

Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -ß over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRß-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -ß over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -ß over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRß-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.

Flexible diaminodihydrotriazine inhibitors of Plasmodium falciparum dihydrofolate reductase: Binding strengths, modes of binding and their antimalarial activities.

A series of flexible diaminodihydrotriazines or cycloguanil (Cyc) analogues are developed and shown to inhibit P. falciparum dihydrofolate reductase (PfDHFR) of the wild type or those carrying either single (S108N), double (C59R + S108N and A16V + S108T), triple (N51I + C59R + S108N and C59R + S108N + I164L) or quadruple (N51I + C59R + S108N + I164L) mutations, responsible for antifolate resistance. The flexibility of the side chain at position N1 has been included in the design so as to avoid unfavourable steric interaction with the side chain of residue 108 of the resistant mutants. The inhibition constants of many inhibitors for the mutant enzymes are in the low nanomolar region. Regaining of drug binding efficacies was achieved with both A16V and S108N series of mutants. X-ray studies of some enzyme-inhibitor complexes designed for optimal interaction with the mutant enzymes reveal the modes of binding in line with the Ki values. A number of these compounds show excellent antimalarial activities against resistant P. falciparum bearing the mutant enzymes, and exhibit low cytotoxicity to mammalian cells, making them good candidates for further development as antimalarial drugs.

Design, synthesis and biological activity of N5-substituted tetrahydropteroate analogs as non-classical antifolates against cobalamin-dependent methionine synthase and potential anticancer agents.

Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell growth and survival. In this study, a novel series of N5-electrophilic substituted tetrahydropteroate analogs without glutamate residue were designed as non-classical antifolates and evaluated for their inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in human tumor cell lines. With N5-chloracetyl as the optimum group, further structure research on the benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC50 value of 12.1 µM against MetH and 0.16-6.12 µM against five cancer cells, acted as competitive inhibitor of MetH. Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G1-phase and then inducted late apoptosis. The molecular docking further explained the structure-activity relationship.

Redox-Responsive and Dual-Targeting Hyaluronic Acid-Methotrexate Prodrug Self-Assembling Nanoparticles for Enhancing Intracellular Drug Self-Delivery.

The clinical translation of methotrexate (MTX) is limited because of low aqueous solubility, poor bioavailability, low uptake efficiency, and toxicity concerns. Herein, dual-acting MTX (not only targeting folate receptors but also killing cells via inhibition of intracellular folate metabolism) and hyaluronic acid (HA, targeting CD44 receptors) were selected to be covalently linked by the redox-responsive disulfide bond. The synthesized prodrug (HA-SS-MTX) as a molecular structural motif could self-assemble into simple yet multifunctional nanoparticles (HA-SS-MTX NPs) in aqueous solution. The HA-SS-MTX NPs displayed an average diameter of ∼110 nm with a uniformly spherical shape and maintained stability in different physiological media. Moreover, the HA-SS-MTX NPs could exhibit a sharp redox-dependent response for rapid structure disassembly and sequential MTX release compared to the redox-irresponsive group (HA-ADH-MTX NPs). Furthermore, the results of confocal microscopy and flow cytometry verified that the nanosystems were selectively uptaken by cancer cells via folate and CD44 receptor-mediated internalization through the dual-active targeting mechanism. In addition, HA-SS-MTX NPs could accumulate within tumor sites for a longer period. Notably, in vitro and in vivo antitumor results demonstrated that HA-SS-MTX NPs significantly promoted the death of cancer cells and enhanced the inhibition of tumor growth while reducing the toxicity as compared to MTX and HA-ADH-MTX NPs. Therefore, the smart HA-SS-MTX NPs as the simple and efficient platform have great potential in tumor-targeting drug delivery and therapy.

Structural basis of inhibition of the human serine hydroxymethyltransferase SHMT2 by antifolate drugs.

Serine hydroxymethyltransferase (SHMT) is the major source of 1-carbon units required for nucleotide synthesis. Humans have cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms, which are upregulated in numerous cancers, making the enzyme an attractive drug target. Here, we show that the antifolates lometrexol and pemetrexed are inhibitors of SHMT2 and solve the first SHMT2-antifolate structures. The antifolates display large differences in their hydrogen bond networks despite their similarity. Lometrexol was found to be the best hSHMT1/2 inhibitor from a panel antifolates. Comparison of apo hSHMT1 with antifolate bound hSHMT2 indicates a highly conserved active site architecture. This structural information offers insights as to how these compounds could be improved to produce more potent and specific inhibitors of this emerging anti-cancer drug target.

Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model.

Methotrexate (MTX) efficacy in autoimmune arthritis is variable and unpredictable resulting in the need for the identification of biomarkers to guide drug therapy. This study utilizes the collagen-induced arthritis mouse model to investigate erythrocyte MTX disposition and anti-folate activity as biochemical markers of efficacy in autoimmune arthritis. Following induction of arthritis, DBA/1J mice were treated with once-weekly subcutaneous MTX at varying doses over a period of 40 days. At the completion of the study tissue samples were analyzed for MTX and folate content and assessed for their relationship with MTX efficacy. MTX treatment resulted in a reduction in disease activity that was variable and dose-dependent. Erythrocyte accumulation of MTX and its polyglutamate metabolites were dose proportionate, however, polyglutamate metabolites represented a mean ±â€¯S.E.M. of 8.9 ±â€¯0.4% of total erythrocyte MTX, which is markedly lower than previously observed in humans and failed to display any significant association with MTX efficacy. MTX treatment resulted in reductions in erythrocyte 5-methyl-tetrahydrofolate (5mTHF) levels that were similar to those previously observed in human studies. Disease induction was associated with a decrease in liver 5mTHF and increased formyl-tetrahydrofolate (fTHF) that was normalized in MTX treated mice. MTX efficacy was associated with reductions in erythrocyte 5mTHF (P = 0.04) and increases in liver 5mTHF (P = 0.0001). Together, these findings demonstrate a relationship between alterations in tissue folate levels and MTX efficacy, and supports erythrocyte levels of 5mTHF as a marker of MTX efficacy in autoimmune arthritis.