Antifolate Activity Research Articles

Abstract 4410: KRAS mutation status is associated with enhanced dependency on purine biosynthesis and related pathways in non small cell lung cancer cells

Abstract KRAS gene mutation is linked to poor prognosis and resistance to oncology therapeutics in Non Small Cell Lung Cancer (NSCLC). We have explored the possibility of exploiting inherent differences in KRAS mutant cell metabolism to enhance the efficacy of treatment. We have identified a greater dependency on purine biosynthesis and related pathways in KRAS mutant compared to KRAS wild type NSCLC cell lines. Purine synthesis requires factors generated from other metabolic reactions including ribose-5-phosphate from the pentose phosphate pathway, THF cofactors from folate metabolism and glycine / amide nitrogen groups from glutamine and aspartate metabolism. In this study, microarray gene expression and biological pathway analysis identified higher expression of purine synthesis and accessory pathways such as folate metabolism, 5-aminoimidazole ribonucleotide biosynthesis and glycine synthesis pathways in KRAS mutant NSCLC cells compared to wildtype counterparts. KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise purine synthesis and folate metabolism pathways. Moreover, pathway analysis and knockdown studies suggest a role for MYC, an oncogene previously associated with KRAS mutant tumors, in the regulation of these pathways in KRAS mutant NSCLC cells. Proliferation studies demonstrated higher responsiveness to antifolates such as methotrexate and pemetrexed in KRAS mutant NSCLC cells, both of which may interfere indirectly with purine biosynthesis. In vivo analysis of NSCLC tumorgraft models in nude mice also identified an association between KRAS mutant tumor status and response to pemetrexed. The expression of a KRAS driven folate/purine synthesis gene, Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2), was also correlated with antifolate activity suggesting its use as a possible biomarker of response to antifolates. We propose that KRAS mutation drives increased purine synthesis activity and as a result an elevated dependency on the factors needed to feed this biosynthetic pathway such as those generated by folate metabolism. Thus, antifolates can indirectly inhibit purine synthesis through the depletion of folate cofactors which may account for the stronger response to these agents in KRAS mutant cells. We are currently expanding this study to examine alternative inhibitors of purine synthesis as possible therapeutics in KRAS mutant NSCLC and other cancers. Collectively, our findings highlight that a better understanding of the molecular mechanisms underlying the dependency of cancer cells on specific metabolic pathways may result in more effective metabolic targeting and new approaches in treating specific cancers. Citation Format: Diarmuid M. Moran, Patricia B. Trusk, Karen Pry, David Sidransky, Keren Paz, Sarah S. Bacus. KRAS mutation status is associated with enhanced dependency on purine biosynthesis and related pathways in non small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2014-4410

Abstract 1784: The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro

Abstract Introduction: Pralatrexate, the 10-propargyl-10-deaza analog of aminopterin, is a much superior substrate for the reduced folate carrier and folylpolyglutamate synthetase than methotrexate (MTX) resulting in the rapid build-up and retention of high levels of the polyglutamate derivatives of this agent within cells. In HeLa cells in vitro, the growth inhibitory effect of a 6h exposure to pralatrexate is two orders of magnitude greater than that of MTX. Yet the drug can be administered weekly at similar doses than MTX, consistent with a high level of selectivity. Nonetheless, mucosites can result in dosage reductions. This has led to the application of “rescue” regimens with the natural isomer of 5-formyltetrahydrofolate, (6S)5-formylTHF. Purpose: To investigate the impact of (6S)5-formylTHF on the antitumor activity of pralatrexate in HeLa cells in vitro, simulating the exposures to this folate as occurs in the clinical setting. These properties were compared to those of MTX. Experimental design: HeLa cells were grown with 25 nM (6S)5-formylTHF as the folate growth source. Experiments evaluated the impact of a 24h exposure to different concentration of (6S)5-formylTHF varying intervals prior to, or after, a 6h exposure to antifolate. The impact on the accumulation of [3H](6S)5-formylTHF and [3H]antifolate was also assessed. Results: A 24h interval between antifolate, followed by (6S)5-formylTHF, was sufficient to sustain the full activities of the antifolates up to a (6S)5-formylTHF level of 4 µM. A 72h interval was required between (6S)5-formylTHF, followed by the antifolate, to sustain full antifolate activities. HeLa cells were then exposed to 4 µM (6S)5-formylTHF for 24h weekly X 4; a concentration of the folate in excess of what is achieved in “rescue” regimens. This had no significant effect on the pralatrexate IC50; however, the MTX IC50 increased 2.5-fold. The four cycles of exposure to 4 µM [3H](6S)5-formylTHF resulted in a 16% increase in the tritiated intracellular folate level (P=0.02) as compared to cells growing continuously in 25 nM [3H](6S)5-formylTHF.There was no significant decrease in total intracellular pralatrexate; there was a 15% decrease in total intracellular MTX (P=0.004). Conclusions: Exposure of HeLa cells to (6S)5-formylTHF either before or after pralatrexate or MTX, simulating the sequence of administration in “rescue” regimens, and at concentrations in excess of what is achieved clinically, had no effect on pralatrexate activity but decreased the activity of MTX. This cumulative effect was associated with a small increase in the intracellular (6S)5-formylTHF level and a small decrease in total intracellular MTX. The data suggests that standard “rescue” regimens should have little or no impact on the antitumor activity of pralatrexate. Citation Format: Michele Visentin, Ersin Selcuk Unal, I David Goldman. The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2014-1784

KRAS Mutation Status Is Associated with Enhanced Dependency on Folate Metabolism Pathways in Non–Small Cell Lung Cancer Cells

KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in non-small cell lung cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS-mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared with KRAS wild-type NSCLC cell lines. Microarray gene expression and biologic pathway analysis identified higher expression of folate metabolism- and purine synthesis-related pathways in KRAS-mutant NSCLC cells compared with wild-type counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS-mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed, and other antifolates in KRAS-mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wild-type and KRAS-mutant cells by antifolates, which may also contribute to higher efficacy of antifolates in KRAS-mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS-mutant tumor among the pemetrexed-responsive tumors and also demonstrated an association between expression of the folate pathway gene, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS-mutant NSCLC cells that may account for higher antifolate activity in this subtype of NSCLC.

Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest

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.

Impact of folate supplementation on the efficacy of sulfadoxine/pyrimethamine in preventing malaria in pregnancy: the potential of 5-methyl-tetrahydrofolate

Malaria remains the leading cause of mortality and morbidity in children under the age of 5 years and pregnant women. To counterbalance the malaria burden in pregnancy, an intermittent preventive treatment strategy has been developed. This is based on the use of the antifolate sulfadoxine/pyrimethamine, taken at specified intervals during pregnancy, and reports show that this approach reduces the malaria burden in pregnancy. Pregnancy is also associated with the risk of neural tube defects (NTDs), especially in women with low folate status, and folic acid supplementation is recommended in pregnancy to lower the risk of NTDs. Thus, in malaria-endemic areas, pregnant women have to take both antifolate medication to prevent malaria and folic acid to lower the risk of NTDs. However, the concomitant use of folate and antifolate is associated with a decrease in antifolate efficacy, exposing pregnant women to malaria. Thus, there is genuine concern that this strategy may not be appropriate. We have reviewed work carried out on malaria folate metabolism and antifolate efficacy in the context of folate supplementation. This review shows that: (i) the folate supplementation effect on antifolate efficacy is dose-dependent, and folic acid doses required to protect pregnant women from NTDs will not decrease antifolate activity; and (ii) 5-methyl-tetrahydrofolate, the predominant form of folate in the blood circulation, could be administered (even at high dose) concomitantly with antifolate without affecting antifolate efficacy. Thus, strategies exist to protect pregnant women from malaria while maintaining adequate folate levels in the body to reduce the occurrence of NTDs.

Factors Influencing the Antifolate Activity of Synthetic Tea-Derived Catechins

Novel tea catechin derivatives have been synthesized, and a structure-activity study, related to the capacity of these and other polyphenols to bind dihydrofolate reductase (DHFR), has been performed. The data showed an effective binding between all molecules and the free enzyme, and the dissociation constants of the synthetic compounds and of the natural analogues were on the same order. Polyphenols with a catechin configuration were better DHFR inhibitors than those with an epicatechin configuration. Antiproliferative activity was also studied in cultured tumour cells, and the data showed that the activity of the novel derivatives was higher in catechin isomers. Derivatives with a hydroxyl group para on the ester-bonded gallate moiety presented a high in vitro binding to DHFR, but exhibited transport problems in cell culture due to ionization at physiologic pHs. The impact of the binding of catechins to serum albumin on their biological activity was also evaluated. The information provided in this study could be important for the design of novel medicinal active compounds derived from tea catechins. The data suggest that changes in their structure to avoid serum albumin interactions and to facilitate plasmatic membrane transport are essential for the intracellular functions of catechins.

Strategies for potentiation of ethionamide and folate antagonists against Mycobacterium tuberculosis

Antifolates inhibit de novo folate biosynthesis, whereas ethionamide targets the mycolate synthetic pathway in Mycobacterium tuberculosis. These antibiotics are effective against M. tuberculosis but their use has been hampered by concerns over toxicity and low therapeutic indexes. With the increasing spread of drug-resistant forms, interest in using old drugs for tuberculosis treatment has been renewed. Specific inhibitors targeting resistance mechanisms could sensitize M. tuberculosis to these available, clinically approved drugs. This review discusses recently developed strategies to boost the antituberculous activity of ethionamide and antifolates. These approaches might help broaden the currently limited chemotherapeutic options of not only drug-resistant but also drug-susceptible tuberculosis, which still remains one of the most common infectious diseases in the developing world.

Antifolates in cancer therapy: Structure, activity and mechanisms of drug resistance

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.

Genetic Polymorphism of γ-Glutamyl Hydrolase in Chinese Acute Leukemia Children and Identification of a Novel Double Nonsynonymous Mutation

γ-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action. Polymorphism in the human GGH gene (GGH) was associated with efficacy and side effects of methotrexate for treatment of acute lymphoblastic leukemia (ALL). This study aimed to identify polymorphisms of GGH in Chinese. Seventy-one children with ALL, 25 children with acute myeloid leukemia (AML), and 132 children with nonmalignancy as control were included. Human GGH cDNAs were prepared and analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR)–denaturing gradient gel electrophoresis. The detected variants were further confirmed by DNA sequencing. Novel variant GGH cDNA was cloned, expressed, and characterized. Allelic frequency of GGH 452C→T polymorphism was determined as 9.2% (CT, 11/71; TT, 1/71) in the ALL group, 8.0% (CT, 4/25; TT, 0/25) in the AML group, and 9.1% (TT, 4/132; T/C, 16/132) in the controls, respectively. The total allelic frequency in the Chinese population (9.0%) was higher than those reported in Japanese (5.6%) and African Americans (4.4%), and was similar to Caucasians (10.0%). Association of 452TT+TC with increased rate of hepatotoxicity and mucositis was observed in the ALL patients. Two novel mutations were determined in the coding region of GGH in 2 boys with ALL: one of the mutations was a double nonsynonymous heterozygote (841AG+845AG, K257E/N258S), the other was a nonsynonymous heterozygote (797AG, K242R). The corresponding double-mutant protein showed unchanged enzymatic activity by functional analysis. Allele frequency of GGH C452T polymorphism is determined for the first time among Chinese. A novel double nonsynonymous mutation of GGH was identified in a boy with ALL.

Treatment of malignant pleural mesothelioma: current status and future directions

Malignant pleural mesothelioma (MPM) is an aggressive tumor, with a poor prognosis and an increasing incidence as a result of widespread exposure to asbestos. Approximately 80% of MPM can be attributed to asbestos fiber exposure. Surgery and radiotherapy have a limited role in highly selected patients and systemic therapy is the only potential treatment option for the majority of patients. Unfortunately, despite some definite activity of the novel antifolates such as pemetrexed and raltitrexed, the results even in combination with platinating agents are still modest. The median survival of these patients remains of approximately 1 year. Improvements in surgical and radiotherapy techniques, in tumor assessment and staging and in the knowledge of the major molecular pathways involved in MPM are needed to increase the survival of these patients.

Abstract 4530: Pralatrexate-induced apoptosis in multiple myeloma cells correlates with RFC-1 and DHFR expression

Abstract Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a novel anti-folate agent that is approved for use in relapsed peripheral T cell lymphoma (PTCL). It is distinguished from the parent drug methotrexate (MTX) by having significantly greater affinity for the reduced folate carrier (RFC)-1, the principle transporter that internalize anti-folates, as well as broader efficacy, which strongly suggests that PDX has mechanisms of action beyond inhibition of nucleotide synthesis. Given the activity of PDX in aggressive lymphoid malignancies such as PTCL, we examined the activity of this agent in preclinical models of multiple myeloma (MM), a plasma cell malignancy that is currently incurable. We examined the cytotoxic activity of anti-folates in vitro against a panel of human multiple myeloma cell lines (HMCL) and showed that PDX induced dose-dependent apoptotic cell death in a subset of these. In sensitive cell lines (MM.1s and ARH-77) PDX demonstrated significantly greater potency at 48 hours compared to MTX (LD50 PDX = 2-3 nM vs. LD50 MTX = 25-30 nM). PDX also induced apoptosis in a polyclonal population of primary myeloma cells isolated from a patient specimen, whereas MTX had no effect on viability in these cells. PDX-induced apoptosis in sensitive HMCL was characterized by cleavage and activation of caspase-3 and -9, accompanied by the loss of the anti-apoptotic Bcl-2 family protein Mcl-1. Quantitative RT-PCR analysis of a panel of folate metabolism genes, including RFC-1, folyl polyglutamate synthetase (FPGS), gamma-glutamyl hydrolase (GGH), thymidylate synthase (TS), and dihydrofolate reductase (DHFR) revealed that RFC-1 expression was significantly higher in sensitive HMCL compared to resistant. Dose-dependent up-regulation of DHFR protein expression was observed by western blot in PDX-resistant cell lines, but not in PDX-sensitive ones. Incubation of HMCL with the proteasome inhibitor bortezomib or the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA), both of which demonstrate activity in MM, did not affect expression of RFC-1 in PDX-resistant HMCL and did not synergize with PDX to induce apoptosis in vitro. These results demonstrate that PDX induces apoptosis in sensitive HMCL, and that RFC-1 expression and DHFR up-regulation are functional biomarkers for susceptibility to this anti-folate. These data support further exploration of PDX therapy in MM, including combination therapy with agents that can increase expression of RFC-1 and/or block up-regulation of DHFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4530. doi:10.1158/1538-7445.AM2011-4530

Mechanism for synergism between sulphonamides and trimethoprim clarified.

Pseudomonas aeruginosa, Escherichia coli, Pseudomonas cepacia and Moraxella catarrhalis were selected for their markedly different resistance patterns to sulphonamides and trimethoprim. In addition, strains of E. coli and P. cepacia were selected having different resistance profiles to the inhibition of dihydropteroate synthetase and dihydrofolate reductase. All inhibitors of dihydropteroate synthetase combined in any combination with inhibitors of dihydrofolate reductase resulted in mutual enhancement of bacterial uptakes of the inhibitors and corresponding increased antibacterial activity of the combinations. High concentrations of sulphonamides or p-aminobenzoic acid plus trimethoprim caused a decrease in overall activity of the combination and indicated that both sulphonamides and p-aminobenzoic acid at high concentrations can interact with dihydrofolate reductase. The antibacterial activity of p-aminobenzoic acid at high concentrations is considered to be a blocking effect on dihydrofolate reductase even though p-aminobenzoic acid at low concentrations is an essential part of the synthesis of dihydrofolic acid. These findings support an alternative hypothesis for the mechanism of antibacterial action of individual antifolates and their mechanism of synergism in combination.

Comparison of a Pair of Synthetic Tea‐Catechin‐Derived Epimers: Synthesis, Antifolate Activity, and Tyrosinase‐Mediated Activation in Melanoma

Despite bioavailability issues, tea catechins have emerged as promising chemopreventive agents because of their efficacy in various animal models. We synthesized two catechin-derived compounds, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin (TMCG) and 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), in an attempt to improve the stability and cellular absorption of tea polyphenols. The antiproliferative and pro-apoptotic activities of both compounds were analyzed with various cancer cell systems, and TMCG, which was easily synthesized in excellent yield, was more active than TMECG in both melanoma and non-melanoma cell lines. TMCG was also a better inhibitor of dihydrofolate reductase and was more efficiently oxidized by tyrosinase, potentially explaining the difference in activity between these epimers.

Para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable (13)C(6)-isotope of pABA (p- amino[aromatic-(13)C(6)]benzoic acid ([(13)C(6)]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[(13)C(6)]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [(13)C(6)]pABA or [(13)C(6)]4HB generate both (13)C(6)-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product (13)C(6)-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ(6). This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ(6) quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.

Insights into antifolate activity of phytochemicals against Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic drug resistant pathogen. Drug interaction studies for phytochemicals (protocatechuic acid (PA), gallic acid (GA), quercetin (QUER), and myricetin (MYR)) in combination with antifolates (sulfamethoxazole (SMX) and trimethoprim (TMP)) are presented. Our results show that the combinations of SMX and phytochemicals are synergistic, whereas TMP in combination with phytochemicals results in additive mode of interaction. Molecular docking of phytochemicals in the active site of modeled P. aeruginosa dihydrofolate reductase (DHFR), an important enzyme in the folic acid biosynthesis pathway, shows that the phytochemicals QUER and MYR dock in the active site of P. aeruginosa DHFR with promoted binding at the NADP site, PA, and GA dock in the active site of P. aeruginosa DHFR with promoted binding at the folate binding site. Possible mode of action of these phytochemicals as anti-DHFR compounds in this bacterium is suggested. Taken together, the above findings provide novel insights to mode of interactions of these phytochemicals with antibiotics and may have significance as prospective leads in the development of antipseudomonal drug developments.

Novel 2‐hydrazino‐pyrimidin‐4(3H)‐one derivatives as potential dihydrofolate reductase inhibitors

Abstract magnified imageNovel substituted 2‐hydrazino‐pyrimidin‐4(3H)‐one derivatives were synthesized and examined for their antifolate activity against DHFR from Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma), and rat liver (rl). A novel, simple, and feasible methodology was developed for the synthesis of the titled compounds. Amongst these, compound 8 6‐phenyl‐2‐(2‐(1‐(thiophen‐2‐yl) ethylidene)hydrazinyl) pyrimidin‐4(3H)‐one exhibited 17.74 μM activity against pcDHFR. J. Heterocyclic Chem., (2010).

Multiple sclerosis

<h3>Abstract</h3> The activity and potency of a drug is inherently affected by the metabolic state of its target cell. Solute Carriers (SLCs) represent the largest family of transmembrane transporters in humans and constitute major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of individual chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in the haploid human cell line HAP1 using a set of 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using a SLC-focused CRISPR/Cas9 lentiviral library, we identified transporters whose absence induced resistance to the drugs tested. Among the hundreds of drug-SLC relationships identified, we confirmed the role of the folate transporter SLC19A1 on the activity of antifolates and of SLC29A1 on several nucleoside analogs. Among the newly discovered dependencies, we identified the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the compounds screened suggested a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provided an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.

Preclinical evaluation of a novel pyrimidopyrimidine for the prevention of nucleoside and nucleobase reversal of antifolate cytotoxicity

Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity in vitro, but its clinical activity is compromised by binding to the plasma protein alpha(1)-acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the in vivo activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 in vitro and in vivo. Plasma NU3153 concentrations commensurate with rescue inhibition in vitro were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity in vivo. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (> or =90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved in vivo using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver.

Metabolism-blocked antifolates as potential anti-rheumatoid arthritis agents: 4-Amino-4-deoxy-5,8,10-trideazapteroyl- d, l-4′-methyleneglutamic acid (CH-1504) and its analogs

4-Amino-4-deoxy-5,8,10-trideazapteroyl- d, l-4′-methyleneglutamic acid (CH-1504) is the prototype of a potentially therapeutically more selective class of antifolates for rheumatoid arthritis treatment. This class is characterized by retention of dihydrofolate reductase (DHFR; EC 1.5.1.3) as their locus of action and transport by the reduced folate carrier (RFC; SLC19A1), but their lack of metabolism by known pathways of antifolate (e.g., methotrexate (MTX)) metabolism. Five new CH-1504 analogs (CHL-001–CHL-005) were synthesized and diastereomers of CH-1504 itself were obtained by preparative chiral HPLC; all were characterized biochemically. The analogs are not metabolized by aldehyde oxidase (EC 1.2.3.1), carboxypeptidase G2 (EC 3.4.17.11), or (excepting CHL-003) folylpolyglutamate synthetase (EC 6.3.2.17) and thus, unlike MTX, are “metabolism-blocked”. All analogs are potent DHFR inhibitors; several are nearly as potent as MTX or CH-1504. Each analog uses the RFC for transport, although with varying apparent affinities. In contrast, each weakly inhibits other enzymes of folate metabolism relevant to rheumatoid arthritis therapy (thymidylate synthase (EC 2.1.1.45), two formyltransferases of purine biosynthesis (EC 2.1.2.2 and EC 2.1.2.3), and 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20)). Biochemical characterization showed one 4′-diastereomer of racemic CH-1504 was significantly more active than the other. Based on literature data concerning the effect of d- and l-glutamic acid substitution on antifolate activity, it is likely that the diastereomer containing l-4′-methylene-glutamic acid is the more active. Because of concern about potential pharmacokinetic and biochemical effects of d-4′-methylene-glutamic acid-containing species, these data suggest that future analogs should contain only l-4′-methylene-glutamic acid. Overall, these data provide several interesting new leads for preclinical development.

The anti-inflammatory and anti-cancer properties of epigallocatechin-3-gallate are mediated by folate cycle disruption, adenosine release and NF-κB suppression

To understand the mechanism by which (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, exerts its anti-inflammatory action. To check our hypothesis that the anti-inflammatory properties of EGCG could be related to its antifolate action and whether adenosine and its receptors are involved in EGCG action, we investigated the EGCG-induced suppression of NF-kappaB in Caco-2 cell monolayer, which acted as a model of the human intestinal epithelium. We demonstrate that the anti-inflammatory properties of EGCG are associated with its antifolate activity. By using a natural stable folate we were able to reverse the EGCG suppression of TNF-alpha-induced NF-kappaB activation, the phosphorylation and degradation of IkappaBalpha and the phosphorylation of Akt in this human colon carcinoma cell line. These suppressions were mediated by the release of adenosine following disruption of the folate cycle by EGCG. By binding to its specific receptors, adenosine can modulate the Akt and NF-kappaB pathway. Moreover, EGCG produces a significant increase in a specific adenosine receptor, which could explain the suppression of the constitutive activation of NF-kappaB in colon cancer cells. The data suggest that by modulating NF-kappaB activation, EGCG might not only combat inflammation, but also cancer.