Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

It was 62 years ago that Sidney Farber and his group at the Children9s Hospital in Boston reported the induction of complete, albeit temporary, remissions in children with acute leukemia achieved with an antifolate, aminopterin (2-amino-folic acid). One year later a second closely related antifolate, amethopterin (2-amino-10-methyl- folic acid; methotrexate [MTX]) was introduced and shortly thereafter replaced aminopterin because of what appeared to be lesser toxicity. Over the following decades an understanding of the mechanism of action of MTX, its cytotoxic determinants, and mechanisms of resistance emerged, a subject that continues to mature and evolve. MTX is a potent inhibitor of dihydrofolate reductase (DHFR) which results in depletion of cellular tetrahydrofolate cofactors required for the synthesis of purines, thymidylate and s-adenosylmethionine. This agent is transformed to polyglutamate derivatives, mediated by folylpolyglutamate synthetase (FPGS), a critical determinant of activity because these congeners are retained within cells and have additional targets as direct inhibitors of tetrahydrofolate-dependent enzymes. A second critical determinant of activity is membrane transport mediated by a facilitative transporter, the reduced folate carrier (RFC; SCL19A1) which is limiting to the formation of polyglutamate derivates. Important determinants of resistance are related to alterations in the structure or expression of RFC and/or FPGS and/or increased expression or amplification of DHFR. The selectivity of these drugs appears to be due to the much lesser build-up of polyglutamate derivatives in the proliferating compartment of bone marrow and intestinal cells (1, 2). As the understanding of the biochemical pharmacology of MTX was developing, structure-activity studies were employed to design new generations of DHFR inhibitors, attempting to design in selectivity based on optimization of transport and polyglutamation. Frank Sirotnak developed 10-propargyl-10-deazaaminopterin, pralatrexate, an agent with high affinity for both RFC and FPGS which results in the rapid build-up of high levels of active polyglutamate derivatives in tumor cells. Pralatrexate was recently approved for the treatment of cutaneous T-cell lymphoma (3). With the recognition that polyglutamylation of MTX alters the spectrum of target enzymes, other synthetic efforts were directed to designing drugs that in their polyglutamate forms are direct inhibitors of purine or thymidylate synthesis culminating in the development of pemetrexed. This agent is an excellent substrate RFC and has a very high affinity for FPGS that promotes rapid metabolism and intracellular accumulation as polyglutamate derivatives. In its polyglutamate forms, pemetrexed is an nM inhibitor of thymidylate synthase, and also has a second mechanism that results in inhibition of purine synthesis. In 2004, pemetrexed received FDA approval for the treatment of mesothelioma and more recently for first-line, second-line and maintenance therapy of the nonsquamous forms of non-small cell lung cancer (1). However, subsequent molecular pharmacological studies by Richard Moran identified the downstream folate-dependent enzyme required for purine synthesis, AICART, as the second site of pemetrexed action. Interestingly, AICART inhibition results in the build-up of ZMP in cells to levels that activate AMP-dependent kinase with subsequent inhibition of MTOR-dependent and independent processes (4). Hence, pemetrexed represents the convergence of inhibition of a classical target of antifolates, thymidylate synthase, with inhibition of a signal transduction pathway frequently dysregulated in carcinomas. One of the unusual properties of pemetrexed is that, despite its high affinity for RFC, when cells become resistant to MTX because of loss of RFC activity they remain fully sensitive to pemetrexed indicative of an additional portal of entry into cells (1, 2). The ensuing search for this route resulted in the discovery of what was named the proton-coupled folate transporter (PCFT; SLC46A1), a facilitative carrier that operates optimally at low pH. Subsequent studies demonstrated that PCFT is the mechanism of folate absorption in the proximal small intestine and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (5). Because of the low pH in the tumor microenvironment, synthetic efforts are currently underway by Larry Matherly and Aleem Gangjee to develop targeted antifolates with high affinity for PCFT and low affinity for the RFC, hence designing in minimization of transport by the route primarily used in normal proliferating tissues (6). Finally, there is a third route for folate-antifolate transport primarily in tumor cells, a folate receptor-mediated endocytic process. This route has been exploited in an attempt to deliver drugs selectively to tumor cells expressing this receptor. In this approach, thymidylate synthase inhibitors and purine synthesis inhibitors have been developed with high affinity for folate receptor but very low affinity for RFC (6, 7). One such thymidylate synthase inhibitor, ONX 0801, developed by Ann Jackman, is currently in a Phase I clinical trial. Another approach, pioneered by Philip Low, exploits the high affinity of folic acid for folate receptors and its ability to deliver structurally unrelated agents into cells by this mechanism (7, 8). Folic acid is linked through an acid-sensitive sulfhydryl bond to a variety of structurally unrelated lipid-soluble agents. These compounds are endocytosed via the folate receptor and when the endosome that forms acidifies, the linkage is broken, the antineoplastic is released and diffuses out of the endosome to reach its target. Folic acid can also be linked to radionuclides to identify occult areas of inflammation or to localize tumors and confirm their capacity to take up the reagent prior to treatment with the folic acid-antineoplastic conjugate. Several folic acid conjugated drugs and imaging reagents are current in clinical trial (7, 8). References: 1. Chattopadhyay S, Moran RG, Goldman ID. Pemetrexed: Biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther. 2007;6:404-17. 2. Zhao R, Matherly LH, Goldman ID. Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues. Expert Rev Mol Med. 2009;11:e4. 3. O9Connor OA, Horwitz S, Hamlin P, Portlock C, Moskowitz CH, Sarasohn D, Neylon E, Mastrella J, Hamelers R, Macgregor-Cortelli B, Patterson M, Seshan VE, Sirotnak F, Fleisher M, Mould DR, Saunders M, Zelenetz AD. Phase II-I-II study of two different doses and schedules of pralatrexate, a high-affinity substrate for the reduced folate carrier, in patients with relapsed or refractory lymphoma reveals marked activity in T-cell malignancies. J Clin Oncol. 2009;27(26):4357-64. 4. Racanelli AC, Rothbart SB, Heyer CL, Moran RG. Therapeutics by cytotoxic metabolite accumulation: pemetrexed causes ZMP accumulation, AMPK activation, and mammalian target of rapamycin inhibition. Cancer Res. 2009;69:5467-74. 5. Qiu A, Jansen M, Sakaris A, Min SH, Chattopadhyay S, Tsai E, Sandoval C, Zhao R, Akabas MH, Goldman ID. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell. 2006;127:917-28. 6. Wang L, Cherian C, Desmoulin SK, Polin L, Deng Y, Wu J, Hou Z, White K, Kushner J, Matherly LH, Gangjee A. Synthesis and antitumor activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry. J Med Chem. 2010;53:1306-18. 7. Leamon CP, Jackman AL. Exploitation of the folate receptor in the management of cancer and inflammatory disease. Vitam Horm. 2008;79:203-33. 8. Low PS, Kularatne SA. Folate-targeted therapeutic and imaging agents for cancer. Curr Opin Chem Biol. 2009;13:256-62. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA6-1.