In recent years, there has been renewed emphasis on folate-based therapeutics for cancer based on capacities for tumor-selective membrane transport. This application explores the therapeutic potential for the human proton-coupled folate transporter (hPCFT), a recently discovered membrane transport system for folates and antifolates which is functionally and (to some degree) anatomically distinct from the ubiquitously expressed reduced folate carrier (RFC), the major tissue folate transporter. We hypothesize that hPCFT represents a novel and selective means for therapeutic targeting small molecule cytotoxins that are not transported by RFC. This concept is based on frequent and high level hPCFT expression in many human solid tumors, the acidic pH optimum for hPCFT vis ` vis RFC which parallels the pH microenvironments of solid tumors, and, most significantly, identification of the first small molecule cytotoxins that are selectively transported by hPCFT but not RFC. For analogs AG17, AG71, and AG94, in vitro cytotoxicities resulted from hPCFT transport and inhibition of de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase. Another cytotoxic hPCFT substrate, AG112, was a potent inhibitor of thymidylate synthase. AG71 was tested in vivo against subcutaneous human hepatoma cells in SCID mice and showed potent hPCFT-targeted activity without significant toxicity. The goal of this project is to develop a new generation of potent tumor-targeted chemotherapy agents based on their selective capacities for cellular uptake by hPCFT over RFC.
In aim 1, we will synthesize novel bicyclic and tricyclic analogs from 22 series of compounds, based on structure-activity profiles for AG17, AG71, AG94 and AG112, and molecular modeling with modifications of the ring systems, the linker domain, and the terminal glutamate.
In aim 2, we test compounds from aim 1 for cytotoxicity in isogenic hamster and human tumor cell line models with established patterns of RFC and hPCFT expression, identify molecular targets by nucleoside protection, in situ metabolic labeling and analysis of intracellular metabolites, and studies with isolated enzymes. Additional studies will characterize transport properties of the novel analogs with hPCFT vis a vis RFC, and metabolism to polyglutamates. Finally, in aim 3, we will evaluate in vivo efficacies of the most potent hPCFT-targeted analogs by in vivo toxicity and efficacy trials in hPCFT-expressing human tumors implanted into SCID mice. Our results will define a comprehensive structure-activity relationship for transport by hPCFT vis a vis RFC and afford optimized analogs with potent and selective antitumor activities against hPCFT-expressing tumors in vitro and in vivo. This study will define mechanisms of action of the novel hPCFT-targeted cytotoxic analogs and potentially provide agents to be used clinically, albeit with a different spectrum of antitumor activities and reduced toxicities than those currently in use.
This application explores potential therapeutic applications of human proton-coupled folate transporter (hPCFT), a recently discovered membrane transport system for folates and antifolates in many human tissues and solid tumors which is functionally and (to some degree) anatomically distinct from the ubiquitously expressed human reduced folate carrier (hRFC), the major tissue folate transport system in human cells and tissues, and the high affinity folate receptors (FRs). We hypothesize that hPCFT represents a novel and selective mechanism for therapeutic targeting small molecule cytotoxins which are not transported by other major (anti)folate transport systems, most notably hRFC. This concept is based on the frequent and high level hPCFT expression in many human solid tumors, the acidic pH optimum for hPCFT vis a vis hRFC which parallels the low pH microenvironments reported for solid tumors, and, most significantly, identification of first small molecule cytotoxins that are selectively transported by hPCFT but not hRFC (typified by compounds AG17, AG71, AG94, and AG112). An important goal of this application is to rationally develop specific transport substrates for hPCFT that will afford antitumor agents without toxicity to normal cells that primarily use hRFC for (anti)folate uptake. To our knowledge, this collaboration between an accomplished biochemist specializing in molecular pharmacology of anticancer drugs and an outstanding medicinal chemist with a demonstrated track record of antifolate drug design represents the only such endeavor capable of generating unique pharmacophores for all the therapeutically relevant (anti)folate transporters, including hRFC and hRFC, and FRs.
|Shah, Khushbu; Raghavan, Sudhir; Hou, Zhanjun et al. (2018) Development and validation of chemical features-based proton-coupled folate transporter/activity and reduced folate carrier/activity models (pharmacophores). J Mol Graph Model 81:125-133|
|Matherly, Larry H; Hou, Zhanjun; Gangjee, Aleem (2018) The promise and challenges of exploiting the proton-coupled folate transporter for selective therapeutic targeting of cancer. Cancer Chemother Pharmacol 81:1-15|
|Ravindra, Manasa; Wilson, Mike R; Tong, Nian et al. (2018) Fluorine-Substituted Pyrrolo[2,3- d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor ? and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis. J Med Chem 61:4228-4248|
|Hou, Zhanjun; Gattoc, Leda; O'Connor, Carrie et al. (2017) Dual Targeting of Epithelial Ovarian Cancer Via Folate Receptor ? and the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates. Mol Cancer Ther 16:819-830|
|Wilson, Mike R; Hou, Zhanjun; Yang, Si et al. (2016) Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates. Mol Pharmacol 89:425-34|
|Deis, Siobhan M; Doshi, Arpit; Hou, Zhanjun et al. (2016) Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase. Biochemistry 55:4574-82|
|Golani, Lalit K; Wallace-Povirk, Adrianne; Deis, Siobhan M et al. (2016) Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine Antifolates with Heteroatom Bridge Substitutions via Cellular Uptake by Folate Receptor ? and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesi J Med Chem 59:7856-76|
|Wang, Yiqiang; Mitchell-Ryan, Shermaine; Raghavan, Sudhir et al. (2015) 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. J Med Chem 58:1479-93|
|Wang, Lei; Wallace, Adrianne; Raghavan, Sudhir et al. (2015) 6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor ? and the Proton-Coupled Folate Transporter in Human Tumors. J Med Chem 58:6938-59|
|Kumar, Vidya P; Cisneros, Jose A; Frey, Kathleen M et al. (2014) Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase. Bioorg Med Chem Lett 24:4158-61|
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