Project Summary/Abstract Cellular one-carbon metabolism (1CM) generates a host of metabolites especially critical to cancer cells. Classical 1CM inhibitors, such as methotrexate and pemetrexed, often primarily target a single cytosolic 1CM enzyme, leading to resistance, and are non-selectively transported into both tumor and normal tissues by the ubiquitously expressed reduced folate carrier (RFC), leading to toxic side effects. Resistance and lack of tumor selectivity of current 1C inhibitors necessitates the development of novel compounds targeting more tumor- specific transporters, such as the proton-coupled folate transporter (PCFT) and multiple/alternate 1CM enzymes, such as those in the mitochondrial matrix. Upregulated in many tumors including non-small cell lung cancer (NSCLC), PCFT is a proton-folate symporter particularly suited to delivering drugs in the acidity of the tumor microenvironment. Among the mitochondrial enzymes, serine hydroxymethyltransferase2 (SHMT2), is strongly correlated with unfavorable prognosis in cancer patients and is the ?gatekeeper? for mitochondrial 1CM, which generates the vast majority of one-carbon (1C) units for cytosolic 1CM, NADPH, and 85% of endogenous glycine. Notably, a 2014 study of over one thousand enzymes spanning nearly two thousand tumors across nineteen different cancer types found SHMT2 to be among the top 5 most upregulated metabolic enzymes, highlighting the key role of mitochondrial 1CM across the entire spectrum of cancers. Unfortunately, no clinically-effective inhibitors of mitochondrial 1CM exist. The development of novel PCFT- selective 5-pyrrolo [2,3-d] and [3,2-d]pyrimidine cytotoxic agents with dual mitochondrial and cytosolic 1CM enzyme targets will potentially overcome resistance to present therapies while decreasing toxic side effects in the treatment of NSCLC. In Aim 1, select series lead compounds among our growing library of 350+ inhibitors will be screened for activity in engineered Chinese hamster ovarian (CHO) cell models which are isogenic except for plasma membrane folate transporter expression (i.e. one expresses PCFT only and the other RFC only). In addition, as induction of purine and glycine auxotrophy implies cytosolic and mitochondrial 1CM targeting respectively, cell rescue by supplementation of these metabolites will establish dual targeted compounds for evaluation in Aim 2. Currently, five promising compounds from CHO studies have progressed to human tumor cell line trials with next-generation compounds constantly being synthesized by a medicinal chemist collaborator. In Aim 2, lead compounds from Aim 1 will be evaluated for efficacy against non-small cell lung cancer. In H460 cells, we will confirm likely transporters and enzyme targets suggested by CHO studies in Aim 1 with in- situ and cell-free assays. Specific effects of targeting mitochondria will also be evaluated and promising analogs will be tested in-vivo with subcutaneous H460 xenografts in severe combined immunodeficient mice.