Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells.Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, b-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure-activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.

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Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6substituted thieno [2,3-d]pyrimidine compounds (3−9) with pyridine (3, 4), fluorine-substituted pyridine (5), phenyl (6, 7), and thiophene side chains (8, 9), for comparison with unsubstituted phenyl (1, 2) and thiophene side chain (10, 11) containing thieno[2,3-d]pyrimidine compounds. Compounds 3−9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or β but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4, 5, 6, and 9 was observed. Replacement of the side-chain 1′,4′-phenyl ring with 2′,5′-pyridyl, or 2′,5′-pyridyl with a fluorine insertion ortho to Lglutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4−9 were highly active (IC 50 's from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17-to 882-fold more potent than previously reported compounds 2, 10, and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1, 2, and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4, 5, 9, and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity.

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide influences the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the wild-type CFTR interactome, silent mutations that disrupt this RNA structure alter how ΔF508 CFTR interacts with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances both the expression and function of ΔF508 CFTR with no impact on wild-type. Finally, we show that disrupting this motif enhances the pharmacological rescue of ΔF508 by Trikafta, which implies ribosomal frameshifting antagonizes the effects of leading CF therapeutics. Together, our results reveal that ribosomal frameshifting selectively reduces the expression and assembly of a misfolded CFTR variant. These findings suggest cotranslational misfolding alters the processivity of translation and potentially the stability of the mRNA transcript through the dynamic modulation of ribosomal frameshifting.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Though most patients initially respond to platinum-based therapy, the likelihood of disease reoccurrence is nearly 100%. Thus, new tumor-selective therapies for EOC are urgently needed. One such treatment option involves targeting tumors via folate receptor α (FRα), which is overexpressed in up to 90% of EOCs and shows increasing expression with higher stage and grade of disease. Our laboratory discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359, AGF362 and AGF363) that inhibit mitochondrial one-carbon (C1) metabolism at serine hydroxymethyltransferase (SHMT) 2, with secondary inhibition at cytosolic enzyme targets including those in de novo purine biosynthesis. Potent inhibition was seen with several FRα-expressing EOC tumor cells. Inhibitory potencies were in order, AGF347 > AGF359 > AGF362 > AGF363. Drug effects were substantially reduced with excess folic acid (FA), confirming FRα-mediated drug uptake. Toward cisplatin resistant SKOV3, TOV112D and A2780 EOC cells, inhibition in the nanomolar range was detected with all compounds. Targeted metabolomics, using L-[2,3,3-2H]serine as a tracer in wild-type or SHMT2 knockdown SKOV3 cells, confirmed all compounds inhibited cytosolic and mitochondrial C1-metabolism (at SHMT2). Apoptosis was detected for all compounds by Annexin V/PI, with partial rescue of apoptosis observed upon addition of glutathione (GSH). Glutathione pools (GSH and total GSH+GSSG) were significantly perturbed by drug treatment with all inhibitors in SKOV3 cells, comparable to GSH levels observed in SHMT2 KD cells. In vivo efficacy studies with SKOV3 xenografts treated with either AGF347 or cisplatin in SCID mice showed cisplatin resistance, while AGF347 demonstrated efficacy and delay in disease progression with a median tumor growth delay of 10 days, with the longest delay being 15 days. Our studies describe a series of novel inhibitors targeting mitochondrial and cytosolic C1-metabolism, selectively delivered via FRα, which show direct cytotoxic effects against cisplatin resistant EOC in vitro and in vivo, and display additional mechanisms of cytotoxicity mediated through glycine depletion. Citation Format: Adrianne C. Wallace-Povirk, Carrie O'Connor, Xun Bao, Jade Katinas, Jennifer Wong-Roushar, Aamod Dekhne, Zhanjun Hou, Md. Junayed Nayeen, Khushbu Shah, Jose Cardiel Nunez, Jing Li, Seongho Kim, Lisa Polin, Charles E. Dann, Aleem Gangjee, Larry H. Matherly. Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2348.