Novel Pyrrolo[3,2-d]pyrimidine Compounds Target Mitochondrial and Cytosolic One-carbon Metabolism with Broad-spectrum Antitumor Efficacy

Abstract

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.

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism

“…, AGF291, AGF320, and AGF347 were synthesized as previously described (Wang et al, 2011;Dekhne et al, 2019).…”

“…Cell proliferation assays in CHO cells were performed as described in complete FFRPMI without glycine. Glycine/adenosine rescue experiments were performed in BxPC-3 and HPAC cells without additions, or in the presence of adenosine (60 mM) and/or glycine (130 mM) (Dekhne et al, 2019).…”

“…We recently described novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds, AGF291 [(4-(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)benzoyl)-L-glutamic acid], AGF320 [(5-(5-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d] pyrimidin-5-yl)pentyl)thiophene-2-carbonyl)-L-glutamic acid], and AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-L-glutamic acid], as anticancer agents (Dekhne et al, 2019). Direct inhibition of SHMT2 in mitochondria and targeting of SHMT1 and de novo purine biosynthesis [at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferases (GARFTase and AICARFTase, respectively)] in cytosol were established in colon and lung cancer cells, as well as in pancreatic cancer (PaC) models.…”

“…Direct inhibition of SHMT2 in mitochondria and targeting of SHMT1 and de novo purine biosynthesis [at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferases (GARFTase and AICARFTase, respectively)] in cytosol were established in colon and lung cancer cells, as well as in pancreatic cancer (PaC) models. For early and late-stage MIA PaCa-2 (an aggressive PaC model) tumor xenograft models treated with AGF347, impressive in vivo antitumor efficacy with curative potential was accompanied by minimal toxicity, and enzyme targets were validated by metabolomics analysis and in vitro enzyme assays (Dekhne et al, 2019). Fig.…”

“…Our studies establish broad-spectrum in vitro antitumor efficacy against a panel of PaC models and demonstrate the critical roles of plasma membrane transport, mitochondrial accumulation, and conversion to polyglutamates in both cytosol and mitochondria in effecting antitumor activity. We also explore mechanisms of antitumor activity of pyrrolo[3,2-d]pyrimidine compounds, including the impact of loss of serine catabolism and purine biosynthesis resulting from AGF347 (Dekhne et al, 2019) on AMP-activated protein kinase (AMPK) activation and mammalian target of rapamycin (mTOR) signaling, mitochondrial respiration, and glutathione pools and levels of reactive oxygen species (ROS). Collectively, our results underscore the unique pharmacology of this novel series and establish AGF347 as a promising new therapeutic for cancer.…”