One-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a host of metabolites critical to tumor propagation. Although drug-targeting of cytosolic 1CM remains a clinically-relevant mainstay, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in lung, colon, breast, glioma, and liver cancer and, overall, is the fifth-most differentially expressed metabolic enzyme in cancer cell versus normal tissue. Despite the unequivocal oncogenic importance and therapeutic potential of SHMT2, there are no clinically relevant (i.e. active in vivo) inhibitors of this enzyme.
In this dissertation work, we sought to design, synthesize, and characterize pharmacodynamics of our 5-substituted pyrrolo[3,2-d]pyrimidine antifolates synergistically dual-targeting mitochondrial SHMT2 and cytosolic 1CM, the latter specifically at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). By depleting SHMT2-derived formate, these compounds potentiated their own inhibition of the downstream formate-dependent GARFTase and AICARFTase. We generated these compounds (AGF291, AGF320, and AGF347) by melding structures of SHMT2 cofactor 5,10-methylene tetrahydrofolate with our previously reported purine inhibitors and confirmed enzyme targets with in vitro targeted metabolomics in H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma) human tumor cell lines as well as in vitro cell-free assays. Transport assays revealed significant uptake by both the proton-coupled folate transporter (narrow physiological niche, but commonly expressed in many solid tumors) and the reduced folate carrier (major tissue folate transporter). Subcellular fractionation of MIA PaCa-2 and GlyB Chinese hamster ovary cells revealed AGF347 to be heavily (>98%) polyglutamylated in both cytosol and mitochondria with mitochondrial uptake partially mediated by the mitochondrial folate transporter. Intracellular glycine depletion secondary to SHMT2 inhibition by all compounds also depleted cellular ROS scavenging capacity as reflected in decreased GSH/GSSG ratio. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice with tumor growth delay (T-C) of 61 days and one out of five treated mice tumor-free 120+ days after treatment. In vivo metabolomics on these xenografts confirmed inhibition of purine biosynthesis. Collectively, the work in this dissertation establishes the exceptional therapeutic potential of dual-targeting mitochondrial and cytosolic 1CM.
|Advisor:||Matherly, Larry H|
|Commitee:||Patrick, Stephan, Ratnam, Manohar, Lash, Lawrence|
|School:||Wayne State University|
|School Location:||United States -- Michigan|
|Source:||DAI-B 81/4(E), Dissertation Abstracts International|
|Keywords:||antifolate, folate, Mitochondria, One-carbon, pyrrolopyrimidine, SHMT2|
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