RESUMEN
We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3-5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., Kis), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate.
RESUMEN
Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange between serine, glycine and formate. In both the cytosol and mitochondria, folates exist as polyglutamates with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets SHMT2 in the mitochondria, and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ~19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (~30-fold) more than cFPGS (~4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the non-classical SHMT2/SHMT1 inhibitor, SHIN1. These results suggest a critical role of mFPGS levels in determining anti-tumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. Significance Statement AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347 Mitochondrial FPGS levels play important roles in determining the anti-tumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.
RESUMEN
One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.
Asunto(s)
Cisplatino , Citosol , Resistencia a Antineoplásicos , Mitocondrias , Neoplasias Ováricas , Humanos , Femenino , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Citosol/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Cisplatino/farmacología , Ratones , Línea Celular Tumoral , Carbono/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Glicina Hidroximetiltransferasa/metabolismo , Glicina Hidroximetiltransferasa/genética , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/genética , Antagonistas del Ácido Fólico/farmacologíaRESUMEN
Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.