Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase

Abstract

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.

Folate receptors and transporters: biological role and diagnostic/therapeutic targets in cancer and other diseases

“…Promising in vivo anti-tumor efficacy with curative potential was demonstrated in an aggressive pancreas cancer model. C. Dann III (Indiana University, USA) presented the “Biochemical and structural analysis of targeted cytotoxic therapeutics on folate utilizing enzymes in C1 metabolism” in which he described the enzymology and structural biology of target enzymes including GARFTase, AICARFTase/inosine monophosphate cyclohydrolase (ATIC), SHMT1, SHMT2 and MTHFD2 with the lead pyrrolo [2,3- d ] pyrimidine inhibitors [ 11 ]. His presentation described efforts to determine crystal structures of these enzyme inhibitor complexes to facilitate future drug design.…”

PvdF of pyoverdin biosynthesis is a structurally unique N10-formyltetrahydrofolate-dependent formyltransferase

“…Here we report structural characterization of a hydroxyornithine transformylase, the PvdF enzyme from Pseudomonas aeruginosa. The structure reveals a core fold common among N 10 -fTHF dependent transformylases, including the glycinamide ribonucleotide transformylases (GART) [15][16][17][18][19] , the methionyl-tRNA transformylase (MTF) 20,21 , and N-sugar transformylases of O-antigen formation [22][23][24][25][26] . However, the structure reveals large, unique insertions that we propose are important for binding the substrate OHOrn, and that place PvdF as the first documented member of a new structural subclass.…”

Oncology Therapeutics Targeting the Metabolism of Amino Acids

“…Despite clinical efficacy, pemetrexed, along with all other antifolates, is cytotoxic due to non-specific transport by the reduced folate carrier (RFC). 2-amino-4-oxo-6-substituted pyrrolo [2¨C-d]pyrimidine derivatives are potent antifolates that inhibit GART in the nanomolar range [195]. They are poor substrates for RFC, and as a result, more effectively reduce growth of folate receptor (FR)-and proton-coupled folate transporter (PCFT)-expressing tumor cells and in human tumor xenografts [196,197].…”