Potent inhibition of human folylpolyglutamate synthetase by a phosphinic acid mimic of the tetrahedral reaction intermediate.

A phosphorous-containing pseudopeptide folate analog (Valiaeva et al., J Org Chem 2001;66:5146-54) was designed to mimic the tetrahedral intermediate formed in the ATP-dependent reaction catalyzed by folylpolyglutamate synthetase (FPGS). This analog, methotrexate-phosphinate (MTX-phosphinate; 4-amino-4-deoxy-10-methylpteroyl-L-Glu-gamma-[psi(P(O)(OH)-CH(2))]glutarate), is a highly potent (K(is), 3.1+/-0.5 nM), competitive inhibitor of recombinant human cytosolic FPGS. Within experimental limits, FPGS inhibition was not time-dependent, and preincubation of FPGS, inhibitor, and ATP did not potentiate the inhibition. These results suggest that slow phosphorylation to produce a more potent inhibitor form is not involved. MTX-phosphinate was not growth inhibitory to human CCRF-CEM leukemia cells at 1 microM (70-fold above the concentration of MTX giving 50% growth inhibition), probably because of poor transport. Because of its exceedingly high potency as an FPGS inhibitor, MTX-phosphinate represents a lead structure from which cell-permeable analogs may be developed to test the hypothesis that FPGS inhibition is therapeutically efficacious.