| Abstract: | Folylpolyglutamate synthetase (FPGS) catalyzes the gamma-glutamylation of both folates and folate antagonists and has been found to be essential for the survival of mammalian cells. Twelve analogs of the antifolates aminopterin (AMT) and methotrexate (MTX) having the -(CH2)2COOH moiety replaced by -(CH2)nX, where X = SO3H,PO3H2 or NH2, were evaluated as inhibitors of FPGS isolated from human liver. The AMT analogs were consistently found to be better inhibitors than their MTX counterparts, following the order of Km values determined for the parent antifolates as FPGS substrates. For the amino and phosphonate (but not for the sulfonate) compounds, inhibitory efficiencies were markedly dependent on the methylene chain length, with the most effective inhibitors having the groups -(CH2)3NH2(Ki = 0.2 microM) and -(CH2)2PO3H2 (Ki = 1.9 microM). Of those compounds exhibiting Ki values less than 200 microM, six were competitive inhibitors whereas three showed mixed inhibition (Ki' = approximately 6 Ki) when analyzed using AMT as the variable substrate. This demonstration of mixed inhibition of FPGS is consistent with the binding of inhibitor to a second site on the enzyme. Very similar Ki values (0.2-0.3 microM) were obtained for the -(CH2)3NH2 analog of AMT when using folic acid, AMT, MTX, and gamma-glutamyl-MTX as variable substrates, suggesting that the same enzymatic site on FPGS is active in the gamma-glutamylation of these four folyl derivatives. These findings serve to identify structural features which are important for inhibition of human liver FPGS and may therefore prove useful for the design of new compounds having potential as chemotherapeutic agents. |
