A strategy for the design of membrane-permeable folypoly-gamma-glutamate synthetase inhibitors: "bay-region"-substituted 2-desamino-2-methyl-5,8-dideazafolate analogs
Sanghani, P.C.; Jackman, A.; Evans, V.R.; Thornton, T.; Hughes, L.; Calvert, A.H.; Moran, R.G.
Molecular Pharmacology 45(2): 341-351
1994
ISSN/ISBN: 0026-895X PMID: 8114682 Document Number: 425655
Previous attempts to design inhibitors of mammalian folylpolyglutamate synthetase (FPGS) have resulted in three classes of active compounds, all of which have charged moieties in the side chain, but structural alteration of the rest of the folate molecule has not seemed. to be an avenue for drug discovery. However, groups in the side chain of folate analogs that bear charge distributions different from that of glutamic acid appear to prevent efficient transport into mammalian cells on the reduced folate carrier system. We now report that substituents at the 7-, 2'-, or 3'-position of 2-desamino-2-methyl-4-hydroxyquinazoline antifolates decrease or prevent the catalysis of diglutamate formation by FPGS but are compatible with efficient binding to the reduced folate carrier system. Thus, 5,8-dideazafolates with a 3'-alkyl group had a lower V-max for FPGS than did the corresponding unsubstituted quinazolines, by a factor of 4-12, but these compounds inhibited the reaction of control FPGS substrates, indicating that the 3'-groups had much larger effects on catalytic activity than on binding to enzyme. A 7-methyl substituent affected the V-max of a series of 5,8-dideazafolate compounds by a factor of 2-8, but this decrease in the catalytic rate was also accompanied by an increase in the K-m of the substituted compounds by a factor of 10-100. The extent of the effect of a 7-methyl substituent on V-max appeared to be dependent on the size of the substituent at N10. Different substituents at the 2'-position affected the kinetics of the FPGS reaction with one of three patterns, i.e., 1) a 2'-fluoro substituent both increased V-max and decreased K-m slightly, 2) either -OH or -NH-2 decreased the V-max without affecting the K-m, and 3) 2'-Cl, -CH-3, -CF-3, or -OCH-3 substituents were found to both decrease V-max and increase K-m. Substitutions at the 7-, 2'-, or 3'-position had only minor effects on the ability of 2-desamino-2-methyl-4-oxoquinazolines to interfere with the transport of (3H)methotrexate into L1210 cells. Hence, these classes of compounds are likely to be efficiently transported by the reduced folate carrier system. We conclude that the region of the folate molecule bounded by the 7-, 6-, 9-, 10-, 3'-, and 2'-positions, the "bay region," is of major importance both for the binding of folates and folate analogs to FPGS and for the assumption of a conformation of the enzyme-substrate complex compatible with catalysis. We also suggest that appropriate substitutions at these positions would lead to membrane-permeable, specific FPGS inhibitors potent enough to allow evaluation of this enzyme as a target for cancer chemotherapy.