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Influence of lipophilicity and carboxyl group content on the rate of hydroxylation of methotrexate derivatives by aldehyde oxidase

1990; Elsevier BV; Volume: 40; Issue: 4 Linguagem: Inglês

10.1016/0006-2952(90)90326-g

1873-2968

A. Rosowsky, Joel E. Wright, Sylvia A. Holden, David J. Waxman,

Vitamin C and Antioxidants Research

The influence of lipophilicity and carboxyl group content on the ability of methotrexate (MTX) derivatives to undergo 7-hydroxylation in vitro by partly purified rabbit hepatic aldehyde oxidase was examined. Addition of two to four γ-glutamyl residues to the MTX molecule caused a progressive decrease in the rate of hydroxylation associated mainly with a decrease in Vmax rather than an increase in Km. These results suggest that the number of carboxyl groups in the side chain has a relatively small effect on affinity for the enzyme active site, but hinders the formation of product. The catalytic efficiency of hydroxylation of MTX tetraglutamate, estimated from VmaxKm ratios, was 36-fold lower than that of the monoglutamate. In contrast, when the number of carboxyl groups was decreased to one, as in 4-amino-4-deoxy-N10-methylpteroic acid, Nα-(4-amino-4-deoxy-N10-methylpteroyl)-l-lysine, and γ-t-butyl-3′-chloromethotexate, enhanced catalytic efficiency was observed, involving both a decrease in Km and an increase in Vmax. The catalytic efficiency of hydroxylation of these three substrates was 88-, 360-and 2100-fold higher than that of MTX. γ-t-Butyl-3′-chloromethotrexate was a better substrate than γ-t-butyl-MTX, demonstrating the strong contribution of a lipophilic Cl atom on the phenyl ring. Nα-(4-Amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine, with two carboxyl groups, showed substrate activity similar to that of MTX. The γ-t-butyl esters of MTX, 3′-chloromethotrexate, and 3′,5′-dichloromethotrexate were compared with the parent acids as inhibitors of the growth of cultured human leukemic lymphoblasts (CEM cells) and an MTX-resistant subline (CEM/MTX) defective in MTX transport and polyglutamylation. Although the esters were less effective than the acids against CEM cells except at high concentrations, they were more effective against CEM/MTX cells. This “collateral sensitivity” of CEM/MTX cells to lipophilic MTX esters is consistent with a decreased ability to take up and utilize reduced folates from the culture medium.