Influence of lipophilicity and carboxyl group content on the rate of hydroxylation of methotrexate derivatives by aldehyde oxidase.
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 gamma-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 Vmax/Km 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, N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-lysine, and gamma-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. gamma-t-Butyl-3'-chloromethotrexate was a better substrate than gamma-t-butyl-MTX, demonstrating the strong contribution of a lipophilic Cl atom on the phenyl ring. N alpha-(4-Amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, with two carboxyl groups, showed substrate activity similar to that of MTX. The gamma-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.
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
A
A
Rosowsky
J E
JE
Wright
S A
SA
Holden
D J
DJ
Waxman
D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000494:pharmacology*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D006900:Hydroxylation; D007700:Kinetics; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011817:Rabbits; D012995:Solubility; D013329:Structure-Activity Relationship
D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D000445:Aldehyde Oxidoreductases; D042931:Aldehyde Oxidase; D008727:Methotrexate