A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-L-lysine analogue of methotrexate, N alpha-(4-amino-4-deoxy-10- methylpteroyl )-N epsilon-(5-[N,N-dimethylamino]-1-naphthalenesulfonyl)-L-lysine, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [3H]methotrexate transport with a Ki of 7.02 microM, a value virtually identical to the Kt for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-L-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.