Structural preferences among folate compounds and their analogues for ATPase-mediated efflux by inside-out plasma membrane vesicles derived from L1210 cells.

Our prior studies with inside-out plasma membrane vesicles from L1210 cells (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) identified an outwardly directed, translocating ATPase as mediating the majority of methotrexate (MTX) efflux in these cells. In the current studies, we examined by competitive inhibition with [3H]MTX as permeant some of the structural features that determine preferences among folate compounds and their analogues as permeants for this ATPase. The results show that folate compounds are preferred over simple quinazolines (5,8-dideaza-pteridines), and IL5-CH3-folateH4, and probably other 5-substituted folates are preferred over folic acid. In the latter regard, the observed equivalence in preference to IL5-CH3-folateH4 of the 4-oxa-pyridopyrimidine, lometrexol (DDATHF), probably relates to its close similarity to folateH4. The results also suggest that the 4-position in the case of folate analogues, but not in the case of the quinazoline analogues, is an important determinant with 4-amino preferred over 4-oxa. They also suggest that the N10 position on the bridge region in both series of compounds, and probably for the pyridopyrimidine lometrexol, is not an important determinant. In contrast to results seen with the simple quinazolines, the 2-CH3 desamino quinazoline ZEN D1694, modified as well by a 2-benzyl to thienyl replacement on the side chain, was highly preferred. The same relative differences seen among some of these analogues as inhibitors of [3H]MTX efflux in inside-out vesicles were documented for their effectiveness as permeants for ATP-dependent efflux in intact L1210 cells.