Quantitative structure-activity relationships of methotrexate and methotrexate analogues transported by the rat...

Quantitative structure-activity relationship (QSAR) computational methods were performed to characterize structural requirements and molecular features for rMrp2-mediated methotrexate (MTX) transport. The compounds used in this analysis included MTX and 24 MTX analogues, with activity assessed by measuring inhibition of (3)H-DNP-SG ((3)H-S-(2,4)-dinitrophenyl glutathione) uptake in rat canalicular membrane vesicles. 2D-QSAR modeling using simulated annealing partial least squares (SA-PLS) method identified octanol/water partition coefficient, hydrophobicity, and negative charge as three important factors for MTX and MTX analogue affinity to rMrp2. Further analysis using 3D-QSAR method identified a pharmacophore model consisting of two hydrophobes, two aromatic rings, and a negative ionizable group as the critical molecular features that predict binding affinity of these compounds to rMRP2. The addition of a benzoyl ornithine group at a 9.3A distance and 136.5 degrees vector from the negative ionizable structure of MTX resulted in a 40-fold more potent inhibition of DNP-SG transport, suggesting that this chemical modification, while not essential for activity, contributes to the transport of MTX analogue by rMrp2. These observations provide important insights to the rationale development of analogues of MTX for the treatment of neoplastic and immunological diseases that may be devoid of hepatotoxicity or lack drug resistance.