Use of In Silico Docking and Peptide Mapping Studies with the Cocaine Analog MFZ 2 24 to Elucidate the Dopamine Transporter Cocaine Binding Site

Abstract

Background Cocaine inhibits the dopamine transporter (DAT), resulting in altered dopaminergic homeostasis, that leads to psychostimulant effects. However, the molecular mechanisms underlying this phenomenon are not fully understood. Objective: Identification of the adduction site of the photoactive cocaine analog MFZ 2 24 through computational docking and biochemical peptide mapping to elucidate the cocaine-binding site on DAT. Results: Computational docking methods RosettaLigand and Induced Fit Docking independently predicted the azide adduction of MFZ 2 24 azide group occurs on residue Asp79 or Leu80. Peptide mapping of CNBr digested fragments obtained through introduction of site-directed methionine substitution mutants between Ile67 and Leu80 of transmembrane domain one (TM1) confirmed the computational results. These findings place the tropane core of MFZ 2 24 in the central binding pocket similar to our results with the cocaine photoaffinity analog RTI 82 which adducts to TM6. Conclusion Computational and biochemical docking analysis reveals that MFZ 2 24 adducts to Asp79 or Leu80 in TM1 of DAT with the tropane core positioned in the central binding site. Grant Support: INBRE Program of the NCRR and NIDA-IRP (AHN), DA027845 (LKH & RAV), P20 RR017699 from the COBRE Program of the NCRR, and P20 RR016741 from ND EPSCoR IIG (RAV & JDF).