Structure-activity relationship studies of propafenone analogs based on P-glycoprotein ATPase activity measurements

Propafenone analogs (PAs) were previously identified as potent inhibitors o f P-glycoprotein (Pgp) mediated toxin efflux. For this as well as other cla sses of Pgp inhibitors, lipophilicity as well as hydrogen bond acceptor str ength are important determinants of biological activity. The question as to whether a direct interaction between PA-type modulators and Pgp takes plac e was addressed by means of Pgp ATPase measurements and transport studies. Propafenone-type modulators stimulated ATPase activity up to 2-fold over ba sal activity in a concentration-dependent biphasic manner. Within a series of structural homologs, K-alpha values of ATPase stimulation strongly corre lated with lipophilicity. Analogs containing a quaternary nitrogen stimulat ed Pgp ATPase activity with lesser efficacy, while K-alpha values were some what higher when compared to corresponding tertiary analogs. Transport stud ies performed in inside-out plasmamembrane (I/O) vesicles demonstrated that analogs containing a tertiary nitrogen rapidly associated with the biomemb rane. Quaternary analogs, which are restricted by a permanent positive char ge in transiting the plasma membrane by diffusion, accumulated in Pgp conta ining I/O vesicles in an ATP-dependent and cyclosporin A-inhibitable manner , which identified them as Pgp substrates. Identical structure-activity rel ationships were found in either Pgp ATPase stimulation experiments in I/O v esicles or in toxin efflux inhibition studies using intact cells. Therefore , differences in membrane transit are not responsible for the observed stru cture-activity relationships. (C) 1999 Elsevier Science Inc.