Quantitative distinctions of active site molecular recognition by P-glycoprotein and cytochrome P450 3A4

The bulk of characterized xenobiotic defense and disposition is conferred by the abundant enzymes cytochrome P450 3A4 and P-glycoprotein. Although expressed in many tissues, these enzymes are most abundant in the liver and intestine and seem to share most substrates and inhibitors, with the apparent synergy between these two promiscuous enzymes asserted because of their extensive overlap of substrates and shared tissue location. Since the broad-spectrum tolerance to lipophilic compounds of various sizes naturally results in a similar pattern of substrate/inhibitor recognition, the cause or mechanism of many drug/drug and drug/herb interactions can be difficult to determine. These two seemingly indiscriminate enzymes, however, do not share some unique inhibitor selectivity. Particularly, we show various potent CYP3A4 inhibitors that do not affect P-gp active transport function. Remarkably, we have also identified several compounds-valinomycin, norverapamil, reserpine, nobiletin, emetine, gallopamil, fluphenazine-that uniquely inhibit P-gp function with affinities comparable to benchmark P-gp inhibitors despite a lack of effect on CYP3A4 function at physiologically relevant concentrations. Indeed, valinomycin inhibits P-gp with an IC(50) similar to cyclosporin A yet apparently does not affect CYP3A4 function, and emetine and nobiletin are also specific for interaction with P-gp. Additionally, norverapamil and reserpine have, respectively, a 60- and 40-fold preference for inhibition of P-gp over CYP3A4. Some striking structural analogies among these compounds are discussed. These distinguishing qualities of substrate recognition between CYP3A4 and P-gp should reveal nuances of active-site architecture unique to each and could serve as tools to probe for the specific discernment of P-gp-mediated drug/drug or drug/herb interactions. Learning more about binding distinctions and quantitative activity relationships of substrate/inhibitor interactions with these two enzymes and the differences between them may indicate how they recognize such a wide variety of molecules as substrates (and/or inhibitors). Moreover, identification of specific inhibitors will allow the determination of which enzyme is responsible for drug interactions and/or the extent of contribution in a multiple exposure situation.