Modulator-Induced Interference in Functional Cross Talk between the Substrate and the ATP Sites of Human P-glycoprotein

Abstract

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.

Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1

“…This might occur through communication between the drug‐binding site(s) and TM12. In fact, mutations in TM12 were demonstrated to affect transport or ATPase activity [32,33], in particular, the stimulation of ATP hydrolysis by vinblastine and nicardipine [15]. These two compounds are known to interact at pharmacologically distinct (allosterically linked) sites in ABCB1 [34], and this supports the notion of TM12 acting as a key conduit.…”

The Monoamine Oxidase A Inhibitor Clorgyline Is a Broad-Spectrum Inhibitor of Fungal ABC and MFS Transporter Efflux Pump Activities Which Reverses the Azole Resistance of Candida albicans and Candida glabrata Clinical Isolates

“…The PCL was selected as the compound library because it is compact (1,200 compounds) and also because published data which would aid interpretation of the results are available on each compound. Of the nine hits identified in the PCL screen, four (flupentixol, prochlorperazine, droperodol, and ethoxyquin) are known inhibitors or inducers of human ABC transporters (15,25,26,36), and four (ebselen, econazole, sulconazole, and disulfiram) have known antifungal activity (2,3,20,49,53). The finding that eight of nine hits detected in the screen, excepting clorgyline, had been shown previously to interact with efflux pumps and/or show antifungal activity validated our assay and this approach to screening.…”

Diagnosis of Fungal Infections

“…As the polyenes span the lipid bilayer, they may not be able to enter the drugbinding pocket of the transporter. Significant hydrophobicity is currently considered a prerequisite for substrate-multidrugtransporter interaction (204), and size is also reported to be a factor determining whether a hydrophobic compound can act as a substrate of at least one fungal ABC transporter, ScPdr5p (117). Echinocandins are large lipopeptide molecules with a molecular weight of about 1,200, and although they have a lipid side chain (Fig.…”