Allosteric Modulation of Human P-glycoprotein

Abstract

The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z) Cellular expression of human P-glycoprotein (Pgp), 1 the product of the MDR1 gene, confers resistance to a broad variety of structurally unrelated chemotherapeutic agents and restricts bioavailability of many therapeutic drugs in experimental models (1, 2). Pgp is a 1280-amino acid plasma membrane protein that has two homologous halves separated by a linker region of about 80 amino acids (3). Each half of the protein contains a hydrophobic region with six putative transmembrane (TM) helices, followed by a cytoplasmic consensus ATPbinding/hydrolysis site (3). The TM regions are presumed to form the drug-translocating pathway (4), whereas the ATP sites, through ATP hydrolysis, provide the necessary driving force for transport (5, 6).-Pgp-mediated drug transport is inhibited by a number of structurally unrelated compounds known as reversing agents or modulators (for review see Ref.2). Although some of the modulators are currently being tested for their clinical effectiveness, there remains a growing need for molecules with higher efficacy (7-9). To develop such compounds, a clear knowledge of the mechanisms of action of the existing repertoire is essential. Some of the Pgp modulators themselves, such as verapamil (10) and cyclosporin A (11), are substrates of the pump and inhibit drug transport in a competitive manner without interrupting the catalytic turnover (catalytic cycle) of Pgp (12-15). However, for many others, the inhibitory mechanisms are yet to be fully understood.Recent studies on the mechanism of action of Pgp modulators indicated an allosteric mode of action for several compounds. Martin et al. (16) demonstrated that inhibition of vinblastine transport by the anthranilic acid derivative XR9576 is not through direct physical competition for the drug translocating pathway, indicating an allosteric effect on substrate recognition or ATP hydrolysis (17). A similar study suggested that the indolizin sulfone SR33557 affected vinblastine binding to Pgp through interaction with a site distinct from the site of substrate recognition (17). Boer et al. (18) and Ferry et al. (19) have shown that modulators like dexniguldipine and prenylamine inhibited vinblastine interaction with Pgp by a non-competitive mechanism. Based on these and other similar studies, drug interaction sites of Pgp have been categorized into the following two discrete types: 1) transport sites, where translocation of drug across the lipid bilayer can occur, and 2) regulatory sites, which modulate Pgp function (20,21). Most of these studies investigated the effect of the modulators on substrate binding in isolated membranes, ...

Allosteric Modulation Bypasses the Requirement for ATP Hydrolysis in Regenerating Low Affinity Transition State Conformation of Human P-glycoprotein

“…Since fluoroaluminate traps both dinucleotide and trinucleotide at the catalytic site (37), the ability of cis-(Z)-flupentixol to regenerate [ 125 I]IAAP binding to fluoroaluminate-trapped Pgp indicates that irrespective of whether the low affinity state is generated by nucleotide hydrolysis (18,19) or by tight nucleotide binding (21), transition to the high affinity state can be induced by the modulator. An argument can be made that the recovery of 125 I]IAAP binding suggested no physical overlap between the interaction sites of the two (18,32). In this report, we demonstrate that in a catalytic transition state conformation of Pgp, which prevents binding of transport substrate […”

“…These results suggested that the [ 125 I]IAAP site remains occluded in the vanadatetrapped conformation and only becomes accessible after a second hydrolytic event (28). We reported that thioxanthene-based Pgp modulators, such as flupentixols, allosterically interact with Pgp through a site that is functionally distinct from the site of substrate ([ 125 I]IAAP) recognition (32). In this study, we demonstrate that in the vanadatetrapped low affinity conformation of Pgp, the allosteric site remains accessible and responsive to allosteric modulation by cis-(Z)-flupentixol (Fig.…”

“…Interestingly, the [ 125 I]IAAP-binding site once transformed to its low affinity state remains unaltered even after dissociation of vanadate and ADP from the catalytic sites of Pgp (27,28 (32). Experimental evidence also suggests that the allosteric site can effectively communicate with the substrate and the ATP sites (31,32).…”

“…Experimental evidence also suggests that the allosteric site can effectively communicate with the substrate and the ATP sites (31,32). In this study, we demonstrate that the allosteric site remains accessible and responsive to modulator interaction, in the vanadate-trapped low affinity conformation of Pgp and mediates regeneration of [ Baculovirus Expression of Human Pgp-Recombinant baculoviruses BV-MDR1-(His 6 ) (19) and BV-MDR1-(His 6 )-F983A (32) harboring the wild type and the Pgp F983A mutant, respectively, were used to infect High Five insect cells grown in Excell 400 medium as described (34).…”

“…Although the photoaffinity labeling does not provide a stoichiometric binding, inhibition studies with other transport substrates (such as vinblastine) and competitive modulators (such as cyclosporin A) suggest that the affinity-labeled fraction is a fair representation of the entire Pgp population. Also, instead of ATP, we used 8-azido-ATP to be consistent with the experiments done in parallel to study [␣- 32 It was not totally clear why the regenerated level of [ 125 I]IAAP binding was higher than that bound to untrapped Pgp. One possible explanation is that since both the untrapped and trapped Pgp were subjected to similar washing and centrifugation steps, some of the untrapped Pgp molecules get inactivated during the procedure, whereas the tightly bound ADP and vanadate at the catalytic site conferred protection to the trapped Pgp from such inactivation.…”