P‐Glycoprotein conformational changes detected by antibody competition

Abstract

Conformational changes accompanying P‐glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp‐expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA94, 12908–12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre‐staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30–40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween‐80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2‐shift, as suggested by the simultaneous assessment of the two phenomena.

Complete Inhibition of P-glycoprotein by Simultaneous Treatment with a Distinct Class of Modulators and the UIC2 Monoclonal Antibody

“…1) (in accordance with Mechetner et al, 1997;Druley et al, 2001). We have previously shown (Nagy et al, 2001(Nagy et al, , 2004) that the incubation of cells with certain Pgp substrates and modulators (referred to as ACT-positive agents) increases the reactivity of UIC2 to Pgp to a large extent in a reproducible fashion as opposed to other substrates and modulators. Close to 100% of all of the cell surface Pgp molecules bind UIC2 in the presence of such ACT-positive agents (e.g., CsA, SDZ PSC 833, vinblastine, ivermectin, and valinomycin) compared with untreated cells or the cells incubated with ACT-negative agents, like verapamil (among many other agents tested).…”

“…At the same time, ACT-negative agents do not induce an inhibitory binding of UIC2 (Fig. 2), even if they elicit a mild increase in UIC2 reactivity (Nagy et al, 2001(Nagy et al, , 2004. As calcein accumulation was increased significantly only at Ն60 to 70% ligation of cell surface Pgp with UIC2 (Fig.…”

“…Previously, applying a novel assay [antibody competition test (ACT)] (Nagy et al, 2001(Nagy et al, , 2004, we have selected a distinct class of Pgp modulators (herein referred to as ACTpositive agents) that elicit a marked increase in UIC2 binding. Here we show that administration of UIC2 in the presence of these modulators (e.g., CsA, SDZ PSC 833, and vinblastine) leads to a near-complete inhibition of Pgp.…”

Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

“…These differences show that verapamil shifts Pgp into at least two distinct conformations and the largest conformational changes occur at low concentrations of verapamil. This observation is consistent with verapamil-induced Pgp conformational changes deduced from cross-linking of [84][85][86], trypsin digestion of [87] and antibody competition with Pgp [88]. The slope of the Stern-Volmer plot for Pgp in the presence of 250 μM digoxin was 2.29 + − 0.12 M − 1 ( Figure 4D).…”

Function-dependent Conformational Changes of the ABCG2 Multidrug Transporter Modify Its Interaction with a Monoclonal Antibody on the Cell Surface

“…Similar antibodies have already been prepared against the human MDR1 multidrug transporter (23,24). In the case of MDR1, several of the mAbs reacting with extracellular epitopes were found to inhibit the transport function of the protein, and the reactivity of one of these antibodies, UIC2, was reported to depend on the conformation of the MDR1 protein (23,(25)(26)(27).…”