Stimulation of P-Glycoprotein ATPase by Analogues of Tetramethylrosamine:  Coupling of Drug Binding at the “R” Site to the ATP Hydrolysis Transition State

Abstract

The multidrug resistance efflux pump P-glycoprotein (Pgp) couples drug export to ATP binding and hydrolysis. Details regarding drug trajectory, as well as the molecular basis for coupling, remain unknown. Nearly all drugs exported by Pgp have been assayed for competitive behavior with rhodamine123 transport at a canonical "R" drug binding site. Tetramethylrosamine (TMR) displays a relatively high affinity for Pgp when compared to other rhodamines. Here, we present the construction and characterization of a library of compounds based upon the TMR scaffold and use this set to assess the determinants of drug binding to the "R" site of Pgp. This set contained modifications in (1) the number, location, and conformational mobility of hydrogen-bond acceptors; (2) the heteroatom in the xanthylium core; and (3) the size of the substituent in the 9-position of the xanthylium core. Relative specificity for coupling to the distal ATP catalytic site was assessed by ATPase stimulation. We found marked ( approximately 1000-fold) variation in the ATPase specificity constant within the library of TMR analogues. Using established methods involving ADP-Vi trapping by wild-type Pgp and ATP binding by catalytic carboxylate mutant Pgp, these effects can be extended to ATP hydrolysis transition-state stabilization and ATP occlusion at a single site. These data support the idea that drugs trigger the engagement of ATP catalytic site residues necessary for hydrolysis. Further, the nature of the drug binding site and coupling mechanism may be dissected by variation of a drug-like scaffold. These studies may facilitate development of novel competitive inhibitors at the "R" drug site.

About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work

“…Using a cysteine-less mutant of Pgp and chemical cross-linking, they show that compounds that stimulate or inhibit ATP hydrolysis do so by increasing and decreasing respectively the distance between the Walker A and the LSGGQ sequences. This would imply that the former favors the formation of the sandwich dimer (see below), a view that is consistent with that of Tombline et al (40).…”

Rhodamine Inhibitors of P-Glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

“…38 Similarly, the addition of N,N -diethyl 2-lithiobenzamide ( 37 ) to 14-S gave chalcogenorhodamine 29-S in 43% isolated yield (Scheme 3). …”

“…Chalcogenorhodamines 1-E, 2-E , and 5-E through 13-S 38-40 were synthesized via the addition of the appropriate Grignard reagent (prepared from the corresponding bromoalkane or arylbromide) to chalcogenoxanthones 14-E 41 or 15-E 42 (Chart 2). The initial addition products were dehydrated in aqueous HPF 6 or HBr to give the indicated salts.…”

Chalcogenopyrylium Dyes as Differential Modulators of Organic Anion Transport by Multidrug Resistance Protein 1 (MRP1), MRP2, and MRP4

“…Modulators have typically been identified by either testing drugs already in clinical practice (or derivatives thereof) or screening libraries of natural product or synthetic chemical entities. As rhodamine derivatives originally developed as photosensitizers and subsequently determined to be modulators of P-glycoprotein and/or MRP1, the CGPs are an example of the latter (Tombline et al, 2006;Sawada et al, 2008;Gannon et al, 2009;Ebert et al, 2012). In the present study, six of the seven molecules comprising a new class of CGPs (class V) tested initially at a single concentration inhibited MRP1-mediated E 2 17bG uptake by .80%, thus identifying class V CGPs with their distinctive methine or trimethine linkage between two disubstituted pyrylium moieties as a particularly effective class of MRP1 modulators (Fig.…”