Stimulation of P-Glycoprotein ATPase by Analogues of Tetramethylrosamine: Coupling of Drug Binding at the “R” Site to the ATP Hydrolysis Transition State ² Gregory Tombline,* ,‡,§ David J. Donnelly, ‡ Jason J. Holt, ‡ Youngjae You, ‡ Mao Ye, ‡ Michael K. Gannon, ‡ Cara L. Nygren, ‡ and Michael R. Detty* ,‡ Department of Chemistry, UniVersity at Buffalo, The State UniVersity of New York, Buffalo, New York 14260-3000, and Department of Biochemistry and Biophysics, UniVersity of Rochester Medical Center, 601 Elmwood AVenue, P.O. Box 607, Rochester, New York 14642 ReceiVed February 19, 2006; ReVised Manuscript ReceiVed May 8, 2006 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 (∼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. P-glycoprotein (Pgp, 1 also known as MDR1 or ABCB1) (1-3), a mammalian plasma membrane protein, is a member of the ATP-binding cassette (ABC) superfamily and was the first efflux protein identified and associated with multidrug resistance (MDR) in cancer chemotherapy. Pgp is also the most studied of a growing family of proteins known to confer MDR (3). Related efflux pumps are associated with resistance in the treatment of AIDS, bacterial, parasitic, and fungal diseases (4-7). The reversal or inhibition of MDR is a clinically important goal and, numerous classes of com- pounds have been investigated for this role (8, 9). Pgp consists of a single polypeptide chain that forms two putative transmembrane domains (TMDs) and two nucle- otide-binding domains (NBDs) alternating along the chain. Binding and hydrolysis of ATP at the two NBDs is coupled to drug export via the TMDs. The alternating sites model for the mechanism of Pgp drug export suggested that hydrolysis at a single NBD is sufficient to facilitate a single transport event and that the two NBDs alternatively hydro- lyze ATP (10). Newer variations of this model suggest that two hydrolysis events are required for each transport cycle (11) and that ATP binding (and not hydrolysis) is most important for the primary drug transport event (12). ATP hydrolysis and drug transport have been shown to share the same rate-limiting transition state, providing formal proof of coupling of the two and that drugs transported at a higher rate bind the transition state more tightly (13). Low-resolution structural analysis has shown that major structural changes occur upon nucleotide binding to Pgp leading to asymmetry in the TMDs (14, 15). Recent structural data of homologues ² This research was supported in part by NIH Grant No. T32 CA09363 (Postdoctoral Training Grant) to G.T. and by the Department of Defense [Breast Cancer Research Program] under Award No. W81XWH-04-1-0708 to M.R.D. and W81XWH-04-1-0368 to D.J.D. Views and opinions of, and endorsements by the author(s), do not reflect those of the U.S. Army or the Department of Defense. * To whom correspondence should be addressed. For G.T.: current address, Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, P.O. Box 607, Rochester, NY 14642; tel., (585)-285-2769; fax, (585)-271-2683; e-mail, Gregory_Tombline@urmc.rochester.edu. For M.R.D.: Tel., (716)-645- 6800 x2200; fax, (716)-645-6963; e-mail, mdetty@buffalo.edu. ‡ University at Buffalo, The State University of New York. § University of Rochester Medical Center. 1 Abbreviations: Pgp, P-glycoprotein; MDR, multidrug resistance; ABC, ATP binding cassette; TMR, tetramethylrosamine; TMR-S, thiotetramethylrosamine; TMR-Se, selenotetramethylrosamine; VER, verapamil; R123, rhodamine 123; R6G, rhodamine 6G; TMD, trans- membrane domain; NBD, nucleotide binding domain; NCI, National Cancer Institute; ATP, adenosine triphosphate; ADP, adenosine diphos- phate; Vi, vanadate anion; Pi, inorganic phosphate; HRMS (EI), high- resolution mass spectrometry (electrospray ionization); THF, tetrahy- drofuran; tert-BuLi, tert-butyllithium; ORTEP, Oak Ridge thermal ellipsoid plot; DMSO, dimethyl sulfoxide; TCEP, tris(2-carboxyethyl)- phosphine hydrochloride; EGTA, ethylene glycol bis-2-aminoethyl ether tetraacetic acid; PEP, phosphoenolpyruvate; DTT, dithiothreitol; CFTR, cystic fibrosis transmembrane conductance regulator. 8034 Biochemistry 2006, 45, 8034-8047 10.1021/bi0603470 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/08/2006