title: 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.
authors.affiliation: Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA. Gregory_Tombline@urmc.rochester.edu
authors.forename: Gregory
authors.initials: G
authors.lastname: Tombline
authors.forename: David J
authors.initials: DJ
authors.lastname: Donnelly
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Holt
authors.forename: Youngjae
authors.initials: Y
authors.lastname: You
authors.forename: Mao
authors.initials: M
authors.lastname: Ye
authors.forename: Michael K
authors.initials: MK
authors.lastname: Gannon
authors.forename: Cara L
authors.initials: CL
authors.lastname: Nygren
authors.forename: Michael R
authors.initials: MR
authors.lastname: Detty
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D012235:Rhodamines; D014961:X-Ray Diffraction
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D006575:Heterocyclic Compounds, 3-Ring; D012235:Rhodamines; C412715:tetramethylrosamine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases