A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands

Abstract

BACKGROUND AND PURPOSEThe human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH Using [3 H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTSKinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor-ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k−2, was 1.2 × 10 −3 min −1. The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONSThe interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc-receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand.

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

“…4 and Table 2). This value is in good correlation to what has been reported in the literature obtained in cellular systems, for the binding of maraviroc to CCR529. As elucidated from the high resolution X-ray structure, maraviroc binds in the bottom of a pocket defined by residues from helices I, II, III, V, VI and VII15.…”

“…The functionality of the reconstituted protein was confirmed by its capacity to bind to the antagonist maraviroc. The measured affinity of maraviroc for CCR5 StaR resembles that reported by cellular studies on binding to wild type CCR529. This demonstrates that the reconstituted receptor is properly folded and functional.…”

Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV

“…Slow off-rate is also a defining feature of Maraviroc (Swinney et al, 2014). However, [5P7]CCL5 may have additional advantages over Maraviroc with respect to the development of HIV drug resistance.…”

Understanding ligand-receptor non-covalent binding kinetics using molecular modeling

“…Despite the comprehension of the importance of the kinetics in drug design, a concrete recipe of kinetic effects remains as an open question due to the intrinsic difficulties of this issue. Experimental investigation of drug-protein binding is usually done by in vitro studies (6, 7), mutagenesis (8, 9), and structural biology (10, 11). …”