In addition to being an important receptor in leukocyte activation and mobilization, CCR5 is the essential coreceptor for human immunodeficiency virus (HIV). A large number of smallmolecule CCR5 antagonists have been reported that show potent activities in blocking chemokine function and HIV entry. To facilitate the design and development of next generation CCR5 antagonists, docking models for major classes of CCR5 antagonists were created by using site-directed mutagenesis and CCR5 homology modeling. Five clinical candidates: maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220 were used to establish the nature of the binding pocket in CCR5. Although the five antagonists are very different in structure, shape, and electrostatic potential, they were able to fit in the same binding pocket formed by the transmembrane (TM) domains of CCR5. It is noteworthy that each antagonist displayed a unique interaction profile with amino acids lining the pocket. Except for TAK-779, all antagonists showed strong interaction with Glu283 in TM 7 via their central basic nitrogen. The fully mapped binding pocket of CCR5 is being used for structure-based design and lead optimization of novel anti-HIV CCR5 inhibitors with improved potency and better resistance profile.Human immunodeficiency virus (HIV) enters the host cell via the interaction of the viral envelope protein gp160 and the receptor/coreceptors on host cell surface. The majority of primary HIV-1 strains use CCR5 as coreceptor (termed R5 virus), whereas some viruses are able to use another chemokine receptor, CXCR4, as coreceptor (termed X4 virus) or use both CCR5 and CXCR4 as coreceptors (termed R5X4 virus). Because CCR5 is the predominant coreceptor for clinical HIV isolates, and the normal physiology within the human genetic knockout population, CCR5 has become a very attractive target for anti-HIV therapy. A number of small molecule CCR5 antagonists have been identified that demonstrated potent antiviral effects both in cell culture and in clinical trials.TAK-779, a quaternary ammonium anilide, was the first small molecule CCR5 antagonist reported (Baba et al., 1999). This compound was terminated as a result of poor oral availability. Two structurally diverse followers TAK-220 and TAK-652 are both in clinical trials (Imamura et al., 2006;Seto et al., 2006). Several other small molecule CCR5 antagonists with good potency and/or pharmacological properties have also been reported by other pharmaceutical companies. These include SCH-C (SCH-351125), vicriviroc (VVC, SCH-D, SCH-417690), aplaviroc (APL, AK602, GW873140), and maraviroc (MVC, UK-427,857). SCH-C is an oximino-piperidino-piperidine amide (Palani et al., 2002) that showed potent antiviral activity in vivo. However, its clinical development was terminated as a result of HERG inhibitory activity. SCH-D is the next generation compound of SCH-C, which is in late stage clinical development. SCH-D showed better oral availability, potency, safety, and pharmacological properties than SCH-C Article, publication d...

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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.

A panel of four CCR5 monoclonal antibodies (mAbs) recognizing different epitopes on CCR5 was examined in CCR5-mediated cell-cell fusion assay, alone or in combination with a variety of small molecule CCR5 antagonists. Although no antagonism was observed between any of the CCR5 inhibitors, surprisingly potent synergy was observed between CCR5 mAbs and antagonists, and the synergistic activity was confirmed in other antiviral assays. Strong synergy was also observed between CCR5 inhibitors and the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide. There was no synergy observed between small molecule CCR5 inhibitors; however, potent synergy was observed between mAbs recognizing different parts of CCR5. In all synergistic combinations, greater synergy was achieved at higher percent inhibition levels. A negative correlation was found between the degree of synergy between the two classes of CCR5 inhibitors and the ability to compete each other for binding to the receptor. For example, the greatest synergy, observed between the mAb ROAb13 and the small molecule inhibitor maraviroc, did not interfere with binding to CCR5 for either inhibitor, whereas no synergy was found between mAb 45523 and maraviroc, which do compete for binding to CCR5. In addition, in contrast to a recent report, the CCR5 inhibitors tested here were found to inhibit the same stage of HIV entry. Based on the data presented here, we hypothesize that CCR5 inhibitors exert synergistic antiviral actions through a cobinding mechanism.

In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly 4 Ser) n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18-to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibodyresistant HIV-1 strains.