16th International AIDS Conference Toronto, Canada - August 13 - 18, 2006

COOPERATIVE INTERACTION BETWEEN A CCR5 ANTAGONIST, APLAVIROC(873140/ONO-4128), AND HUMAN CCR5 WITH ALLOSTERIC MECHANISM

Int Conf AIDS. 2006 Aug 13-18;16 Abstract No. ThAa0305

Shibayama S.¹, Tanihiro T.¹, Takeda K.¹, Watanabe N.¹, Hayashi T.¹, Sagawa K.¹, Demarest J.², Mcdanal C.², Sparks S.², Takaoka Y.³, Fukushima D.^1
1 Ono Pharmaceutical Co., LTD., Exploratory Research Laboratories, Tsukuba, Japan, ² GlaxoSmithKline, Research & Development, Research Triangle Park, United States, ³ Ono Pharmaceutical Co., LTD., Medicinal Chemistry Research Laboratories, Osaka, Japan

BACKGROUND: CCR5 antagonists have been shown to inhibit protein-protein (the HIV-1 gp120 and CCR5) interaction via a proposed allosteric mechanism. CCR5 receptor occupancy (RO) of aplaviroc (873140/ONO-4128) was nicely correlated to its anti-HIV activity. We have studied the relationship of aplaviroc's slow off-set rate with anti-HIV activity, and assessed its receptor binding mechanism using CCR5 mutants and [3H]aplaviroc in binding experiments.

METHODS: Flow cytometric analysis was used to determine duration of in vivo blood RO after intraperitoneal administration of aplaviroc in mice injected with CSFE-labeled murine cells expressing human CCR5 (huCCR5). The half-life in vitro for RO decline was also calculated by dissociation experiments of [3H]aplaviroc. We assessed if the binding of aplaviroc follows the law of mass-action using Scatchard plot and the Hill equation based on the [3H]aplaviroc-saturation binding assay.

RESULTS: The RO of aplaviroc in the mouse system was sustained (t_1/2=13.7 hours) more than the decline of its blood concentrations. For in vitro studies, [3H]aplaviroc dissociated from huCCR5 with 0.0004 min-1 of a koff (t_1/2=1708 min) and from a huCCR5(Ile198Met) mutant with 0.004 min-1 of a koff. The Scatchard plot of the binding of [3H]aplaviroc to human CCR5 showed a convex curve and the Hill coefficient was 1.7.

CONCLUSIONS: [3H]aplaviroc showed persistent binding to human CCR5 both in vivo and in vitro, but was dissociated 10-times faster from the huCCR5 (Ile198Met) mutant despite a similar kon value in both CCR5s. Ile198 in transmembrane region-5 is a key residue to confer the property. The binding of aplaviroc does not appear to follow the law of mass-action simply according to the analysis of Scatchard plot and Hill plot. The results imply that involvement of positive co-operativity and CCR5 dimerization in the binding mechanism of aplaviroc is associated to persistent RO.

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2006-08-13
ThAa0305

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