A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy

Ilze Bot,Daniël Van Der Velden,Dirk-Jan Van Der Berg,Henk De Vries,Dean Stamos,Laura H Heitman,Natalia V Ortiz Zacarías,Johan Kuiper,Mara J Kröner,Elizabeth C M De Lange,Peter J Van Santbrink,Wilhelmus E A De Witte,Adriaan P Ijzerman

doi:10.1038/s41598-017-00104-z

Abstract

CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were performed to determine affinity and binding kinetics of 15a on murine CCR2. To assess the in vivo efficacy, western-type diet fed apoE−/− mice were treated daily with 15a or vehicle as control. Treatment with 15a reduced the amount of circulating CCR2+ monocytes and the size of the atherosclerotic plaques in both the carotid artery and the aortic root. We then showed that the long pharmacokinetic half-life of 15a combined with the high drug concentrations ensured prolonged CCR2 occupancy. These data render 15a a promising compound for drug development and confirms high receptor occupancy as a key parameter when targeting chemokine receptors.

Highlights

The chemokine system comprises more than 20 different chemokine receptors, which belong to the class A or rhodopsin-like family of G protein-coupled receptors (GPCRs)
We aimed to evaluate the binding kinetics and pharmacokinetics of 15a for murine Chemokine Receptor 2 (CCR2) and determine whether this CCR2 antagonist is effective in an apoE−/− mouse model of atherosclerosis
Association curves of [3H]INCB3344 in the presence of 15a in mouse CCR2 (mCCR2) resulted in the typical overshoot characteristic for long residence time (RT) compounds (Fig. 3b) and a kinetic rate index (KRI) > 1.0 (KRI = 1.6)

Summary

Introduction

The chemokine system comprises more than 20 different chemokine receptors, which belong to the class A or rhodopsin-like family of G protein-coupled receptors (GPCRs). The combination of structure-affinity and structure-kinetics optimization resulted in the discovery of compound 15a (Fig. 1), an orthosteric CCR2 small molecule antagonist with high affinity of 2.4 nM and a RT of 714 min for human CCR2 (hCCR2)[24] With such a prolonged CCR2 inhibition, 15a emerges as a potential candidate to evaluate the in vivo effects of long RT. Using a combination of in vitro radioligand binding assays and in vivo studies, we show that prolonged CCR2 antagonism with 15a, due to high target occupancy, is linked to a robust and significant inhibition of atherogenesis in mice These results support the need of achieving more than 90% continuous inhibition when developing chemokine receptor antagonists[26]. This study highlights the importance of in vitro characterization of drug candidates in all relevant species for in vivo pre-clinical studies, in order to improve the translational value of animal models, and to reduce the attrition in drug discovery programs

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