Background:  CCR5 antagonists, such as maraviroc (Selzentry, MVC) represent a new class of recently approved HIV entry inhibitors. However, viral resistance has emerged as mutations in gp120 permits HIV entry via antagonist-bound receptor. An alternate strategy impervious to such resistance is to remove CCR5 from the cell surface. Here, we demonstrate that a novel and patented, small-molecule CCR5 agonist effectively inhibits HIV infection in peripheral blood mononuclear cells (PBMC) by its selective internalization of the CCR5 receptor.

Methods:  High throughput screening of a proprietary library of 140,000 small molecules was performed using the AequoScreen assay on CCR5:CHO-KI cells and agonist hit compounds were selected for optimization. Compound affinity for CCR5 was measured using both [¹²⁵I]MIP-1b and [¹²⁵I]RANTES radioligand binding assays. Agonist-induced receptor internalization was visualized using YFP-tagged CCR5 receptor whereas agonist-induced receptor desensitization was measured using the cAMP-HTRF assay. The efficacy of compounds to inhibit HIV infection was monitored in a reporter gene assay, and optimized compounds were evaluated in an infection assay using phytohemagglutinin-stimulated PBMC from healthy donors. IC₅₀ values for the inhibition of viral replication were calculated from p24 antigen production after 10 to 12 days of infection.

Results:  The high throughput screening campaign identified CCR5 agonists with EC₅₀ values in the µM range. ESN-196 is derived from original structures identified in high throughput screening whereupon ~1000-fold improvement in agonist potency has been achieved. ESN-196 demonstrates potent and selective affinity for the CCR5 receptor (Ki, 0.8 nM). This compound potently internalized YFP-tagged CCR5 and, correspondingly, desensitized CCR5 signalling. Furthermore, ESN-196 demonstrated a high potency of inhibition in viral entry in the reporter assay (IC₅₀:  90 nM), as well as in PBMC (IC₅₀ between 50 nM and 230 nM depending on the HIV strain ).

Conclusions:  ESN-196 is a small molecule CCR5 agonist with potent activity in inhibiting HIV replication in PBMC. Small-molecule CCR5 agonists offer significant advantages over the known peptide agonists where clinical utility is limited, in part, due to pharmacokinetic liabilities. Moreover, the concept of agonist-induced CCR5 receptor internalization may offer significant advantages over the receptor-blockade (i.e. antagonist) approach by reducing the frequency of emergence of viral resistance.