Combining AlphaFold with Focused Virtual Library Design in the Development of Novel CCR2 and CCR5 Antagonists

CC chemokine receptor (CCR) 2 and 5 are G protein-coupled receptors that play a crucial role in immunohomeostasis. Accordingly, overactivation of their signaling pathways is involved in various immunopathologies and cancer. Extensive research focusing on discovering CCR2 and CCR5 orthosteric antagonists, ultimately resulted in some clinical success, but the area of intracellular allosteric modulators is still underexplored and the move from orthosteric to allosteric modulation could be an interesting paradigm shift. To this end, we document the development of novel CCR2 and CCR5 intracellular allosteric antagonists through a virtual screen on a small combinatorial library derived from existing CCR2, CCR5, and CCR4 ligands. Using a molecular docking approach, the created library was screened in its entirety utilizing a refined AlphaFold model of CCR5 based on the crystal structure of its close homologue, CCR2. The screening resulted in the identification of several virtual hits, out of which one was developed further by in-house synthesis. In total, 18 analogues were prepared and experimentally evaluated for their binding affinity for CCR2 and functional inhibition on CCR5. This expeditious and simple workflow beginning from docking to compound evaluation identified 3 hits for CCR2 (<i>K</i>_i = 1.3-6 μM) and 1 hit (IC₅₀ = 10.8 μM) for CCR5. The obtained structure-activity relationships were also further rationalized using structural information available for both CCR5 and CCR2 providing valuable insights for future development of intracellular allosteric ligands.