New insights from the inside – Novel approaches for the discovery of intracellular GPCR modulators

G protein-coupled receptors (GPCRs) are one of the most relevant protein families in drug discovery, as they are targeted by approximately one third of all available medications. Recently, intracellular allosteric binding sites (IABS) were identified for several GPCRs. Targeting such IABS with small molecules opens new opportunities to modulate receptor activity, receptor selectivity, and functional selectivity. Several small molecule modulators targeting the IABS of GPCRs have already entered clinical trials, thus highlighting the high therapeutic potential of intracellular GPCR modulation. To further explore the druggability of the IABS of GPCRs and to leverage the discovery of new intracellular GPCR modulators with improved pharmacodynamics and pharmacokinetics, we are developing fluorescently labeled ligands as molecular tools for studying ligand binding to the IABS of GPCRs in a direct and straightforward manner. Targeting the chemokine receptor CCR1 with our fluorescent ligand LT166, we were able to identify the first intracellular CCR1 inhibitors that feature significant CCR1 over CCR2 selectivity.[1] Interestingly, for one of these newly identified intracellular CCR1 ligands, we demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist with respect to β-arrestin recruitment.[1] Further, by using the squaramide based fluorescent tracer LT221, we provided profound evidence for the existence of a druggable intracellular allosteric binding site at the chemokine receptors CXCR1 and CCR6.[2] In addition, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894,[3] currently being evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby highlighting the pharmacological relevance of a druggable IABS at CCR6.[2] In the case of CXCR2, our fluorescent ligand Mz438[4] enabled the development of the first 18F-CXCR2-targeting radiotracer for PET imaging of neutrophils,[5] and with another fluorescent ligand targeting an intracellular binding pocket at the neurotensin receptor subtype 1 (NTSR1), we develop the first fluorescent ligand that is based on an intracellular positive allosteric modulator. As a consequence of cooperativity between the ligands binding to the allosteric and orthosteric pocket, our fluorescent NTSR1 ligand can also be used to investigate orthosteric NTSR1 agonists and antagonists.[6] Finally, our CCR7-targeted fluorescent tracer Mz437 enabled the discovery of a new intracellular CCR7 antagonist that shows a tenfold increase in affinity compared to the prior gold standard in the field (i.e., cmp2105).[7] Thus, our fluorescent ligands targeting GPCRs at their IABS are highly promising tools for future studies of GPCR-targeted pharmacology and drug discovery.