Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.

Pharmacological inhibition of HDAC6 suppresses NLRP3 inflammasome-mediated IL-1ß release.

The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is an important regulator of inflammation and immune responses. Histone deacetylase 6 (HDAC6) has been implicated in the assembly and activation of the NLRP3 inflammasome in mouse cells, however, the role in human immune cells remains poorly understood. Here, we investigated the effect of HDAC6 deficiency on NLRP3-mediated interleukin (IL)-1ß release using proteolysis targeting chimeras (PROTAC) technology. We designed an HDAC6 PROTAC (A6) composed of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the E3 ligase ligand thalidomide and a control PROTAC (non-degrading control, nc-A6) that binds to HDAC6 but lacks the ability to induce HDAC6 degradation. A6 but not nc-A6 reduced HDAC6 levels in THP-1 macrophages without affecting cell viability. PROTAC A6 and nc-A6 significantly reduced the release of IL-1ß in a concentration-dependent manner, suggesting that HDAC6 deficiency is not necessary for inhibition of NLRP3 inflammasome-mediated IL-1ß release. We found that inhibition of the catalytic domain with HDAC inhibitor SAHA or the specific HDAC6 inhibitor tubastatin A is sufficient to reduce IL-1ß release indicating that the enzymatic activity of HDAC6 is critical for NLRP3 inflammasome function. Mechanistically, the observed effects of HDAC6 inhibition on NLRP3-mediated inflammatory responses could be attributed to its interaction with Toll-like receptor (TLR) signaling. Tubastatin A did not affect IL-1ß levels when added after TLR-mediated priming. Collectively, our findings indicate that HDAC6 inhibitors show potent anti-inflammatory activity and suppress IL-1ß release by human macrophages, independent of NLRP3 assembly and activation.

Development of the first geldanamycin-based HSP90 degraders.

Despite the early clinical promise, adverse events such as acquired resistance and dose-limiting toxicities have barred the widespread use of HSP90 inhibitors as anticancer drugs. A new approach involving proteolysis-targeting chimeras (PROTACs) to degrade the protein instead of inhibiting it may overcome these problems. In this work, we describe the design, synthesis, and evaluation of cereblon-recruiting geldanamycin-based HSP90 degraders based on the PROTAC technology. Our best degrader, 3a, effectively decreased HSP90α and HSP90ß levels in cells utilizing the ubiquitin-proteasome pathway.

Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2.

Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (1), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9a) as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and high-throughput manner. Further, we show that 9a can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small-molecule-based fluorescent CXCR2 ligand 9a represents a promising tool for future studies of CXCR2 pharmacology.