Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2

Abstract

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1

“…Their complex pharmacology and probe dependence often require labor-intensive testing in various functional assays, which complicate high-throughput screening. Moreover, well-established target engagement methodologies such as radioligand binding may not be available, e.g., because of the lack of high-affinity probes. , Here, the emerging fluorescence-based binding assays represent a valuable alternative, as they do not require extremely high affinities and can be performed in a mix-and-measure setup. , Bioluminescence resonance energy transfer (BRET) ligand binding assays based on fluorescent probes and the optimized nanoluciferase (Nluc) have been established for structurally diverse GPCRs. − Especially, the combination of a receptor tagged to Nluc at its C-terminus and a TAMRA- or bodipy-labeled ligand was shown to be suitable for nanoBRET target engagement assays at the intracellular binding pockets of the chemokine receptor family. − …”

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1

“…The latter is also an important reason why radioligands are often not well-suited for continuous assay readouts, the detection of low-affinity binders, and cellular target engagement studies investigating their binding to intracellular target sites . Recently, we and others reported the development of fluorescent tracers targeting the IABS of CCR2, CCR9, and CXCR2. ,,, These molecular tools were successfully applied for cell-free and cellular binding studies using the nonradioactive NanoBRET technology. In the course of the development of our biarylsulfonamide-based CCR2 tracer 7 (Figure ), which showed three digit nanomolar binding to CCR2 in a membrane-based ( K D (CCR2) = 266 nM) and cellular setup ( K D (CCR2) = 114 nM), respectively, but no relevant binding to CCR1, we have already described the pyrrolone-based fluorescent probes 8 and 9 (Figure ).…”