TY - JOUR
T1 - Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1
AU - Toy, Lara
AU - Huber, Max E.
AU - Lee, Minhee
AU - Bartolomé, Ana Alonso
AU - Ortiz Zacarías, Natalia V.
AU - Nasser, Sherif
AU - Scholl, Stephan
AU - Zlotos, Darius P.
AU - Mandour, Yasmine M.
AU - Heitman, Laura H.
AU - Szpakowska, Martyna
AU - Chevigné, Andy
AU - Schiedel, Matthias
N1 - Funding
M.S. (Li 204/04) was supported by the Verband der Chemischen Industrie (VCI). A.C., M.S., and A.A.B. were supported by the Luxembourg Institute of Health (LIH) through the NanoLux Platform and the Luxembourg National Research Fund (INTER/FNRS grants INTER 20/15084569, CORE C23/BM/18068832, and PRIDE-16749720 “NextImmune2”).
Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.
PY - 2024/7/12
Y1 - 2024/7/12
N2 - In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.
AB - In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.
KW - click chemistry
KW - drug discovery
KW - fluorescent probes
KW - GPCRs
KW - medicinal chemistry
KW - target engagement
UR - http://www.scopus.com/inward/record.url?scp=85196936869&partnerID=8YFLogxK
UR - https://pubmed.ncbi.nlm.nih.gov/39022357/
U2 - 10.1021/acsptsci.4c00182
DO - 10.1021/acsptsci.4c00182
M3 - Article
C2 - 39022357
AN - SCOPUS:85196936869
SN - 2575-9108
VL - 7
SP - 2080
EP - 2092
JO - ACS Pharmacology and Translational Science
JF - ACS Pharmacology and Translational Science
IS - 7
ER -