Two distinct antagonist-bound structures of CysLT ₁ R reveal unique ligand-binding modes and signaling mechanisms.

Cysteinyl leukotriene G protein-coupled receptors CysLT₁ and CysLT₂ regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT₁R has been used for treating asthma and associated diseases for over two decades, CysLT₂R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT₂R in complex with three dual CysLT₁R/CysLT₂R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants.

Cysteinyl leukotriene G protein-coupled receptors, CysLT1R and CysLT2R, regulate bronchoconstrictive and pro-inflammatory effects and play a key role in allergic disorders, cardiovascular diseases, and cancer. CysLT1R antagonists have been widely used to treat asthma disorders, while CysLT2R is a potential target against uveal melanoma. However, very few selective antagonist chemotypes for CysLT receptors are available, and the design of such ligands has proved to be challenging. To overcome this obstacle, we took advantage of recently solved crystal structures of CysLT receptors and an ultra-large Enamine REAL library, representing a chemical space of 680 M readily available compounds. Virtual ligand screening employed 4D docking models comprising crystal structures of CysLT1R and CysLT2R and their corresponding ligand-optimized models. Functional assessment of the candidate hits yielded discovery of five novel antagonist chemotypes with sub-micromolar potencies and the best Ki = 220 nM at CysLT1R. One of the hits showed inverse agonism at the L129Q constitutively active mutant of CysLT2R, with potential utility against uveal melanoma.

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A_2Aadenosine receptor (A_2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A_2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A_2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A_2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Introduction: X-ray crystallography has made important contributions to modern drug development but its application to many important drug targets has been extremely challenging. The recent emergence of X-ray free electron lasers (XFELs) and advancements in serial femtosecond crystallography (SFX) have offered new opportunities to overcome limitations of traditional crystallography to accelerate the structure-based drug discovery (SBDD) process. Areas covered: In this review, the authors describe the general principles of X-ray generation and the main properties of XFEL beams, outline details of SFX data collection and processing, and summarize the progress in the development of associated instrumentation for sample delivery and X-ray detection. An overview of the SFX applications to various important drug targets such as membrane proteins is also provided. Expert opinion: While SFX has already made clear advancements toward the understanding of the structure and dynamics of several major drug targets, its robust application in SBDD still needs further developments of new high-throughput techniques for sample production, automation of crystal delivery and data collection, as well as for processing and storage of large amounts of data. The expansion of the available XFEL beamtime is a key to the success of SFX in SBDD.