Structural diversity of leukotriene G-protein coupled receptors.

Dihydroxy acid leukotriene (LTB4) and cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are inflammatory mediators derived from arachidonic acid via the 5-lipoxygenase pathway. While structurally similar, these two types of leukotrienes (LTs) exert their functions through interactions with two distinct G protein-coupled receptor (GPCR) families, BLT and CysLT receptors, which share low sequence similarity and belong to phylogenetically divergent GPCR groups. Selective antagonism of LT receptors has been proposed as a promising strategy for the treatment of many inflammation-related diseases including asthma and chronic obstructive pulmonary disease, rheumatoid arthritis, cystic fibrosis, diabetes, and several types of cancer. Selective CysLT1R antagonists are currently used as antiasthmatic drugs, however, there are no approved drugs targeting CysLT2 and BLT receptors. In this review, we highlight recently published structures of BLT1R and CysLTRs revealing unique structural features of the two receptor families. X-ray and cryo-EM data shed light on their overall conformations, differences in functional motifs involved in receptor activation, and details of the ligand-binding pockets. An unexpected binding mode of the selective antagonist BIIL260 in the BLT1R structure makes it the first example of a compound targeting the sodium-binding site of GPCRs and suggests a novel strategy for the receptor activity modulation. Taken together, these recent structural data reveal dramatic differences in the molecular architecture of the two LT receptor families and pave the way to new therapeutic strategies of selective targeting individual receptors with novel tool compounds obtained by the structure-based drug design approach.

Rational Design of Drugs Targeting G-Protein-Coupled Receptors: A Structural Biology Perspective.

G protein-coupled receptors (GPCRs) play a key role in the transduction of extracellular signals to cells and regulation of many biological processes, which makes these membrane proteins one of the most important targets for pharmacological agents. A significant increase in the number of resolved atomic structures of GPCRs has opened the possibility of developing pharmaceuticals targeting these receptors via structure-based drug design (SBDD). SBDD employs information on the structure of receptor-ligand complexes to search for selective ligands without the need for an extensive high-throughput experimental ligand screening and can significantly expand the chemical space for ligand search. In this review, we describe the process of deciphering GPCR structures using X-ray diffraction analysis and cryoelectron microscopy as an important stage in the rational design of drugs targeting this receptor class. Our main goal was to present modern developments and key features of experimental methods used in SBDD of GPCR-targeting agents to a wide range of specialists.

Rational Design of Drugs Targeting G-Protein-Coupled Receptors: Ligand Search and Screening.

G protein-coupled receptors (GPCRs) are transmembrane proteins that participate in many physiological processes and represent major pharmacological targets. Recent advances in structural biology of GPCRs have enabled the development of drugs based on the receptor structure (structure-based drug design, SBDD). SBDD utilizes information about the receptor-ligand complex to search for suitable compounds, thus expanding the chemical space of possible receptor ligands without the need for experimental screening. The review describes the use of structure-based virtual screening (SBVS) for GPCR ligands and approaches for the functional testing of potential drug compounds, as well as discusses recent advances and successful examples in the application of SBDD for the identification of GPCR ligands.

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET.

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 A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor's constitutive activity. For the agonist-bound A2AAR, 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.

Functional GPCR Expression in Eukaryotic LEXSY System.

G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A2A adenosine receptor (A2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors.

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family.

Structural insights into ligand recognition and activation of angiotensin receptors.

G protein-coupled angiotensin II receptors, AT1R and AT2R, are integral components of the renin-angiotensin system (RAS) that regulates blood pressure and fluid balance in humans. While AT1R is a well-established target of angiotensin receptor blockers (ARBs) for managing hypertension and a prime system for studying biased signaling, AT2R has been recognized as a promising target against neuropathic pain and lung fibrosis. In this review, we discuss how recent structural advances illuminate ligand-binding modes and subtype selectivity, shared and distinct features of the receptors, their transducer-coupling patterns, and downstream signaling responses. We also underscore the key ATR aspects that require further studies to fully appreciate the mechanistic framework that fine-tunes their cellular and physiological functions, providing untapped potential for drug discovery.

Modulation of Toll-like receptor 1 intracellular domain structure and activity by Zn2+ ions.

Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation.

Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.

Beyond structure: emerging approaches to study GPCR dynamics.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins that are involved in regulation of sensory and physiological processes and implicated in many diseases. The last decade revolutionized the GPCR field by unraveling multiple high-resolution structures of many different receptors in complexes with various ligands and signaling partners. A complete understanding of the complex nature of GPCR function is, however, impossible to attain without combining static structural snapshots with information about GPCR dynamics obtained by complementary spectroscopic techniques. As illustrated in this review, structure and dynamics studies are now paving the way for understanding important questions of GPCR biology such as partial and biased agonism, allostery, oligomerization, and other fundamental aspects of GPCR signaling.

Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs.

Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing.

Structure-Based Virtual Screening of Ultra-Large Library Yields Potent Antagonists for a Lipid GPCR.

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.

Author Correction: Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs.

A Correction to this paper has been published: https://doi.org/10.1038/s41597-020-00759-w.

An outlook on using serial femtosecond crystallography in drug discovery.

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.

Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors.

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.

Structure-based mechanism of cysteinyl leukotriene receptor inhibition by antiasthmatic drugs.

The G protein-coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue-coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs.