Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical ...scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-351.39, Trp-842.60, and Arg-167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N1113.35A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the β-arrestin-biased peptide TRV120027.
Background: Angiotensin receptor (AT1R) blockers are critical therapeutics used to treat cardiovascular disease.
Results: We solved the AT1R-olmesartan structure and identified specific interactions for olmesartan derivatives with different functions.
Conclusion: Our results identified residues critical for the binding of different ligands and allosteric modulation by sodium ion.
Significance: Our results provide new insights into the structural basis for ligand recognition and functional selectivity at AT1R.
The angiotensin II receptors AT
R and AT
R serve as key components of the renin-angiotensin-aldosterone system. AT
R has a central role in the regulation of blood pressure, but the function of AT
R ...is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT
R bound to an AT
R-selective ligand and to an AT
R/AT
R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the ...regulation of blood pressure and core body temperature, oncogenesis, and immune function. Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness by activating two high-affinity G-proteincoupled receptors, type 1A (MT1) and type 1B (MT2). Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids, and is one of the most popular supplements in the United States. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine. The structure of MT2 is described in an accompanying paper. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
The human MT1 and MT2 melatonin receptors are G-proteincoupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns. Drug development efforts have targeted both receptors for ...the treatment of insomnia, circadian rhythm and mood disorders, and cancer, and MT2 has also been implicated in type 2 diabetes. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in 3Hmelatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.
Melatonin receptors MT
and MT
are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large ...scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound
reaching EC
of 0.36 nM. Six of these molecules displayed selectivity for MT
over MT
. Moreover, two most potent agonists, including
and a close derivative of melatonin,
, had dramatically reduced arrestin recruitment at MT
, while compound
was devoid of G
signaling at MT
, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.
Cysteinyl leukotriene G protein-coupled receptors CysLT
and CysLT
regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT
R has been used for ...treating asthma and associated diseases for over two decades, CysLT
R 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 CysLT
R in complex with three dual CysLT
R/CysLT
R 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.
Understanding the mechanism by which ligands affect receptor conformational equilibria is key in accelerating membrane protein structural biology. In the case of G protein-coupled receptors (GPCRs), ...we currently pursue a brute-force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis. The nociceptin/orphanin FQ peptide receptor (NOP) is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening. We observed that antagonist potency is correlated with a ligand's ability to induce receptor stability (Tm) and crystallogenesis. Using this screening strategy, we solved two structures of NOP in complex with top candidate ligands SB-612111 and C-35. Docking studies indicate that while potent, stabilizing antagonists strongly favor a single binding orientation, less potent ligands can adopt multiple binding modes, contributing to their low Tm values. These results suggest a mechanism for ligand-aided crystallogenesis whereby potent antagonists stabilize a single ligand-receptor conformational pair.
•A correlation is demonstrated between receptor stability and BRET functional data•Two antagonist-bound crystal structures of the N/OFQ peptide receptor are reported•Docking indicates degenerate binding modes contribute to poor receptor stabilization•A mechanism for antagonist-induced receptor stabilization is proposed
Miller et al. identify a correlation between antagonist-induced receptor thermal stability and G-protein recruitment inhibition via BRET, and use this strategy to obtain two co-crystal structures of the opioid receptor NOP. Docking studies point to a mechanism for antagonist-induced receptor stabilization.
The G protein-coupled cysteinyl leukotriene receptor CysLT
R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, ...and cancer. Selective CysLT
R 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 CysLT
R 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.
Tryptophan indole 15N–1H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15N–1H signals therefore enables one to obtain site-specific ...information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15N–1H resonances is challenging. Here we exploit the unique indole 15N–1H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native A2AAR global protein fold and ligand binding activity are preserved in these A2AAR variants. The indole 15N–1H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15N–1H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the GαS intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.
Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures
and ultra-large libraries of virtual ...compounds
. However, to keep pace with the rapid growth of virtual libraries, such as readily available for synthesis (REAL) combinatorial libraries
, new approaches to compound screening are needed
. Here we introduce a modular synthon-based approach-V-SYNTHES-to perform hierarchical structure-based screening of a REAL Space library of more than 11 billion compounds. V-SYNTHES first identifies the best scaffold-synthon combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This hierarchical combinatorial approach enables the rapid detection of the best-scoring compounds in the gigascale chemical space while performing docking of only a small fraction (<0.1%) of the library compounds. Chemical synthesis and experimental testing of novel cannabinoid antagonists predicted by V-SYNTHES demonstrated a 33% hit rate, including 14 submicromolar ligands, substantially improving over a standard virtual screening of the Enamine REAL diversity subset, which required approximately 100 times more computational resources. Synthesis of selected analogues of the best hits further improved potencies and affinities (best inhibitory constant (K
) = 0.9 nM) and CB
/CB
selectivity (50-200-fold). V-SYNTHES was also tested on a kinase target, ROCK1, further supporting its use for lead discovery. The approach is easily scalable for the rapid growth of combinatorial libraries and potentially adaptable to any docking algorithm.
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