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The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-Å crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. The crystal structure of the human κ-opioid receptor in complex with an antagonist, JDTic, is determined, with potential importance for the design of new therapeutic agents. Where opiates hit home Four papers in this issue of Nature present the long-awaited high-resolution crystal structures of the four known opioid receptors in ligand-bound conformations. These G-protein-coupled receptors are the targets of a broad range of drugs, including painkillers, antidepressants, anti-anxiety agents and anti-addiction medications. Brian Kobilka’s group reports the crystal structure of the µ-opioid receptor bound to a morphinan antagonist and the δ-opioid receptor bound to naltrindole. Raymond Stevens’ group reports on the κ-opioid receptor bound to the selective antagonist JDTic, and the nociceptin/orphanin FQ receptor bound to a peptide mimetic. In an associated News and Views, Marta Filizola and Lakshmi Devi discuss the implications of these landmark papers for research on the mechanisms underlying receptor function and drug development.

BackgroundChemokine receptor 8 (CCR8) is selectively upregulated on immunosuppressive regulatory T cells (Tregs) within the tumor microenvironment (TME) across multiple solid tumors, including breast, colon, and lung cancers. CCR8+ Tregs contribute significantly to immune evasion and resistance to immune checkpoint blockade, posing a hurdle for effective cancer immunotherapy. Existing therapeutic strategies target CCR8+ Tregs through Fc-mediated Treg depletion mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP). Antibodies lacking effector function have failed to demonstrate preclinical efficacy, highlighting an incomplete understanding of CCR8’s role in Treg biology. Abilita’s novel approach that addresses the role of CCR8 signaling in Tregs delivered an antibody that acts as a potent inverse agonist to block both CCL1-dependent and basal receptor signaling. Leveraging this new MOA, ABT-863 demonstrated anti-tumor efficacy without the need for Treg depletion.MethodsABT-863 is a bivalent VHH-Fc fusion antibody discovered using Abilita’s proprietary Enabled Membrane Protein (EMP) platform. ABT-863’s binding affinity, epitope, selectivity, pharmacology, and developability were extensively characterized in vitro. In vivo efficacy and immune modulation were evaluated using human CCR8 knock-in mice implanted with lung (CMT167) or colon (MC38) carcinoma. Studies included monotherapy and combination with anti-PD1, complemented by comprehensive immune phenotyping and cytokine profiling.ResultsABT-863 displayed potent and highly selective CCR8 binding with novel pharmacological properties. Revealed by cryo-EM analysis, ABT-863’s VHH warhead engages deep within the transmembrane domain orthosteric pocket. This not only blocks activation by CCL1 but also prevents basal, ligand-independent activation. Treatment with ABT-863 led to tumor growth inhibition in vivo. Remarkably, an Fc-effector null variant retained anti-tumor efficacy despite lacking Treg depleting activity, challenging the prevalent paradigm. When combined with anti-PD1, ABT-863 showed synergistic significant tumor suppression. Immune phenotyping of ABT-863 Fcnull treated mice revealed a significant increase in activated CD8+ T cells and early exhausted CD8+ T cells in both blood and tumors. Cytokine and chemokine blood analysis demonstrated elevated circulating levels of IL-10, previously linked to reinvigoration of exhausted CD8+ T cells and enhanced anti-tumor immunity.ConclusionsABT-863 is a first-in-class CCR8 inverse agonist antibody that promotes the anti-tumor response, modulating Treg suppressive activity, without cell depletion, offering a novel and potentially safer MoA. ABT-863 could represent a transformative immunotherapy approach targeting CCR8+ Tregs in solid tumors by coupling its robust anti-tumor activity, and its synergy with PD-1 blockade, with the potential differentiation for a better safety profile and a broader therapeutic window compared to other clinical candidates.Ethics ApprovalThe animal studies have been conducted with a CRO, and have been approved by their internal IACUC.

Methods to measure affinities of membrane proteins and soluble ligands are cumbersome and often rely on truncations or other modifications of the membrane protein or ligand. Baksh et al . show that backscattering interferometry is a sensitive and accurate technology for the label-free quantification of ligand–membrane receptor interactions. Although membrane proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show that backscattering interferometry (BSI) can accurately quantify ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.

Opioid receptorsmediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulationof pain, respiratory drive,mood, and-in the case of κ-opioid receptor (κ-OR)-dysphoria andpsychotomimesis. Here wereport the crystal structure of the human κ-ORin complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9A resolution.The structure reveals important features of the liganδ-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 59-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directedmutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. [PUBLICATION ABSTRACT]

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and--in the case of κ-opioid receptor (κ-OR)--dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 A resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR.

The quinol:fumarate reductase (QFR) is the terminal reductase of anaerobic fumarate respiration, the most commonly occurring type of anaerobic respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. The three-dimensional crystal structure of the QFR from Wolinella succinogenes has previoulsy been solved at 2.2 Å resolution.Although the diheme-containing QFR from W. succinogenes is known to catalyze an electroneutral process, structural and functional characterization of parental and variant enzymes has revealed active site locations which indicate electrogenic catalysis across the membrane. A solution to this apparent controversy was proposed with the so-called “Epathway hypothesis”. According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180, and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role for the former constituent. One aim of this thesis is to investigate by a combination of specific 13C-heme propionate labeling and FTIR difference spectroscopy whether the ring C propionate of the distal heme is involved in redox-coupled proton transfer in the QFR from W. succinogenes.In addition to W. succinogenes, the primary structures of the QFR enzymes of two other e- proteobacteria are known. These are Campylobacter jejuni and Helicobacter pylori, which unlike W. succinogenes are human pathogens. The QFR from H. pylori has previously been established to be a potential drug target, and the same is likely for the QFR from C. jejuni. The two pathogenic species colonize mucosal surfaces causing several diseases. The possibility of studying these QFRs from these bacteria and creating more efficient drugs specifically active for this enzyme depends substantially on the availability of large amounts of high-quality protein. Further, biochemical and structural studies on QFR enzymes from e- proteobacteria species other than W. succinogenes can be valuable to enlighten new aspects or corroborate the current understanding of this class of membrane proteins.