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1. Characterization of Subtype Selective Cannabinoid CB2 Receptor Agonists as Potential Anti-Inflammatory Agents

   https://doi.org/10.3390/ph14040378  

   Tang, Yaliang; Wolk, Barbara; Nolan, Ryan; Scott, Caitlin E.; Kendall, Debra A. (April 2021, Pharmaceuticals)

   null (Ed.)

   Activation of the CB2 receptor has been shown to have anti-inflammatory and antinociceptive effects without causing psychoactive effects. Previously, we reported that the compound ethyl 2(2-(N-(2,3-dimethylphenyl) phenylsulfonamido)acetamido)benzoate (ABK5) is a CB2 subtype selective agonist with anti-inflammatory and antinociceptive effects. In the present study, we tested four ABK5 derivatives, ABK5-1, ABK5-2, ABK5-5, and ABK5-6, to analyze the structure of ABK5 to obtain CB2-selective agonists with higher affinity and efficacy. Affinity, subtype selectivity, and G-protein coupling were determined by radioligand binding assays. Selected compounds were then subjected to evaluation of anti-inflammatory effects using two different cell lines, Jurkat (ABK5-1 and 5-2) and BV-2 cells (ABK5-1), which are models of T cells and microglia, respectively. ABK5-1, ABK5-2, and ABK5-6 had comparable CB2 binding affinity with ABK5 (and stimulated G-protein coupling), while only ABK5-1 and ABK5-2 maintained CB2-subtype selectivity. ABK5-5 did not bind CB2 in the detectable range. RT-PCR and ELISA analysis showed that the two compounds also inhibit IL-2 and TNF-α production, and they were more efficacious than ABK5 in inhibiting TNF-α production. CXCL-12 mediated chemotaxis was also evaluated by the transwell migration assay, and both ABK5-1 and ABK5-2 inhibited chemotaxis with a stronger effect observed in ABK5-1. In the microglia cell line BV-2, ABK5-1 inhibited IL-1β and IL-6 production, which suggests this compound has anti-inflammatory effects through targeting multiple immune cells, and may be a candidate for treatment of inflammation. 

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2. Structural insights into melatonin receptors

   https://doi.org/10.1111/febs.15128  

   Stauch, Benjamin; Johansson, Linda C.; Cherezov, Vadim (November 2019, The FEBS Journal)

   The long‐anticipated high‐resolution structures of the human melatonin G protein‐coupled receptors MT₁and MT₂, involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti‐insomnia and antidepression drugs using X‐ray free‐electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane‐buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT₂from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT₂structure allowed accurate mapping of type 2 diabetes‐related single‐nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein–membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT₁and MT₂with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next‐generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects. 

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3. Structure-based discovery of potent and selective melatonin receptor agonists

   https://doi.org/10.7554/elife.53779  

   Patel, Nilkanth; Huang, Xi Ping; Grandner, Jessica M; Johansson, Linda C; Stauch, Benjamin; McCorvy, John D; Liu, Yongfeng; Roth, Bryan; Katritch, Vsevolod (March 2020, eLife)

   Melatonin receptors MT1 and MT2 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 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, 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. 

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4. Probing the Ni ²⁺ ‐selective Response of Fluorescent Probe NiSensor‐1 with the NiCast Photocaged Complex ^{† ‡}

   https://doi.org/10.1111/php.13567  

   Hickey, Erin E.; Kennedy, Daniel P.; Gwizdala, Celina; Basa, Prem N.; Müller, Peter; MacDonald, John; Burdette, Shawn C. (December 2021, Photochemistry and Photobiology)

   Abstract CTEA (N,N‐bis[2‐(carboxylmethyl)thioethyl]amine) is a mixed donor ligand that has been incorporated into multiple fluorescent sensors such as NiSensor‐1 that was reported to be selective for Ni²⁺. Other metal ions such as Zn²⁺do not produce an emission response in aqueous solution. To investigate the coordination chemistry and selectivity of this receptor, we prepared NiCast, a photocage containing the CTEA receptor. Cast photocages undergo a photoreaction that decreases electron density on a metal‐bound aniline nitrogen atom, which shifts the binding equilibrium toward unbound metal ion. The unique selectivity of CTEA was examined by measuring the binding affinity of NiCast and the CTEA receptor for Ni²⁺, Zn²⁺, Cd²⁺and Cu²⁺under different conditions. In aqueous solution, Ni²⁺binds more strongly to the aniline nitrogen atom than Cd²⁺; however, in CH₃CN, the change in affinity virtually disappears. The crystal structure of [Cu(CTEA)], which exhibits a Jahn–Teller–distorted square pyramidal structure, was also analyzed to gain more insight into the underlying coordination chemistry. These studies suggest that the fluorescence selectivity of NiSensor‐1 in aqueous solution is due to a stronger interaction between the aniline nitrogen atom and Ni²⁺compared to other divalent metal ions except Cu²⁺. 

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5. Analogs of α‐conotoxin PnIC selectively inhibit α7β2‐ over α7‐only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism

   https://doi.org/10.1096/fj.202302079  

   George, Andrew_A; John, Sabin_J; Lucero, Linda_M; Eaton, J_Brek; Jaiswal, Ekta; Christensen, Sean_B; Gajewiak, Joanna; Watkins, Maren; Cao, Yiwei; Olivera, Baldomero_M; et al (December 2023, The FASEB Journal)

   Abstract This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and β2 subunits (α7β2‐nAChR subtype). Basal forebrain cholinergic neurons express α7β2‐nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid‐β associated with early Alzheimer's disease. Additional work indicates that α7β2‐nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7β2‐nAChR from the closely related and more widespread homomeric α7‐only‐nAChR subtype. Functional screening using two‐electrode voltage‐clamp electrophysiology identified a family of α7β2‐nAChR‐selective analogs of α‐conotoxin PnIC (α‐CtxPnIC). A combined electrophysiology, functional kinetics, site‐directed mutagenesis, and molecular dynamics approach was used to further characterize the α7β2‐nAChR selectivity and site of action of these α‐CtxPnIC analogs. We determined that α7β2‐nAChR selectivity of α‐CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist‐binding site shared between α7β2‐ and α7‐only‐nAChR. As numerous previously identified α‐Ctx ligands are competitive antagonists of orthosteric agonist‐binding sites, this study profoundly expands the scope of use of α‐Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α‐CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7β2‐nAChR and detailed investigations of their physiological roles. 

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