State-Dependent Mapping of GlyR-Cholesterol Interactions by Coupling Crosslinking with Mass Spectrometry

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol: lipid ratios below and above the cholesterol activity threshold with aliquots of azi-cholesterol. State-dependent crosslinking studies of receptors primarily in its resting (no glycine), desensitized (10mM glycine) and open (F207A/A288G, 30nM ivermectin) states were then performed at elevated cholesterol levels necessary for activity. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized, mass fingerprinted by tandem mass spectrometry (MS-MS), and sites of cholesterol crosslinks in peptides were refined by targeted MS-MS. Within the GlyR apo state, cholesterol interactions differed as a function of membrane cholesterol concentration correlating to the chemical activity of cholesterol, suggesting two distinct conformations. Differential cholesterol crosslinking patterns between resting, desensitized, and open states were observed, highlighting state-dependent differences in GlyR lipid accessibility. Distinct state-dependent crosslinking patterns indicative of alterations in either the lipid environment and/or channel structure were observed throughout GlyR, most prominently observed in the M4 transmembrane helix, extracellular domain loops and regions nearing the bilayer interface, and the large intracellular M3-M4 loop. The changes in M4 accessibility (transition from surface-mapped crosslinking to regions of the helix less exposed when mapped) suggest an outward twisting motion and translocation towards the bilayer/lipids as GlyR allosterically transitions. Strikingly, crosslinking patterns within the M3-M4 loop offer insight into the generalized structure of this unresolved region in all current pLGIC structural models, by suggesting the crosslinked regions of this intracellular loop are intimately associated or buried within the lipid bilayer. Taken together, crosslinking coupled with MS-MS has the capability to accurately probe and define physiological protein frameworks which can aid in the refinement of allosteric modulation and current structural models

Synthetic cannabinoid receptor agonists and antagonists: implication in CNS disorders

Since the discovery of the cannabinoid receptors, numerous studies associate the endocannabinoid system with several physiological and pathological processes including cancer, appetite, fertility, memory, neuropathic and inflammatory pain, obesity, and neurodegenerative diseases. Over the last two decades, several researches have been dedicated extensively on the cannabinoid receptors ligands since the direct activation of cannabinoid receptors results in several beneficial effects, in the brain and in the periphery

Mind the Gap - Deciphering GPCR Pharmacology Using 3D Pharmacophores and Artificial Intelligence

G protein-coupled receptors (GPCRs) are amongst the most pharmaceutically relevant and well-studied protein targets, yet unanswered questions in the field leave significant gaps in our understanding of their nuanced structure and function. Three-dimensional pharmacophore models are powerful computational tools in in silico drug discovery, presenting myriad opportunities for the integration of GPCR structural biology and cheminformatics. This review highlights success stories in the application of 3D pharmacophore modeling to de novo drug design, the discovery of biased and allosteric ligands, scaffold hopping, QSAR analysis, hit-to-lead optimization, GPCR de-orphanization, mechanistic understanding of GPCR pharmacology and the elucidation of ligand–receptor interactions. Furthermore, advances in the incorporation of dynamics and machine learning are highlighted. The review will analyze challenges in the field of GPCR drug discovery, detailing how 3D pharmacophore modeling can be used to address them. Finally, we will present opportunities afforded by 3D pharmacophore modeling in the advancement of our understanding and targeting of GPCRs

The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity.

The role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here we use a combination of medicinal chemistry, electrophysiology, and computational modelling to develop a rational structure activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length and double bond position promote enhanced inhibition. Overall, the L-stereoisomer is generally a better inhibitor than the D-stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment

Discovery of RAR-related Orphan Receptor γt Inverse Agonists

BACKGROUND Technological advances have provided modern approaches for drug discovery and development. Its application, computer-aided drug design (CADD), enables solving complex drug design challenges practically unsolvable by traditional methods. Here, CADD was applied for a drug target retinoic acid receptor-related orphan receptor gamma t (RORγt). RORγt is a nuclear receptor that mediates inflammation and is connected to multiple chronic inflammatory diseases. RORγt inhibition proposes anti-inflammatory effects and, as such, RORγt has been identified as an appealing drug target. The highest inhibition is achieved with inverse agonist -inhibitors. Due to unspecificity and complexity of RORγt, no effective drugs targeting RORγt have been developed so far. Accordingly, CADD approach can be justified to address this challenge. UNMET MEDICAL NEED Some patients suffering from inflammatory diseases do not respond sufficiently to the current therapies. Hence, search for novel therapies can be justified, and RORγt inhibition is a promising option for this purpose. AIM OF THE STUDY Modern CADD approaches were executed to discover novel RORγt inverse agonists. MATERIALS AND METHODS Several CADD methods were utilized including ligand- and structure-based drug design. RESULTS 29 novel potential RORγt inverse agonists were discovered. CONCLUSION CADD protocol was demonstrated suitable for discovering novel potential RORγt inverse agonists

Insect-selective spider toxins targeting voltage-gated sodium channels

The voltage-gated sodium (Nav) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Nav channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Nav channel conductance. Magi 2 and Tx4(6-1) slow Nav channel inactivation via an interaction with site-3. The δ-palutoxins, and most likely μ-agatoxins and curtatoxins, target site-4. However, their action is complex with the μ-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion β-toxins, but with both δ-palutoxins and μ-agatoxins slowing Nav channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as δ-atracotoxins, are known to target both insect and vertebrate Nav channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped. © 2006 Elsevier Ltd. All rights reserved

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes–6

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials that is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules. In these Editorials, we highlight in brief reports (of about one hundred words) a number of recently published articles that describe crucial findings, such as the discovery of novel drug targets and mechanisms of action or novel classes of drugs, which may inspire future medicinal chemistry endeavors devoted to addressing prime unmet medical needs

Neuroactive steroids, nociception and neuropathic pain: a flashback to go forward

The present review discusses the potential role of neurosteroids / neuroactive steroids in the regulation of nociceptive and neuropathic pain, and recapitulates the current knowledge on the main mechanisms involved in the reduction of pain, especially those occurring at the dorsal horn of the spinal cord, a crucial site for nociceptive processing. We will make special focus on progesterone and its derivative allopregnanolone, which have been shown to exert remarkable actions in order to prevent or reverse the maladaptive changes and pain behaviors that arise after nervous system damage in various experimental neuropathic conditions.Fil: Coronel, Maria Florencia. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Labombarda, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaFil: Gonzalez, Susana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentin

Breakthroughs in medicinal chemistry: New targets and mechanisms, new drugs, new hopes-6

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials that is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules [...]

The Trifluoromethyl Group as a Bioisosteric Replacement of the Aliphatic Nitro Group in CB1 Receptor Positive Allosteric Modulators (PAMs)

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00252. Experimental procedures, characterization of all intermediates and target compounds, and copies of NMR spectra of compounds 1, 39-57. Molecular formula strings of target compounds are available. ACKNOWLEDGEMENTS. We gratefully thank Signal Pharma and the Canadian Institutes of Health Research Proof of Principle grants PPP-125784 and PP2-139101 for financial support and fellowship (C.C.T), NIH grants R01DA039942, P30DA033934 and VCU School of Pharmacy start-up funds (A.H.L.). We thank the EPSRC National Crystallography Service (University of Southampton) for the X-ray data collection. We are grateful to Dr Monica Sani (CNR-ICRM, Milan, Italy) and Mr Massimo Frigerio (Politecnico di Milano, Italy) for the synthesis of two tetrazole-substituted indoles (Het-1 and Het-2)Peer reviewedPostprin