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Background and Purpose: Voltage-gated sodium channels (Na(V) channels) are key players in the generation and propagation of action potentials, and selective blockade of these channels is a promising strategy for clinically useful suppression of electrical activity. The conotoxin µ-CnIIIC from the cone snail Conus consors exhibits myorelaxing activity in rodents through specific blockade of skeletal muscle (Na(V) 1.4) Na(V) channels.Experimental Approach: We investigated the activity of µ-CnIIIC on human Na(V) channels and characterized its inhibitory mechanism, as well as the molecular basis, for its channel specificity.Key Results: Similar to rat paralogs, human Na(V) 1.4 and Na(V) 1.2 were potently blocked by µ-CnIIIC, the sensitivity of Na(V) 1.7 was intermediate, and Na(V) 1.5 and Na(V) 1.8 were insensitive. Half-channel chimeras revealed that determinants for the insensitivity of Na(V) 1.8 must reside in both the first and second halves of the channel, while those for Na(V) 1.5 are restricted to domains I and II. Furthermore, domain I pore loop affected the total block and therefore harbours the major determinants for the subtype specificity. Domain II pore loop only affected the kinetics of toxin binding and dissociation. Blockade by µ-CnIIIC of Na(V) 1.4 was virtually irreversible but left a residual current of about 5%, reflecting a 'leaky' block; therefore, Na(+) ions still passed through µ-CnIIIC-occupied Na(V) 1.4 to some extent. TTX was excluded from this binding site but was trapped inside the pore by µ-CnIIIC.Conclusion and Implications: Of clinical significance, µ-CnIIIC is a potent and persistent blocker of human skeletal muscle Na(V) 1.4 that does not affect activity of cardiac Na(V) 1.5. [ABSTRACT FROM AUTHOR]

Background and Purpose: The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant.Experimental Approach: µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data.Key Results: Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V)1.4 (IC(50) = 1.3 nM) and Na(V)1.2 channels in a long-lasting manner. Cardiac Na(V)1.5 and DRG-specific Na(V)1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3β2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs.Conclusion and Implications: µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels. [ABSTRACT FROM AUTHOR]

Abstract: Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst3 receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5μM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily. [Copyright &y& Elsevier]

The chemokine receptor CCR5 is known to exist in cell surface subpopulations that differ in their capacity to engage ligands. One proposed explanation for this phenomenon is the presence of CCR5 species with different levels of post-translational modifications (PTMs). Tyrosine sulfation and O-glycan sialylation are PTMs that add negative charges to the extracellular domain of CCR5 and make strong contributions to chemokine binding but it is not known whether cellular mechanisms to control their levels exist. In this study we used a combination of sulfation-sensitive and sulfation-insensitive CCR5 ligands to show that the rate of turnover of CCR5 tyrosine sulfation is more rapid than the rate of turnover of the receptor itself. This suggests that the steady state level of CCR5 sulfation is maintained through the combination of tyrosine protein sulfotransferase (TPST), the trans-Golgi network (TGN)-resident 'source enzyme, and a 'sink' activity that removes tyrosine sulfation from CCR5. By measuring the effects on ligand binding of knockdown and overexpression experiments, we provided evidence that non-lysosomal cellular arylsulfatases, particularly ARSG, ARSI and ARSJ, are CCR5 sulfation 'sink' enzymes. We also used targeted knockdown and sialylation-sensitive and insensitive chemokines to identify the sialidase NEU3 as a candidate 'sink' enzyme for CCR5 O-glycan sialylation. This study provides the first experimental evidence of activity of sulfatase and sialidase 'sink' enzymes on CCR5, providing a potential mechanism for cells to control steady-state levels of these PTMs and thereby exert dynamic control over receptor-ligand interactions at the cell surface and during receptor desensitization., (© 2024. The Author(s).)

Numerous members of the human G protein-coupled receptor (GPCR) superfamily are receptors of therapeutic interest. GPCRs are considered to be highly tractable for drug discovery, representing the targets of approximately one-third of currently licensed drugs. These successful drug discovery outcomes cover only a relatively small subset of the superfamily, however, and many other attractive receptors have proven to present significant challenges. Among these difficult GPCRs are those whose natural ligands are peptides and proteins. In this review we explain the obstacles faced by GPCR drug discovery campaigns, with particular focus on those related to peptide and protein GPCRs. We describe a novel and promising approach for these targets based on engineering of their natural ligands and describe an integrated discovery platform that allows potent ligand analogs to be discovered rapidly and efficiently. Finally, we present a case study involving the chemokine receptor CCR5 to show that this approach can be used to generate new drugs for peptide and protein GPCR targets combining best-in-class potency with tunable signaling activity.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric G i protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size., (© 2018 Paolini-Bertrand et al.)

Conotoxins are venom peptides from cone snails with multiple disulfide bridges that provide a rigid structural scaffold. Typically acting on ion channels implicated in neurotransmission, conotoxins are of interest both as tools for pharmacological studies and as potential new medicines. δ-Conotoxins act by inhibiting inactivation of voltage-gated sodium channels (Nav). Their pharmacology has not been extensively studied because their highly hydrophobic character makes them difficult targets for chemical synthesis. Here we adopted an acid-cleavable solubility tag strategy that facilitated synthesis, purification, and directed disulfide bridge formation. Using this approach we readily produced three native δ-conotoxins from Conus consors plus two rationally designed hybrid peptides. We observed striking differences in Nav subtype selectivity across this group of compounds, which differ in primary structure at only three positions: 12, 23, and 25. Our results provide new insights into the structure-activity relationships underlying the Nav subtype selectivity of δ-conotoxins. Use of the acid-cleavable solubility tag strategy should facilitate synthesis of other hydrophobic peptides with complex disulfide bridge patterns., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)