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Venomous animals have evolved diverse molecular mechanisms to incapacitate prey and defend against predators. Most venom components disrupt nervous, locomotor, and cardiovascular systems or cause tissue damage. The discovery that certain fish-hunting cone snails use weaponized insulins to induce hypoglycemic shock in prey highlights a unique example of toxins targeting glucose homeostasis. Here, we show that, in addition to insulins, the deadly fish hunter, Conus geographus, uses a selective somatostatin receptor 2 (SSTR 2 ) agonist that blocks the release of the insulin-counteracting hormone glucagon, thereby exacerbating insulin-induced hypoglycemia in prey. The native toxin, Consomatin nG1, exists in several proteoforms with a minimized vertebrate somatostatin-like core motif connected to a heavily glycosylated N-terminal region. We demonstrate that the toxin's N-terminal tail closely mimics a glycosylated somatostatin from fish pancreas and is crucial for activating the fish SSTR 2 . Collectively, these findings provide a stunning example of chemical mimicry, highlight the combinatorial nature of venom components, and establish glucose homeostasis as an effective target for prey capture., (© 2024. The Author(s).)

Persistent pain affects one in five people worldwide, often with severely debilitating consequences. Current treatment options, which can be effective for mild or acute pain, are ill-suited for moderate-to-severe persistent pain, resulting in an urgent need for new therapeutics. In recent years, the somatostatin receptor 4 (SSTR 4 ), which is expressed in sensory neurons of the peripheral nervous system, has emerged as a promising target for pain relief. However, the presence of several closely related receptors with similar ligand-binding surfaces complicates the design of receptor-specific agonists. In this study, we report the discovery of a potent and selective SSTR 4 peptide, consomatin Fj1, derived from extensive venom gene datasets from marine cone snails. Consomatin Fj1 is a mimetic of the endogenous hormone somatostatin and contains a minimized binding motif that provides stability and drives peptide selectivity. Peripheral administration of synthetic consomatin Fj1 provided analgesia in mouse models of postoperative and neuropathic pain. Using structure-activity studies, we designed and functionally evaluated several Fj1 analogs, resulting in compounds with improved potency and selectivity. Our findings present a novel avenue for addressing persistent pain through the design of venom-inspired SSTR 4 -selective pain therapeutics., One Sentence Summary: Venom peptides from predatory marine mollusks provide new leads for treating peripheral pain conditions through a non-opioid target.

Serotonin is a neurotransmitter regulating numerous physiological processes also modulated by drugs, for example, schizophrenia, depression, migraine, and obesity. However, these drugs typically have adverse effects caused by promiscuous binding across 12 serotonin and more than 20 homologous receptors. Recently, structures of the entire serotonin receptor family uncovered molecular ligand recognition. Here, we present a map of 19 'selectivity hotspots', that is, nonconserved binding site residues governing selectivity via favorable target interactions or repulsive 'off-target' contacts. Furthermore, we review functional rationale from observed ligand-binding affinities and mutagenesis effects. Unifying knowledge underlying specific probes and drugs is critical toward the functional characterization of different receptors and alleviation of adverse effects., Competing Interests: Declaration of interests D.E.G. has a part-time employment at Kvantify. A.A.J. is co-founder and co-owner of the company Lophora ApS., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The development of tool compounds for the ionotropic glutamate receptors (iGluRs) remains an important research objective, as these are essential for the study and understanding of the roles of these receptors in health and disease. Herein, we report on the pharmacological characterization of ( S )-2-hydroxyhistidine ( 2a ) and ( S )-2-mercaptohistidine ( 2b ) as mediators of glutamatergic neurotransmission. While 2a displayed negligible binding affinity or activity at all glutamate receptors and transporters investigated, 2b displayed selectivity for homomeric GluK3 with binding affinities in the low micromolar range ( K i = 6.42 ± 0.74 μM). The iGluR subtype selectivity ratio for 2b was calculated at ∼30-fold for GluK1/GluK3, GluA3/GluK3, and GluA4/GluK3 and >100-fold for GluK2/GluK3, GluA1/GluK3, and GluA2/GluK3. Unexpectedly, functional characterization of 2b revealed that the compound is an antagonist ( K b = 7.6 μM) at homomeric GluK3 receptors while exhibiting only weak agonist activity at GluA2 (EC 50 = 3.25 ± 0.55 mM). The functional properties of 2b were explored further in electrophysiological recordings of mouse hippocampal neurons.

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator-prey SSRP sequences available, we show that although the cone snail's signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.

Conotoxins form a diverse group of peptide toxins found in the venom of predatory marine cone snails. Decades of conotoxin research have provided numerous measurable scientific and societal benefits. These include their use as a drug, diagnostic agent, drug leads, and research tools in neuroscience, pharmacology, biochemistry, structural biology, and molecular evolution. Human envenomations by cone snails are rare but can be fatal. Death by envenomation is likely caused by a small set of toxins that induce muscle paralysis of the diaphragm, resulting in respiratory arrest. The potency of these toxins led to concerns regarding the potential development and use of conotoxins as biological weapons. To address this, various regulatory measures have been introduced that limit the use and access of conotoxins within the research community. Some of these regulations apply to all of the ≈200,000 conotoxins predicted to exist in nature of which less than 0.05% are estimated to have any significant toxicity in humans. In this review we provide an overview of the many benefits of conotoxin research, and contrast these to the perceived biosecurity concerns of conotoxins and research thereof.

In the present study, screening of a library of 49,087 compounds at the excitatory amino acid transporter subtype 3 (EAAT3) led to the identification of 2-(furan-2-yl)-8-methyl- N -( o -tolyl)imidazo[1,2- a ]pyridin-3-amine ( 3a ) which showed a >20-fold preference for inhibition of EAAT3 (IC 50 = 13 μM) over EAAT1,2,4 (EAAT1: IC 50 ∼ 250 μM; EAAT2,4: IC 50 > 250 μM). It was shown that a small lipophilic substituent (methyl or bromine) at the 7- and/or 8-position was essential for activity. Furthermore, the substitution pattern of the o -tolyl group (compound 5b ) and the chemical nature of the substituent in the 2-position (compound 7b ) were shown to be essential for the selectivity toward EAAT3 over EAAT1,2. The most prominent analogues to come out of this study are 3a and 3e that display ∼35-fold selectivity for EAAT3 (IC 50 = 7.2 μM) over EAAT1,2,4 (IC 50 ∼ 250 μM).

Development of pharmacological tools for the ionotropic glutamate receptors (iGluRs) is imperative for the study and understanding of the role and function of these receptors in the central nervous system. We report the synthesis of 18 analogues of (2 S,3 R)-2-carboxy-3-pyrrolidine acetic acid (3a), which explores the effect of introducing a substituent on the ε-carbon (3c-q). A new synthetic method was developed for the efficient synthesis of racemic 3a and applied to give expedited access to 13 racemic analogues of 3a. Pharmacological characterization was carried out at native iGluRs, cloned homomeric kainate receptors (GluK1-3), NMDA receptors (GluN1/GluN2A-D), and excitatory amino acid transporters (EAAT1-3). From the structure-activity relationship studies, several new ligands emerged, exemplified by triazole 3p-d1, GluK3-preferring (GluK1/GluK3 K i ratio of 15), and the structurally closely related tetrazole 3q-s3-4 that displayed 4.4-100-fold preference as an antagonist for the GluN1/GluN2A receptor ( K i = 0.61 μM) over GluN1/GluN2B-D ( K i = 2.7-62 μM).

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT 2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT 2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT 2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT 2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phenylpropanoids, such as flavonoids and stilbenoids, are of great commercial interest, and their production in Saccharomyces cerevisiae is a very promising strategy. However, to achieve commercially viable production, each step of the process must be optimised. We looked at carbon loss, known to occur in the heterologous flavonoid pathway in yeast, and identified an endogenous enzyme, the enoyl reductase Tsc13, which turned out to be responsible for the accumulation of phloretic acid via reduction of p-coumaroyl-CoA. Tsc13 is an essential enzyme involved in fatty acid synthesis and cannot be deleted. Hence, two approaches were adopted in an attempt to reduce the side activity without disrupting the natural function: site saturation mutagenesis identified a number of amino acid changes which slightly increased flavonoid production but without reducing the formation of the side product. Conversely, the complementation of TSC13 by a plant gene homologue essentially eliminated the unwanted side reaction, while retaining the productivity of phenylpropanoids in a simulated fed batch fermentation., (© FEMS 2016.)

Screening of a small compound library at the three excitatory amino acid transporter subtypes 1-3 (EAAT1-3) resulted in the identification of compound (Z)-4-chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoic acid (1a) that exhibited a distinct preference as an inhibitor at EAAT1 (IC 50 20 μM) compared to EAAT2 and EAAT3 (IC 50 > 300 μM). This prompted us to subject 1a to an elaborate structure-activity relationship study through the purchase and synthesis and subsequent pharmacological characterization of a total of 36 analogues. Although this effort did not result in analogues with substantially improved inhibitory potencies at EAAT1 compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently available pharmacological tools in the EAAT field but also substantiates the notion that EAAT ligands not derived from α-amino acids hold considerable potential in terms of subtype-selective modulation of the transporters.

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC 50 values ∼1 μM at all three subtypes, this elaborate structure-activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue 4y that displayed an IC 50 of 0.8 μM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF 3 -phenyl analogue 4r was a potent selective EAAT2-inhibitor (IC 50 = 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles.

The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3., (Published by Elsevier Ltd.)

Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH-101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research field, its inability to penetrate the blood-brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg(-1) ) of the closely related analogue UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH-102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH-102 (10 μm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH-102 (20 mg kg(-1) ) did not induce acute effects or any visible changes in behavior., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, we made further investigations on the structure-activity requirements of the selective excitatory amino acid transporter 1 (EAAT1) inhibitor, 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101), by exploring 15 different substituents (R(1) ) at the 7-position in combination with eight different substituents (R(2) ) at the 4-position. Among the 63 new analogues synthesized, we identified a number of compounds that unexpectedly displayed inhibitory activities at EAAT1 in light of understanding the structure-activity relationship (SAR) of this inhibitor class extracted from previous studies. Moreover, the nature of the R(1) and R(2) substituents were observed to contribute to the functional properties of the various analogues in additive and non-additive ways. Finally, separation of the four stereoisomers of analogue 14 g (2-amino-4-([1,1'-biphenyl]-4-yl)-3-cyano-7-isopropyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene) was carried out, and in agreement with a study of a related scaffold, the R configuration at C4 was found to be mandatory for inhibitory activity, while both the C7 diastereomers were found to be active as EAAT1 inhibitors. A study of the stereochemical stability of the four pure stereoisomers 14 g-A-D showed that epimerization takes places at C7 via a ring-opening, C-C bond rotation, ring-closing mechanism., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The metabotropic glutamate (Glu) receptors (mGluRs) play key roles in modulating excitatory neurotransmission in the brain. In all, eight subtypes have been identified and divided into three groups, group I (mGlu1,5), group II (mGlu2,3), and group III (mGlu4,6-8). In this article, we present a L-2,4-syn-substituted Glu analogue, 1d, which displays selective agonist activity at mGlu2 over the remaining mGluR subtypes. A modeling study and redesign of the core scaffold led to the stereoselective synthesis of four new conformationally restricted Glu analogues, 2a-d. Most interestingly, 2a retained a selective agonist activity profile at mGlu2 (EC50 in the micromolar range), whereas 2c/2d were both selective agonists at group III, subtypes mGlu4,6,8. In general, 2d was 20-fold more potent than 2c and potently activated mGlu4,6,8 in the low-mid nanomolar range.

The five excitatory amino acid transporters (EAAT1-5) mediating the synaptic uptake of the major excitatory neurotransmitter glutamate are differently expressed throughout the CNS and at the synaptic level. Although EAATs are crucial for normal excitatory neurotransmission, explorations into the physiological functions mediated by the different transporter subtypes and their respective therapeutic potential have so far been sparse, in no small part due to the limited selection of pharmacological tools available. In the present update, we outline important new insights into the molecular compositions of EAATs and their intricate transport process, the novel approaches to pharmacological modulation of the transporters that have emerged, and interesting new perspectives in EAAT as drug targets proposed in recent years., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the "short loop" recycling marker Rab4, and little co-localization with the marker of "long loop" recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the beta(2)-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.