Discovery and development of macrocyclic peptide modulators of the cannabinoid 2 receptor.

The cannabinoid-type 2 receptor (CB2R), a G protein-coupled receptor (GPCR), is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB2R is typically targeted by small molecules, including endo-, phyto- and synthetic cannabinoids, peptides - owing to their size - may offer a different interaction space to facilitate differential interactions with the receptor. Here we explore plant-derived cyclic cystine-knot peptides as ligands of the CB2R. Cyclotides are known for their exceptional biochemical stability. Recently they gained attention as GPCR modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB2R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics we identified cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB2R with an affinity (Ki) of 1µM and a potency (EC50) of 8µM. Leveraging deep learning networks we verified the structural topology of vodo-C1 and modelled its molecular volume in comparison to the CB2R ligand binding pocket. In a fragment-based approach we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB2R. This study introduces a macrocyclic peptide phytocannabinoid, which served as template for the development of synthetic CB2R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

Role of G protein-coupled receptor kinases (GRKs) in ß2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the ß2 -AR, transfection of ßARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the ß2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant ß2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by ß2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and ß2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between ß2 -AR and ß-arrestin2 or between ß2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to ß2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to ß2 -AR agonists occurred in CHO-GLUT4myc cells expressing ß2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type ß2 -AR. However, ß2 -ARs lacking phosphorylation sites failed to recruit ß-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the ß2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

Nature-inspired dimerization as a strategy to modulate neuropeptide pharmacology exemplified with vasopressin and oxytocin.

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V1aR, V1bR, V2R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V1aR. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature.

AM833 Is a Novel Agonist of Calcitonin Family G Protein-Coupled Receptors: Pharmacological Comparison with Six Selective and Nonselective Agonists.

Obesity and associated comorbidities are a major health burden, and novel therapeutics to help treat obesity are urgently needed. There is increasing evidence that targeting the amylin receptors (AMYRs), heterodimers of the calcitonin G protein-coupled receptor (CTR) and receptor activity-modifying proteins, improves weight control and has the potential to act additively with other treatments such as glucagon-like peptide-1 receptor agonists. Recent data indicate that AMYR agonists, which can also independently activate the CTR, may have improved efficacy for treating obesity, even though selective activation of CTRs is not efficacious. AM833 (cagrilintide) is a novel lipidated amylin analog that is undergoing clinical trials as a nonselective AMYR and CTR agonist. In the current study, we have investigated the pharmacology of AM833 across 25 endpoints and compared this peptide with AMYR selective and nonselective lipidated analogs (AM1213 and AM1784), and the clinically used peptide agonists pramlintide (AMYR selective) and salmon CT (nonselective). We also profiled human CT and rat amylin as prototypical selective agonists of CTR and AMYRs, respectively. Our results demonstrate that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation. SIGNIFICANCE STATEMENT: AM833 is a novel nonselective agonist of calcitonin family receptors that has demonstrated efficacy for the treatment of obesity in phase 2 clinical trials. This study demonstrates that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation when compared with other selective and nonselective calcitonin receptor and amylin receptor agonists. The present data provide mechanistic insight into the actions of AM833.

I8-arachnotocin-an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor.

The neuropeptides oxytocin (OT) and vasopressin (VP) and their G protein-coupled receptors OTR, V1aR, V1bR, and V2R form an important and widely-distributed neuroendocrine signaling system. In mammals, this signaling system regulates water homeostasis, blood pressure, reproduction, as well as social behaviors such as pair bonding, trust and aggression. There exists high demand for ligands with differing pharmacological profiles to study the physiological and pathological functions of the individual receptor subtypes. Here, we present the pharmacological characterization of an arthropod (Metaseiulus occidentalis) OT/VP-like nonapeptide across the human OT/VP receptors. I8-arachnotocin is a full agonist with respect to second messenger signaling at human V2R (EC50 34 nM) and V1bR (EC50 1.2 µM), a partial agonist at OTR (EC50 790 nM), and a competitive antagonist at V1aR [pA2 6.25 (558 nM)]. Intriguingly, I8-arachnotocin activated the Gαs pathway of V2R without recruiting either ß-arrestin-1 or ß-arrestin-2. I8-arachnotocin might thus be a novel pharmacological tool to study the (patho)physiological relevance of ß-arrestin-1 or -2 recruitment to the V2R. These findings furthermore highlight arthropods as a novel, vast and untapped source for the discovery of novel pharmacological probes and potential drug leads targeting neurohormone receptors.

The TRPV4 Agonist GSK1016790A Regulates the Membrane Expression of TRPV4 Channels.

TRPV4 is a non-selective cation channel that tunes the function of different tissues including the vascular endothelium, lung, chondrocytes, and neurons. GSK1016790A is the selective and potent agonist of TRPV4 and a pharmacological tool that is used to study the TRPV4 physiological function in vitro and in vivo. It remains unknown how the sensitivity of TRPV4 to this agonist is regulated. The spatial and temporal dynamics of receptors are the major determinants of cellular responses to stimuli. Membrane translocation has been shown to control the response of several members of the transient receptor potential (TRP) family of ion channels to different stimuli. Here, we show that TRPV4 stimulation with GSK1016790A caused an increase in [Ca2+]i that is stable for a few minutes. Single molecule analysis of TRPV4 channels showed that the density of TRPV4 at the plasma membrane is controlled through two modes of membrane trafficking, complete, and partial vesicular fusion. Further, we show that the density of TRPV4 at the plasma membrane decreased within 20 min, as they translocate to the recycling endosomes and that the surface density is dependent on the release of calcium from the intracellular stores and is controlled via a PI3K, PKC, and RhoA signaling pathway.

Discovery of peptide probes to modulate oxytocin-type receptors of insects.

The oxytocin/vasopressin signalling system is conserved across the animal kingdom. In insects, the role of oxytocin-type (inotocin) neuropeptides has only been studied in locusts, beetles and ants, but their physiology continues to be poorly understood. One reason for this knowledge deficit is the lack of available research tools to complement functional genomics efforts. Consequently, ligands to probe insect inotocin receptors are essential. In this study, we sought to identify novel agonists and antagonists of the inotocin receptor from the representative model species Tribolium castaneum and Lasius niger. Drawing upon known ligands of the human receptors, we examined the pharmacology of the plant-derived cyclotide kalata B7 and the synthetic oxytocin analogue atosiban. Kalata B7 is a weak partial agonist of both inotocin receptors. This is the first reported direct interaction of cyclotides with an insect receptor, an observation that may explain their presumed role in herbivore defence. Furthermore, we discovered atosiban is an antagonist of the Tribolium receptor, which may provide a useful probe to investigate the functionality of inotocin signalling in beetles and related insect species. Our findings will enable further examination of insect inotocin receptor pharmacology and physiology, and may trigger studies to comprehend the interaction of plant cyclotides and insects.

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 °C. Strips develop spontaneous contractions within 2-3 h under set tension and remain stable for many hours (>6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions. This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor.

Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants' uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules.

Development of a human vasopressin V1a-receptor antagonist from an evolutionary-related insect neuropeptide.

Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

Molecular mechanisms of bitopic ligand engagement with the M1 muscarinic acetylcholine receptor.

TBPB and 77-LH-28-1 are selective agonists of the M1 muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M1 mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.

Molecular determinants of allosteric modulation at the M1 muscarinic acetylcholine receptor.

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M1 muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M1 mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85(2.64) in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397(7.32) and Trp-400(7.35) in TMVII as residues that contribute to the BQCA binding pocket at the M1 mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described "common" allosteric site in the extracellular vestibule of the M1 mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102(3.29) and Asp-105(3.32)) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.

Reverse engineering of the selective agonist TBPB unveils both orthosteric and allosteric modes of action at the M1 muscarinic acetylcholine receptor.

Recent interest in the M1 muscarinic acetylcholine (ACh) receptor (mAChR) has led to the discovery of various selective agonists for the receptor. The novel selective agonist 1-(1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (TBPB) displays unprecedented functional selectivity at the M1 mAChR. This functional selectivity has been described to stem from sole interaction with an allosteric site, although the evidence for such a mechanism is equivocal. To delineate TBPB's mechanism of action, several truncated variants of TBPB were synthesized and characterized. Binding experiments with [³H]N-methylscopolamine at the M1, M2, M3, and M4 mAChRs revealed radioligand displacement in a manner consistent with a competitive binding mode at the orthosteric site by TBPB and fragment derivatives. Cell-based functional assays of fragment derivatives of TBPB identified both agonistic and antagonistic moieties, one of which, 1-(1-cyclohexylpiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (VCP794), lost agonistic selectivity for the M1 mAChR. Further interaction experiments between TBPB or its antagonist fragments with ACh also indicated a mechanism consistent with competitive binding at mAChRs. However, interaction with an allosteric site by an antagonist fragment of TBPB was demonstrated via its ability to retard radioligand dissociation. To reconcile this dual orthosteric/allosteric pharmacological behavior, we propose that TBPB is a bitopic ligand, interacting with both the orthosteric site and an allosteric site, at the M1 mAChR. This mechanism may also be the case for other selective agonists for mAChRs, and should be taken into consideration in the profiling and classification of new novel selective agonists for this receptor family.

Allosteric modulation of G protein-coupled receptors: a pharmacological perspective.

G protein-coupled receptor (GPCR)-based drug discovery has traditionally focused on targeting the orthosteric site for the endogenous agonist. However, many GPCRs possess allosteric sites that offer enormous potential for greater selectivity in drug action. The complex behaviors ascribed to allosteric ligands also present challenges to those interested in preclinical lead discovery. These challenges include the need to detect and quantify various phenomena when screening for allosteric ligands, such as saturability of effect, probe dependence, differential effects on orthosteric ligand affinity vs. efficacy, system-dependent allosteric agonism, stimulus-bias (functional selectivity), and the potential existence of bitopic (hybrid orthosteric/allosteric) ligands. These issues are also critical when interpreting structure-function studies of allosteric GPCR modulators because mutations in receptor structure, either engineered or naturally occurring, can differentially affect not only modulator affinity, but also the nature, magnitude and direction of the allosteric effect on orthosteric ligand function. The ever-expanding array of allosteric modulators arising from both academic and industrial research also highlights the need for the development of a uniform approach to nomenclature of such compounds.