Structure-based design of a chemical probe set for the 5-HT5A serotonin receptor

ABSTRACTThe 5-HT5A receptor (5-HT5AR), for which no selective agonists and only a few antagonists exist, remains the least understood serotonin (5-HT) receptor. A single commercial antagonist (SB-699551) has been widely used to investigate central nervous system (CNS) 5-HT5AR function in neurological disorders, including pain. However, because SB-699551 has affinity for many 5-HTRs, lacks inactive property-matched controls, and has assay interference concerns, it has liabilities as a chemical probe. To better illuminate 5-HT5AR function, we developed a probe set through iterative rounds of molecular docking, pharmacological testing, and optimization. Docking over six million lead-like molecules against a 5-HT5AR homology model identified five mid-μM ligand starting points with unique scaffolds. Over multiple rounds of structure-based design and testing, a new quinoline scaffold with high affinity and enhanced selectivity for the 5-HT5AR was developed, leading to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a much more restricted off-target profile and decreased assay liabilities vs. SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678, which was also consistent with recent published 5-HTR structures. Surprisingly, property-matched analogs of UCSF678 that were either inactive across 5-HTRs or retained affinity for UCSF678’s off-targets revealed that 5-HT5AR engagement is nonessential for alleviating pain in a mouse model, contrary to previous studies using less-selective ligands. Relative to SB-699551, these molecules constitute a well-characterized and more selective probe set with which to study the function of the 5-HT5A receptor. Table of Contents Graphic

Synthetic Retinoids Beyond Cancer Therapy

While the uses of retinoids for cancer treatment continue to evolve, this review focuses on other therapeutic areas in which retinoids [retinol (vitamin A), all- trans retinoic acid (RA), and synthetic retinoic acid receptor (RAR)α-, β-, and γ-selective agonists] are being used and on promising new research that suggests additional uses for retinoids for the treatment of disorders of the kidneys, skeletal muscles, heart, pancreas, liver, nervous system, skin, and other organs. The most mature area, in terms of US Food and Drug Administration–approved, RAR-selective agonists, is for treatment of various skin diseases. Synthetic retinoid agonists have major advantages over endogenous RAR agonists such as RA. Because they act through a specific RAR, side effects may be minimized, and synthetic retinoids often have better pharmaceutical properties than does RA. Based on our increasing knowledge of the multiple roles of retinoids in development, epigenetic regulation, and tissue repair, other exciting therapeutic areas are emerging. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 62 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The Contribution of Phospholipase C in Vomiting in the Least Shrew (Cryptotis Parva) Model of Emesis

Gq and Gβγ protein-dependent phospholipase C (PLC) activation is extensively involved in G protein-coupled receptor (GPCR)-mediated signaling pathways which are implicated in a wide range of physiological and pathological events. Stimulation of several GPCRs, such as substance P neurokinin 1-, dopamine D2/3-, histamine H1- and mu-opioid receptors, can lead to vomiting. The aim of this study was to investigate the role of PLC in vomiting through assessment of the emetic potential of a PLC activator (m-3M3FBS), and the antiemetic efficacy of a PLC inhibitor (U73122), in the least shrew model of vomiting. We find that a 50 mg/kg (i.p.) dose of m-3M3FBS induces vomiting in ∼90% of tested least shrews, which was accompanied by significant increases in c-Fos expression and ERK1/2 phosphorylation in the shrew brainstem dorsal vagal complex, indicating activation of brainstem emetic nuclei in m-3M3FBS-evoked emesis. The m-3M3FBS-evoked vomiting was reduced by pretreatment with diverse antiemetics including the antagonists/inhibitors of: PLC (U73122), L-type Ca2+ channel (nifedipine), IP3R (2-APB), RyR receptor (dantrolene), ERK1/2 (U0126), PKC (GF109203X), the serotoninergic type 3 receptor (palonosetron), and neurokinin 1 receptor (netupitant). In addition, the PLC inhibitor U73122 displayed broad-spectrum antiemetic effects against diverse emetogens, including the selective agonists of serotonin type 3 (2-Methyl-5-HT)-, neurokinin 1 receptor (GR73632), dopamine D2/3 (quinpirole)-, and muscarinic M1 (McN-A-343) receptors, the L-type Ca2+ channel (FPL64176), and the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. In sum, PLC activation contributes to emesis, whereas PLC inhibition suppresses vomiting evoked by diverse emetogens.

Adrenoceptors in GtoPdb v.2021.3

The nomenclature of the Adrenoceptors has been agreed by the NC-IUPHAR Subcommittee on Adrenoceptors [60, 186]. Adrenoceptors, α1 The three α1-adrenoceptor subtypes α1A, α1B and α1D are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. -(-)phenylephrine, methoxamine and cirazoline are agonists and prazosin and doxazosin antagonists considered selective for α1- relative to α2-adrenoceptors. [3H]prazosin and [125I]HEAT (BE2254) are relatively selective radioligands. S(+)-niguldipine also has high affinity for L-type Ca2+ channels. Fluorescent derivatives of prazosin (Bodipy FLprazosin- QAPB) are used to examine cellular localisation of α1-adrenoceptors. α1-Adrenoceptor agonists are used as nasal decongestants; antagonists to treat symptoms of benign prostatic hyperplasia (alfuzosin, doxazosin, terazosin, tamsulosin and silodosin, with the last two compounds being α1A-adrenoceptor selective and claiming to relax bladder neck tone with less hypotension); and to a lesser extent hypertension (doxazosin, terazosin). The α1- and β2-adrenoceptor antagonist carvedilol is used to treat congestive heart failure, although the contribution of α1-adrenoceptor blockade to the therapeutic effect is unclear. Several anti-depressants and anti-psychotic drugs are α1-adrenoceptor antagonists contributing to side effects such as orthostatic hypotension. Adrenoceptors, α2 The three α2-adrenoceptor subtypes α2A, α2B and α2C are activated by (-)-adrenaline and with lower potency by (-)-noradrenaline. brimonidine and talipexole are agonists and rauwolscine and yohimbine antagonists selective for α2- relative to α1-adrenoceptors. [3H]rauwolscine, [3H]brimonidine and [3H]RX821002 are relatively selective radioligands. There are species variations in the pharmacology of the α2A-adrenoceptor. Multiple mutations of α2-adrenoceptors have been described, some associated with alterations in function. Presynaptic α2-adrenoceptors regulate many functions in the nervous system. The α2-adrenoceptor agonists clonidine, guanabenz and brimonidine affect central baroreflex control (hypotension and bradycardia), induce hypnotic effects and analgesia, and modulate seizure activity and platelet aggregation. clonidine is an anti-hypertensive (relatively little used) and counteracts opioid withdrawal. dexmedetomidine (also xylazine) is increasingly used as a sedative and analgesic in human [31] and veterinary medicine and has sympatholytic and anxiolytic properties. The α2-adrenoceptor antagonist mirtazapine is used as an anti-depressant. The α2B subtype appears to be involved in neurotransmission in the spinal cord and α2C in regulating catecholamine release from adrenal chromaffin cells. Although subtype-selective antagonists have been developed, none are used clinically and they remain experimental tools. Adrenoceptors, β The three β-adrenoceptor subtypes β1, β2 and β3 are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. Isoprenaline is selective for β-adrenoceptors relative to α1- and α2-adrenoceptors, while propranolol (pKi 8.2-9.2) and cyanopindolol (pKi 10.0-11.0) are relatively selective antagonists for β1- and β2- relative to β3-adrenoceptors. (-)-noradrenaline, xamoterol and (-)-Ro 363 show selectivity for β1- relative to β2-adrenoceptors. Pharmacological differences exist between human and mouse β3-adrenoceptors, and the 'rodent selective' agonists BRL 37344 and CL316243 have low efficacy at the human β3-adrenoceptor whereas CGP 12177 (low potency) and L 755507 activate human β3-adrenoceptors [88]. β3-Adrenoceptors are resistant to blockade by propranolol, but can be blocked by high concentrations of bupranolol. SR59230A has reasonably high affinity at β3-adrenoceptors, but does not discriminate between the three β- subtypes [320] whereas L-748337 is more selective. [125I]-cyanopindolol, [125I]-hydroxy benzylpindolol and [3H]-alprenolol are high affinity radioligands that label β1- and β2- adrenoceptors and β3-adrenoceptors can be labelled with higher concentrations (nM) of [125I]-cyanopindolol together with β1- and β2-adrenoceptor antagonists. Fluorescent ligands such as BODIPY-TMR-CGP12177 can be used to track β-adrenoceptors at the cellular level [8]. Somewhat selective β1-adrenoceptor agonists (denopamine, dobutamine) are used short term to treat cardiogenic shock but, chronically, reduce survival. β1-Adrenoceptor-preferring antagonists are used to treat cardiac arrhythmias (atenolol, bisoprolol, esmolol) and cardiac failure (metoprolol, nebivolol) but also in combination with other treatments to treat hypertension (atenolol, betaxolol, bisoprolol, metoprolol and nebivolol) [507]. Cardiac failure is also treated with carvedilol that blocks β1- and β2-adrenoceptors, as well as α1-adrenoceptors. Short (salbutamol, terbutaline) and long (formoterol, salmeterol) acting β2-adrenoceptor-selective agonists are powerful bronchodilators used to treat respiratory disorders. Many first generation β-adrenoceptor antagonists (propranolol) block both β1- and β2-adrenoceptors and there are no β2-adrenoceptor-selective antagonists used therapeutically. The β3-adrenoceptor agonist mirabegron is used to control overactive bladder syndrome. There is evidence to suggest that β-adrenoceptor antagonists can reduce metastasis in certain types of cancer [189].

Retinoic Acid Receptors and the Control of Positional Information in the Regenerating Axolotl Limb

We know little about the control of positional information (PI) during axolotl limb regeneration, which ensures that the limb regenerates exactly what was amputated, and the work reported here investigates this phenomenon. Retinoic acid administration changes the PI in a proximal direction so that a complete limb can be regenerated from a hand. Rather than identifying all the genes altered by RA treatment of the limb, we have eliminated many off-target effects by using retinoic acid receptor selective agonists. We firstly identify the receptor involved in this respecification process as RARα and secondly, identify the genes involved by RNA sequencing of the RARα-treated blastemal mesenchyme. We find 1177 upregulated genes and 1403 downregulated genes, which could be identified using the axolotl genome. These include several genes known to be involved in retinoic acid metabolism and in patterning. Since positional information is thought to be a property of the cell surface of blastemal cells when we examine our dataset with an emphasis on this aspect, we find the top canonical pathway is integrin signaling. In the extracellular matrix compartment, we find a MMP and several collagens are upregulated; several cell membrane genes and secretory factors are also upregulated. This provides data for future testing of the function of these candidates in the control of PI during limb regeneration.

Pharmacological Properties, Therapeutic Potential and Molecular Mechanisms of JWH133, a CB2 Receptor-Selective Agonist

The endocannabinoid system has attracted attention as a pharmacological target for several pathological conditions. Cannabinoid (CB2)-selective agonists have been the focus of pharmacological studies because modulation of the CB2 receptor (CB2R) can be useful in the treatment of pain, inflammation, arthritis, addiction, and cancer among other possible therapeutic applications while circumventing CNS-related adverse effects. Increasing number of evidences from different independent preclinical studies have suggested new perspectives on the involvement of CB2R signaling in inflammation, infection and immunity, thus play important role in cancer, cardiovascular, renal, hepatic and metabolic diseases. JWH133 is a synthetic agonist with high CB2R selectivity and showed to exert CB2R mediated antioxidant, anti-inflammatory, anticancer, cardioprotective, hepatoprotective, gastroprotective, nephroprotective, and immunomodulatory activities. Cumulative evidences suggest that JWH133 protects against hepatic injury, renal injury, cardiotoxicity, fibrosis, rheumatoid arthritis, and cancer as well as against oxidative damage and inflammation, inhibits fibrosis and apoptosis, and acts as an immunosuppressant. This review provides a comprehensive overview of the polypharmacological properties and therapeutic potential of JWH133. This review also presents molecular mechanism and signaling pathways of JWH133 under various pathological conditions except neurological diseases. Based on the available data, this review proposes the possibilities of developing JWH133 as a promising therapeutic agent; however, further safety and toxicity studies in preclinical studies and clinical trials in humans are warranted.

Selective Somatostatin 5 (SST5) and Somatostatin 2 (SST2) Nonpeptide Agonists Potently Suppress Glucose- and Tolbutamide-Stimulated Dynamic Insulin Secretion From Isolated Human Islets

Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in newborns and infants and arises from dysregulated insulin secretion. Rapid recognition and treatment are vital to prevent seizures, permanent developmental delays, coma, or even death. Very few medical options exist to treat congenital HI patients: the KATP channel activator diazoxide, the injectable somatostatin receptor peptide agonists octreotide and lanreotide, or chronic glucose infusions. However, side effects and/or limited efficacy render these therapies inadequate for many patients. Somatostatin is a 14-amino acid peptide hormone with a broad spectrum of biological actions, which are regulated through five somatostatin receptor subtypes (SST1-SST5). Somatostatin’s common physiological role is to down-regulate secretion of other hormones in various tissues. Its role in the maintenance of euglycemia is to regulate insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Somatostatin regulates insulin secretion by decreasing the intracellular levels of cAMP, inhibition of voltage-gated calcium channels (VGCC), activation of the G protein-activated inward rectifier K+ channel (GIRK), and direct inhibition of insulin exocytosis. Several studies have evaluated the effect of somatostatin, somatostatin peptide analogs, and a limited number of nonpeptide somatostatin receptor agonists on insulin secretion in static assays using isolated human islets. However, the lack of highly selective agonists has made the interpretation of the contribution of SST receptor sub-types difficult to discern. Our programs for the treatment of hyperinsulinism, acromegaly, and other indications have led to the development of selective nonpeptide SST2, SST3, SST4, and SST5 agonists, possessing EC50s < 1 nM in cell-based assays of receptor activation and selectivity > 130 times over the other members of the family. The ability of these selective nonpeptide agonists to regulate glucose- and tolbutamide-stimulated dynamic insulin secretion from human islets was evaluated using a perifusion system (Biorep, FL). We found that selective SST2 and SST5 agonists potently suppressed dynamic insulin secretion in contrast to SST3 or SST4 selective agonists. Importantly, SST5 agonists were shown to have a greater effect than selective SST2 agonists or diazoxide, demonstrating their potential utility in human conditions such as congenital HI. In addition, SST5 activation is also known to have a smaller effect on glucagon secretion and is also less prone to agonist-driven desensitization than SST2 activation. Taken together, these studies support our program to identify, characterize, and develop potent, nonpeptide, orally-bioavailable, selective SST5 agonists with appropriate pharmaceutical and safety characteristics for the treatment of congenital HI.