Receptor Subtype Selectivity Research Articles

Oestradiol influences on dopamine release from the nucleus accumbens shell: sex differences and the role of selective oestradiol receptor subtypes.

Females are more sensitive than males to both the acute and prolonged effects of psychomotor stimulants. In females, this is regulated by oestradiol, which enhances dopamine release in the dorsal striatum. In this study, we tested the acute effect of oestradiol on dopamine release in the nucleus accumbens (NAc) shell after cocaine administration and investigated which oestradiol receptors (ERs) contribute to sex differences in the response to cocaine. The ability of oestradiol benzoate (EB) to acutely modulate the effect of cocaine on phasic dopamine release in the NAc shell was measured by fast-scan cyclic voltammetry in anaesthetized male and female rats. The roles of ER subtypes, ERα and ERβ, was determined with selective agonists. EB acutely enhanced the effect of cocaine on stimulated dopamine release from the NAc shell in females but not in male rats only at levels of stimulation expected to optimally saturate dopamine transporters. Enhanced dopamine release after cocaine administration was also observed in females after selective activation of ERβ but not ERα. EB attenuated the effect of cocaine on NAc shell dopamine reuptake in males but not in females. Oestradiol acutely and rapidly regulates dopamine release in females and dopamine reuptake in males. In females, oestradiol rapidly enhances the effect of cocaine on dopamine release, likely via activation of ERβ. The effect of oestradiol in males is not seen with selective receptor subtype activation, a topic deserving of further study. This article is part of a themed section on The Importance of Sex Differences in Pharmacology Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.21/issuetoc.

6-Phenylpyrimidin-4-ones as Positive Allosteric Modulators at the M1 mAChR: The Determinants of Allosteric Activity.

Targeting allosteric sites of the M1 muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer's disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M1 mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M1 mAChR PAMs.

Human pharmacology of positive GABA-A subtype-selective receptor modulators for the treatment of anxiety.

Anxiety disorders arise from disruptions among the highly interconnected circuits that normally serve to process the streams of potentially threatening stimuli. The resulting imbalance among these circuits can cause a fundamental misinterpretation of neural sensory information as threatening and can lead to the inappropriate emotional and behavioral responses observed in anxiety disorders. There is considerable preclinical evidence that the GABAergic system, in general, and its α2- and/or α5-subunit-containing GABA(A) receptor subtypes, in particular, are involved in the pathophysiology of anxiety disorders. However, the clinical efficacy of GABA-A α2-selective agonists for the treatment of anxiety disorders has not been unequivocally demonstrated. In this review, we present several human pharmacological studies that have been performed with the aim of identifying the pharmacologically active doses/exposure levels of several GABA-A subtype-selective novel compounds with potential anxiolytic effects. The pharmacological selectivity of novel α2-subtype-selective GABA(A) receptor partial agonists has been demonstrated by their distinct effect profiles on the neurophysiological and neuropsychological measurements that reflect the functions of multiple CNS domains compared with those of benzodiazepines, which are nonselective, full GABA(A) agonists. Normalizing the undesired pharmacodynamic side effects against the desired on-target effects on the saccadic peak velocity is a useful approach for presenting the pharmacological features of GABA(A)-ergic modulators. Moreover, combining the anxiogenic symptom provocation paradigm with validated neurophysiological and neuropsychological biomarkers may provide further construct validity for the clinical effects of novel anxiolytic agents. In addition, the observed drug effects on serum prolactin levels support the use of serum prolactin levels as a complementary neuroendocrine biomarker to further validate the pharmacodynamic measurements used during the clinical pharmacological study of novel anxiolytic agents.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor

Human muscarinic receptor, M2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptor compared to M3 receptor leads to subtype selectivity of AF-DX 384.

Altered colonic sensory and barrier functions by CRF: roles of TLR4 and IL-1

Visceral allodynia and increased colonic permeability are considered to be crucial pathophysiology of irritable bowel syndrome (IBS). Corticotropin-releasing factor (CRF) and immune-mediated mechanisms have been proposed to contribute to these changes in IBS, but the precise roles have not been determined. We explored these issues in rats in vivo. The threshold of visceromotor response, i.e., abdominal muscle contractions induced by colonic balloon distention was electrophysiologically measured. Colonic permeability was estimated by quantifying the absorbed Evans blue in colonic tissue. Intraperitoneal injection of CRF increased the permeability, which was blocked by astressin, a non-selective CRF receptor antagonist, but astressin2-B, a selective CRF receptor subtype 2 (CRF2) antagonist did not modify it. Urocortin 2, a selective CRF2 agonist inhibited the increased permeability by CRF. Eritoran, a toll-like receptor 4 (TLR4) antagonist or anakinra, an interleukin-1 receptor antagonist blocked the visceral allodynia and the increased gut permeability induced by CRF. Subcutaneous injection of lipopolysaccharide (immune stress) or repeated water avoidance stress (WAS, psychological stress), 1 h daily for 3 days induced visceral allodynia and increased gut permeability (animal IBS models), which were also blocked by astressin, eritoran or anakinra. In conclusion, stress-induced visceral allodynia and increased colonic permeability were mediated via peripheral CRF receptors. CRF induced these visceral changes via TLR4 and cytokine system, which were CRF1 dependent, and activation of CRF2 inhibited these CRF1-triggered responses. CRF may modulate immune system to alter visceral changes, which are considered to be pivotal pathophysiology of IBS.

Structural Basis for ( S)-3,4-Dicarboxyphenylglycine (DCPG) As a Potent and Subtype Selective Agonist of the mGlu8 Receptor.

( S)-3,4-Dicarboxyphenylglycine (DCPG) was first reported in 2001 as a potent orthosteric agonist with high subtype selectivity for the mGlu8 receptor, but the structural basis for its high selectivity is not well understood. We have solved a cocrystal structure of recombinant human mGlu8 amino terminal domain (ATD) protein bound to ( S)-DCPG, which possesses the largest lobe opening angle observed to date among known agonist-bound mGlu ATD crystal structures. The binding conformation of ( S)-DCPG observed in the crystal structure is significantly different from that in the homology model built from an l-glutamate-bound rat mGlu1 ATD crystal structure, which has a smaller lobe opening angle. This highlights the importance of considering various lobe opening angles when modeling mGlu ATD-ligand complex. New homology models of other mGlu receptors based on the ( S)-DCPG-bound mGlu8 ATD crystal structure were explored to rationalize ( S)-DCPG's high mGlu8 receptor subtype selectivity.

Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist.

Salmeterol is a partial agonist for the β2 adrenergic receptor (β2AR), and the first long-acting β2AR agonist (LABA) to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol has been controversial both for its safety and mechanism of action. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β2AR in complex with an active-state stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β1AR and β2AR explain the high receptor subtype selectivity. A structural comparison with the β2AR bound to the full agonist epinephrine reveals differences in the hydrogen bond network involving residues Ser 2045.43 and Asn 2936.55. Mutagenesis and biophysical studies suggest that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G protein activation and the limited β-arrestin recruitment for salmeterol.

Optimization of Preclinical Metabolism for Somatostatin Receptor Subtype 5-Selective Antagonists.

A series of structurally diverse azaspirodecanone and spirooxazolidinone analogues were designed and synthesized as potent and selective somatostatin receptor subtype 5 (SSTR5) antagonists. Four optimized compounds each representing a subseries showed improvement in their metabolic stability and pharmacokinetic profiles compared to those of the original lead compound 1 while maintaining pharmacodynamic efficacy. The optimized cyclopropyl analogue 13 demonstrated efficacy in a mouse oral glucose tolerance test and an improved metabolic profile and pharmacokinetic properties in rhesus monkey studies. In this Communication, we discuss the relationship among structure, in vitro and in vivo activity, metabolic stability, and ultimately the potential of these compounds as therapeutic agents for the treatment of type 2 diabetes. Furthermore, we show how the use of focused libraries significantly expanded the structural class and provided new directions for structure-activity relationship optimization.

International Union of Basic and Clinical Pharmacology. CVI: GABAA Receptor Subtype- and Function-selective Ligands: Key Issues in Translation to Humans.

GABAA receptors are the major inhibitory transmitter receptors in the brain. They are ligand-gated chloride channels and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. GABAA receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 homologous subunits and their distinct regional, cellular, and subcellular distribution gives rise to a large number of GABAA receptor subtypes with distinct pharmacology, which modulate different functions of the brain. A variety of compounds have been identified that were claimed to modulate selectively individual GABAA receptor subtypes. However, many of these compounds have only incompletely been investigated or, in addition to a preferential modulation of a receptor subtype, also modulate other subtypes at similar concentrations. Although their differential efficacy at distinct receptor subtypes reduced side effects in behavioral experiments in rodents, the exact receptor subtypes mediating their behavioral effects cannot be unequivocally delineated. In addition, the discrepant in vivo effects of some of these compounds in rodents and man raised doubts on the applicability of the concept of receptor subtype selectivity as a guide for the development of clinically useful drugs. Here, we provide an up-to-date review on the currently available GABAA receptor subtype-selective ligands. We present data on their actual activity at GABAA receptor subtypes, discuss the translational aspect of subtype-selective drugs, and make proposals for the future development of ligands with better anxioselectivity in humans. Finally, we discuss possible ways to strengthen the conclusions of behavioral studies with the currently available drugs.

Neurotoxic peptides and proteins: biomarkers for screening and identifying ion channel subtypes in healthy/diseased conditions and for drug design

Cys-loop receptors comprising nicotinic acetylcholine receptors (nAChRs), glycine-, ionotropic GABA and serotonin receptors exist as several subtypes differing in localization and role in normal functioning/pathologies. Contrary to antibodies, their identification is more efficient with neurotoxic peptides and proteins from animal venoms, highly selectivity for a particular receptor subtype. Naturally-occurring neurotoxins are used in electrophysiology, while their radioactive/fluorescent derivatives help to measure the receptor subtypes at normal state and diseases. Such measurements are important, for example, for α4β2 nAChRs involved in nicotine addiction, for α7 neuronal nAChRs implicated in β-amyloid peptide binding and for “non-neuronal” α7 nAChR regulating anti-inflammatory response and lung cancer growth. X-ray analysis of the neurotoxin complexes with the receptor models shed light on the binding sites and on possible functional effects of certain disease-associated mutations. Neurotoxins give hints for new drugs or become drugs themselves: like a strong analgesic ω-conotoxin (Prialt) blocking one Ca2+ channel subtype. Several α-conotoxins blocking α9/α10 nAChRs are considered as possible analgesics emphasizing that fundamental research of ligand-receptor interactions is important for solving practical medical tasks. Our recent work on (1) nAChRs and other Cys-loop receptors will be illustrated with novel high-affinity α-conotoxins, (2) snake venom α-neurotoxins (3) human endogenous proteins that have the same three-finger folding as α-neurotoxins and regulate the nAChR activities.

Synthetic peripherally-restricted cannabinoid suppresses chemotherapy-induced peripheral neuropathy pain symptoms by CB1 receptor activation

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and dose-limiting side effect of cancer treatment that affects millions of cancer survivors throughout the world and current treatment options are extremely limited by their side effects. Cannabinoids are highly effective in suppressing pain symptoms of chemotherapy-induced and other peripheral neuropathies but their widespread use is limited by central nervous system (CNS)-mediated side effects. Here, we tested one compound from a series of recently developed synthetic peripherally restricted cannabinoids (PRCBs) in a rat model of cisplatin-induced peripheral neuropathy. Results show that local or systemic administration of 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI) dose-dependently suppressed CIPN mechanical and cold allodynia. Orally administered PrNMI also dose-dependently suppressed CIPN allodynia symptoms in both male and female rats without any CNS side effects. Co-administration with selective cannabinoid receptor subtype blockers revealed that PrNMI's anti-allodynic effects are mediated by CB1 receptor (CB1R) activation. Expression of CB2Rs was reduced in dorsal root ganglia from CIPN rats, whereas expression of CB1Rs and various endocannabinoid synthesizing and metabolizing enzymes was unaffected. Daily PrNMI treatment of CIPN rats for two weeks showed a lack of appreciable tolerance to PrNMI's anti-allodynic effects. In an operant task which reflects cerebral processing of pain, PrNMI also dose-dependently suppressed CIPN pain behaviors. Our results demonstrate that PRCBs exemplified by PrNMI may represent a viable option for the treatment of CIPN pain symptoms.

Potential role for histone deacetylation in chronic diazepam-induced downregulation of α1-GABAA receptor subunit expression.

Corroborating evidence indicate that the downregulation of GABAA receptor subunit expression may underlie tolerance to the anticonvulsant and anxiolytic actions of benzodiazepine (BZ) ligands that act as full allosteric modulators (FAMs) of GABA actions at a variety of GABAA receptor subtypes. We and others have shown that 10‐14 days treatment with increasing doses of diazepam (a FAM) resulted in anticonvulsant tolerance and decreased the expression of the α1 GABAA receptor subunit mRNA and protein in frontal cortex. In addition, we have also shown that long‐term treatment with imidazenil, a partial allosteric modulator of GABA action at selective GABAA receptor subtypes, fail to change the expression of the α1 subunit mRNA or induce tolerance to its anticonvulsant or anxiolytic action. However, little is known regarding the potential role of epigenetic mechanisms on long‐term BZ‐induced downregulation of GABAA receptor subunit. Therefore, we examined the role of histone acetylation and DNA methylation mechanisms on long‐term diazepam‐induced downregulation of the α1 subunit mRNA expression in rat frontal cortex. We found that 10 days treatment with increasing doses of diazepam but not imidazenil decreased the expression of the α1 GABAA receptor subunit mRNA and promoter acetylation in frontal cortex. In addition, we also found that 10 days treatment with diazepam but not imidazenil increased the expression of histone deacetylase (HDAC) 1 and 2 in frontal cortex. Thus, the increased expression of HDAC1 and HDAC2 (class 1 HDACs) and consequently increased histone deacetylation mechanism of this class 1 HDACs, may underlie long‐term diazepam‐induced decreased expression of the α1 GABAA receptor subunit mRNA in frontal cortex.

Chemoprevention of LA7-Induced Mammary Tumor Growth by SM6Met, a Well-Characterized Cyclopia Extract.

Breast cancer (BC) is the leading cause of cancer-related deaths in women. Chemoprevention of BC by using plant extracts is gaining attention. SM6Met, a well-characterized extract of Cyclopia subternata with reported selective estrogen receptor subtype activity, has shown tumor suppressive effects in a chemically induced BC model in rats, which is known to be estrogen responsive. However, there is no information on the estrogen sensitivity of the relatively new orthotopic model of LA7 cell-induced mammary tumors. In the present study, the potential chemopreventative and side-effect profile of SM6Met on LA7 cell-induced tumor growth was evaluated, as was the effects of 17β-estradiol and standard-of-care (SOC) endocrine therapies, such as tamoxifen (TAM), letrozole (LET), and fulvestrant (FUL). Tumor growth was observed in the tumor-vehicle control group until day 10 post tumor induction, which declined afterward on days 12–14. SM6Met suppressed tumor growth to the same extent as TAM, while LET, but not FUL, also showed substantial anti-tumor effects. Short-term 17β-estradiol treatment reduced tumor volume on days prior to day 10, whereas tumor promoting effects were observed during long-term treatment, which was especially evident at later time points. Marked elevation in serum markers of liver injury, which was further supported by histological evaluation, was observed in the vehicle-treated tumor control, TAM, LET, and long-term 17β-estradiol treatment groups. Alterations in the lipid profiles were also observed in the 17β-estradiol treatment groups. In contrast, SM6Met did not augment the increase in serum levels of liver injury biomarkers caused by tumor induction and no effect was observed on lipid profiles. In summary, the results from the current study demonstrate the chemopreventative effect of SM6Met on mammary tumor growth, which was comparable to that of TAM, without eliciting the negative side-effects observed with this SOC endocrine therapy. Furthermore, the results of this study also showed some responsiveness of LA7-induced tumors to estrogen and SOC endocrine therapies. Thus, this model may be useful in evaluating potential endocrine therapies for hormone responsive BC.

Binding, Thermodynamics, and Selectivity of a Non-peptide Antagonist to the Melanocortin-4 Receptor.

The melanocortin-4 receptor (MC4R) is a potential drug target for treatment of obesity, anxiety, depression, and sexual dysfunction. Crystal structures for MC4R are not yet available, which has hindered successful structure-based drug design. Using microsecond-scale molecular-dynamics simulations, we have investigated selective binding of the non-peptide antagonist MCL0129 to a homology model of human MC4R (hMC4R). This approach revealed that, at the end of a multi-step binding process, MCL0129 spontaneously adopts a binding mode in which it blocks the agonistic-binding site. This binding mode was confirmed in subsequent metadynamics simulations, which gave an affinity for human hMC4R that matches the experimentally determined value. Extending our simulations of MCL0129 binding to hMC1R and hMC3R, we find that receptor subtype selectivity for hMC4R depends on few amino acids located in various structural elements of the receptor. These insights may support rational drug design targeting the melanocortin systems.

Chemical space screening around Phe3 in opioid peptides: Modulating µ versus δ agonism by Suzuki-Miyaura cross-couplings

In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1–4] (i.e. Dmt-d-Ala-Phe-GlyNH2, Dmt = 2′,6′-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe3 side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere.

Antidepressant Efficacy and Tolerability of Ketamine and Esketamine: A Critical Review.

Ketamine and its enantiomer S-ketamine (esketamine) are promising candidates to produce a rapid-onset antidepressant effect in treatment-resistant depression. Ketamine causes continued blockade of the glutamate N-methyl-D-aspartate (NMDA) receptor, though this might not primarily mediate the antidepressant effect. Alternative hypotheses include selectivity for the NMDA receptor subtype containing the NMDA receptor subunit 2B (NR2B), inhibition of the phosphorylation of the eukaryotic elongation factor 2 (eEF2) kinase, increased expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrKB), and activation of the mammalian target of rapamycin (mTOR) signaling pathway, alongside other independent actions attributed to the ketamine metabolism to R-hydroxynorketamine (R-HNK). The enantiomer S-ketamine (esketamine) displays approximately fourfold greater affinity for the glutamate NMDA receptor in vitro than R-ketamine. Proof-of-concept single-dose and repeat-dose studies with intravenous ketamine show a significant antidepressant and probably antisuicidal effect in the short term, with response rates over 60% as early as 4.5h after a single dose, with a sustained effect after 24h, and over 40% after 7days. This response can be further sustained over several weeks with repeated doses (two to three doses per week). Tolerability seems acceptable in the short term, with transient elevation of blood pressure and mild and transient dissociative and psychotomimetic effects. Intranasal esketamine has shown a comparable antidepressant effect, which has resulted in the US FDA granting the drug a "breakthrough therapy" designation, and theoretically it may offer an improved tolerability profile. However, major concerns remain regarding an effective protocol to maintain the clinical antidepressant effect of ketamine seen with acute administration and the safety of ketamine and esketamine in the long term, specifically related to potential neurocognitive and urologic toxicity, together with the potential induction of substance use disorders. Ketamine and esketamine are not currently approved treatments for depression, but the clinical use of ketamine is increasing in a variety of practice settings internationally.

Basic and clinical aspects of antimuscarinic agents used to treat overactive bladder.

Antimuscarinic agents are now widely used as the pharmacological therapy for overactive bladder (OAB) because neuronal (parasympathetic nerve) and non-neuronal acetylcholine play a significant role for the bladder function. In this review, we will highlight basic and clinical aspects of eight antimuscarinic agents (oxybutynin, propiverine, tolterodine, solifenacin, darifenacin, trospium, imidafenacin, and fesoterodine) clinically used to treat urinary dysfunction in patients with OAB. The basic pharmacological characteristics of these eight antimuscarinic agents include muscarinic receptor subtype selectivity, functional bladder selectivity, and muscarinic receptor binding in the bladder and other tissues. The measurement of drug-receptor binding after oral administration of these agents allows for clearer understanding of bladder selectivity by the integration of pharmacodynamics and pharmacokinetics under in vivo conditions. Their central nervous system (CNS) penetration potentials are also discussed in terms of the feasibility of impairments in memory and cognitive function in elderly patients with OAB. The clinical aspects of efficacy focus on improvements in the daytime urinary frequency, nocturia, bladder capacity, the frequency of urgency, severity of urgency, number of incontinence episodes, OAB symptom score, and quality of life (QOL) score by antimuscarinic agents in patients with OAB. The safety of and adverse events caused by treatments with antimuscarinic agents such as dry mouth, constipation, blurred vision, erythema, fatigue, increased sweating, urinary retention, and CNS adverse events are discussed. A dose-dependent relationship was observed with adverse events, because the risk ratio generally increased with elevations in the drug dose of antimuscarinic agents. Side effect profiles may be additive to or contraindicated by other medications.

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

Structure‐activity relationships of a distinct family of selective agonists of α7‐nAChRs

Nicotinic acetylcholine receptors (nAChRs) are lead members of a pentameric ligand‐gated ion channel family; they are excitatory receptors, activated by the endogenous neurotransmitter, acetylcholine. nAChRs are present in the peripheral and central nervous system, where they mediate and modulate neuromuscular, autonomic and CNS functions. Receptor‐selective ligands that cross the blood‐brain barriers may form useful therapies for disorders of aging, such as Alzheimers disease and disorders of development, such as schizophrenia. In previous work, we developed a small sub‐group of 4,6 di‐substituted 2‐aminopyrimidines that interact with the acetylcholine binding protein (AChBP) and activate a7‐nAChRs. These agents show Kd and EC50 values for both AChBP binding and a7‐nAChR activation between 10 and 70nM. Moreover, they are structurally distinct from classical nicotinic agonists that are quaternary amines or typically contain a strong base separated from a weaker base or hydrogen bonding acceptor, often on separate ring systems. Selectivity for the a7‐AChR and high affinity for AChBP was achieved by substituting a dipicolyl group at the 4 position of the pyrimidine. In contrast to classical nicotinic agonists, the nitrogens on the pyrimidine and pyridine rings are weakly basic. Moreover, X‐ray crystallographic studies show that these ligands bind to the classical agonist‐antagonist site, but with a distinctive orientation of the core pyrimidine ring and the associated picolyl moiety. We have expanded our structural landscape of these AChBP binding and a7‐selective ligands, based on our previous leads by combining the pyrimidine system with the di‐picolyl substitution at the 4‐position. To examine receptor interactions of this family of compounds on nAChRs and related pentameric ligand‐gated ion channels, we employ HEK cells transfected with cDNA's encoding one of three requisite receptor subtypes: α7‐nAChR, α4β2‐nAChR and 5HT3AR, along with a fluorescent reporter. To assess rapid activation of lead candidates we use an oocyte system, revealing an agonist behavior distinguishing them from silent agonists and ago‐PAM's. To examine the molecular determinants and ligand poses involved, we have crystallized the higher affinity complexes with AChBP. Accordingly, we have a structure‐guided approach to novel ligand design for nAChRs. In conclusion, we find: 1) a di‐picolyl moiety in the 4‐position is fundamental for the activation of the a7‐nAChR, 2) the 2‐amino group tolerates only certain structural changes controlled by steric and bonding constraints, 3) substitutions at the 6‐position affect both the potency and receptor subtype selectivity of the ligands.Support or Funding InformationCONACYT Mexico graduate fellowship to GACHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of Novel Melanocortin Receptor Agonists Based on the Cyclic Peptide Framework of Sunflower Trypsin Inhibitor-1.

Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3-5R. Double N-methylated compound 18 showed a p Ki of 8.73 ± 0.08 ( Ki = 1.92 ± 0.34 nM) and a pEC50 of 9.13 ± 0.04 (EC50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.