Discovery of dual positive allosteric modulators (PAMs) of the metabotropic glutamate 2 receptor and CysLT1 antagonists for treating migraine headache

Pyridylmethylsulfonamide series were the first reported example of positive allosteric modulators (PAM) of the mGlu₂ receptor. The hydroxyacetophenone scaffold is a second series of mGlu₂ PAMs we have identified. This series of molecules are potent mGlu₂ potentiators and possess significant CysLT1 (cysteinyl leukotriene receptor 1) antagonist activity, showing in vivo efficacy in a dural plasma protein extravasation (PPE) model of migraine. In this paper, we describe the dual SAR, pharmacokinetics and preclinical in vivo efficacy data for a tetrazole containing hydroxyacetophenone scaffold.     

4-aryl piperazine and piperidine amides as novel mGluR5 positive allosteric modulators

Positive allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) is regarded as a potential novel treatment for schizophrenic patients. Herein we report the synthesis and SAR of 4-aryl piperazine and piperidine amides as potent mGluR5 positive allosteric modulators (PAMs). Several analogs have excellent activity and desired drug-like properties. Compound 2b was further characterized as a PAM using several in vitro experiments, and produced robust activity in several preclinical animal models.     

New positive allosteric modulators of the metabotropic glutamate receptor 2 (mGluR2): identification and synthesis of N-propyl-8-chloro-6-substituted isoquinolones

A series of N-propyl-8-chloro-6-substituted isoquinolones was identified as positive allosteric modulators of metabotropic glutamate receptor 2 (mGluR2 PAM) via high throughput screening (HTS). The subsequent synthesis and initial SAR exploration that led to the identification of compound 28 is described.     

Allosteric activation of metabotropic glutamate receptor 5

Abstract

Metabotropic glutamate receptor 5 (mGluR5) is a class C G protein-coupled receptor (GPCR) with both an extracellular ligand binding site and an allosteric intrahelical chamber located similarly to the orthosteric ligand binding site of Class A GPCRs. Ligands binding to this ancestral site of mGluR5 can act as positive (PAM), negative (NAM) or silent (SAM) allosteric modulators, and their medicinal chemistry optimization is notoriously difficult, as subtle structural changes may cause significant variation in activity and switch in the functional response. Here we present all atom molecular dynamics simulations of NAM, SAM and PAM complexes formed by closely related ligands and analyse the structural differences of the complexes. Several residues involved in the activation are identified and the formation of a continuous water channel in the active complex but not in the inactive ones is recognized. Our results suggest that the mechanism of mGluR5 activation is similar to that of class A GPCRs.

Communicated by Ramaswamy H. Sarma     

Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators

The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.     

Design and synthesis of substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides as positive allosteric modulators of the metabotropic glutamate receptor subtype 5

Based on SAR in the alkyne class of mGlu5 receptor negative allosteric modulators and a set of amide-based positive allosteric modulators, optimized substitution of the aryl ‘b’ ring was used to create substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides. Results from an mGlu5 receptor functional assay, using calcium fluorescence, revealed varying efficacies and potencies that provide evidence that subtle changes in compounds within a close structural class can have marked effects on functional activity including switches in modes of efficacy (i.e., negative to positive allosteric modulation).     

Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABAA receptors

Biphenylic compounds related to the natural products magnolol and 4′-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA_A receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5′-hexylbiphenyl-2,2′-diol (45) and the honokiol analogs 4′-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4′-methoxybiphenyl-2-ol (62) and 5-hexyl-4′-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3 μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA_A receptor agonists.     

Identification of multiple allosteric sites on the M1 muscarinic acetylcholine receptor

Staurosporine and four staurosporine derivatives were docked on the rhodopsin-based homology model of the M1 muscarinic acetylcholine receptor in order to localize the possible allosteric sites of this receptor. It was found that there were three major allosteric sites, two of which are located at the extracellular face of the receptor, and one in the intracellular domain of the receptor. In the present study, the localization of these binding sites is described for the first time. The present study confirms the existence of multiple allosteric sites on the M1 muscarinic receptor, and lays the ground for further experimental and computational analysis to better understand how muscarinic receptors are modulated via their allosteric sites. These findings will also help to design and develop novel drugs acting as allosteric modulators of the M1 receptor, which can be used in the treatment of the Alzheimer's disease.     

Discovery of a class of calcium sensing receptor positive allosteric modulators; 1-(benzothiazol-2-yl)-1-phenylethanols

1-(Benzothiazol-2-yl)-1-(4-chlorophenyl)ethanol (1) was identified as a positive allosteric modulator (PAM) of the CaSR in a functional cell-based assay. This compound belongs to a class of compounds that is structurally distinct from other known positive allosteric modulators, for example, the phenylalkylamines cinacalcet, a modified analog (13) potently suppressed parathyroid hormone (PTH) release in rats, consistent with its profile as a PAM of CaSRs.     

Chemical conversion of nicotinamide into type I positive allosteric modulator of α7 nAChRs

Structural modifications of nicotinamide, a form of vitamin B3, gave rise to a series of compounds (8aa–8ce) that exhibit activities as type I positive allosteric modulators (PAMs) of human α7 nAChR expressed in Xenopus oocytes in two-electrode voltage clamp assay. The compound 8ai was a potent and efficacious PAM with an EC₅₀ = 3.34 ± 1.13 μM and the maximum activation effect of α7 current over 1474 ± 246% in the presence of acetylcholine (100 μM). It is highly specific to α7 nAChR over other subtypes of nAChR and 5-HT_3A receptors. The structure–activity relationship analysis identified a key skeleton of nicotinamide nucleus critical for biological activity. Taken together, the 8ai as a type I PAM of α7 nAChR may be beneficial for improvement of cognitive deficit.     

4-(1-Phenyl-1H-pyrazol-4-yl)quinolines as novel, selective and brain penetrant metabotropic glutamate receptor 4 positive allosteric modulators

4-(1-Phenyl-1H-pyrazol-4-yl)quinoline (1) was identified by screening the Lundbeck compound collection, and characterized as having mGlu4 receptor positive allosteric modulator properties. Compound 1 is selective over other mGlu receptors and a panel of GPCRs, ion channels and enzymes, but has suboptimal lipophilicity and high plasma and brain non-specific binding. In view of the challenges at the hit-to-lead stage previously reported in the development of mGlu4 receptor positive allosteric modulators (PAMs), a thorough structure-mGlu4 PAM activity relationship study was conducted to interrogate the chemical tractability of this chemotype. The central pyrazole ring tolerates the addition of one or two methyl groups. The C-7 position of the quinoline ring provides a site tolerant to hydrophilic substituents, enabling the design of diverse analogs with good in vitro mGlu4 PAM potency and efficacy, as well as improved microsomal turnover in vitro, compared to 1. In spite of the excellent ligand efficiency of 1 (LE=0.43), optimization of in vitro potency for this series reached a plateau around EC(50)=200 nM.     

Challenges in the development of an M4 PAM in vivo tool compound: The discovery of VU0467154 and unexpected DMPK profiles of close analogs

This letter describes the chemical optimization of a novel series of M₄ positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).     

Mechanistic Insights into Allosteric Structure-Function Relationships at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M₁ muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M₁ mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M₁ mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400^7.35 in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M₁ mAChR-targeting PAMs.     

Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.     

Screening for positive allosteric modulators: assessment of modulator concentration-response curves as a screening paradigm

Targeting allosteric binding sites represents a powerful mechanism for selectively modulating receptor function. The advent of functional assays as the screening method of choice is leading to an increase in the number of allosteric modulators identified. These include positive allosteric modulators that can increase the affinity of the orthosteric agonist and potentiate the evoked response. A common method for screening for positive allosteric modulators is to examine a concentration-response (C/R) curve to the putative modulator in the presence of a single, low concentration of agonist. The study reported here has used data simulations for positive allosteric modulators according to the allosteric ternary complex model to generate modulator C/R curves. The results are then compared to the mechanistic parameters used to simulate the data. It is clear from the simulations that the potency of a positive modulator C/R curve in a screening assay is the product of both its affinity and positive cooperativity. However, it is often difficult to tell which parameter dominates the response; not knowing the actual affinity or cooperativity of a ligand may have consequences for receptor selectivity. Further modeling demonstrates that the use and choice of single agonist concentration, as well as changes in the agonist curve Hill slope, can have significant effects on the modulator C/R curve. Finally, the quantitative relationship between modulator C/R curves and the allosteric ternary complex model is explored. These simulations emphasize the importance of careful interpretation of screening data and of conducting full mechanism of action studies for positive allosteric modulators.     

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.     

Discovery and SAR of a novel series of non-MPEP site mGlu5 PAMs based on an aryl glycine sulfonamide scaffold

Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu₅) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site mGlu₅ PAM chemotype.     

Differing pharmacological activities of thiazolidinone analogs at the FSH receptor

The follicle-stimulating hormone is critical to reproductive success and is an important target for development of novel reproductive therapies. We have recently reported the development of thiazolidinone positive allosteric modulators of the follicle-stimulating hormone receptor. Here, we demonstrate that discrete modifications in the chemical structure of the thiazolidinone agonists produced compounds with different pharmacological properties. Positive allosteric modulators activated adenylate cyclase signaling (Gs). Using an ADP-ribosylation assay we found that both differing glycosylated variants of human FSH (hFSH) and selected thiazolidinone allosteric modulators of the FSHR induce activation of the Gi signaling pathway. Additionally, we observed that some analogs of this class could activate both pathways. These data suggest that the pharmacological activity of thiazolidinone modulators to the FSHR may be due to the ability of these compounds to induce association of the FSHR with either Gs or Gi signaling pathways in an analog-specific manner.     

ELIC-α7 Nicotinic Acetylcholine Receptor (α7nAChR) Chimeras Reveal a Prominent Role of the Extracellular-Transmembrane Domain Interface in Allosteric Modulation

The native α7 nicotinic acetylcholine receptor (α7nAChR) is a homopentameric ligand-gated ion channel mediating fast synaptic transmission and is of pharmaceutical interest for treatment of numerous disorders. The transmembrane domain (TMD) of α7nAChR has been identified as a target for positive allosteric modulators (PAMs), but it is unclear whether modulation occurs through changes entirely within the TMD or changes involving both the TMD and the extracellular domain (ECD)-TMD interface. In this study, we constructed multiple chimeras using the TMD of human α7nAChR and the ECD of a prokaryotic homolog, ELIC, which is not sensitive to these modulators, and for which a high resolution structure has been solved. Functional ELIC-α7nAChR (EA) chimeras were obtained when their ECD-TMD interfaces were modified to resemble either the ELIC interface (EA_ELIC) or α7nAChR interface (EA_α7). Both EA_α7 and EA_ELIC show similar activation response and desensitization characteristics, but only EA_α7 retained the unique pharmacology of α7nAChR evoked by PAMs, including potentiation by ivermectin, PNU-120596, and TQS, as well as activation by 4BP-TQS. This study suggests that PAM modulation through the TMD has a more stringent requirement at the ECD-TMD interface than agonist activation.     

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (E_max(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding E_max(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.