Kyle A. Emmitte

Allosteric modulation of seven transmembrane spanning receptors: theory, practice, and opportunities for central nervous system drug discovery.

1.1 Allosteric Modulation – A Historical Perspective nEarly ideas regarding allosterism emerged over 50 years ago, but gained little traction in the receptor field due to limitations in molecular pharmacology and screening technology.1,2 Allosterism is a critical biochemical mechanism, as it enables proteins to sense changes in their environment and respond to them; Fenton has recently referred to this as ‘second secret of life’, preceded only by the genome.1–3 The term allostery comes from the Greek allos ( λλoς), “other”, and stereos (στeρeOς), “solid (object)”, meaning that an allosteric site of a regulatory protein is physically distinct from the classic, active, site.1–4–8 In terms of receptor-based small molecule drug discovery, the binding site for the endogenous ligand is referred to as the orthosteric site.1,4–8 In this setting, an allosteric modulator is a small molecule that binds at a topographically distinct allosteric site, and either potentiates or inhibits the binding and/or signaling of an orthosteric ligand.1,4–8 Fueled by the clinical success of the first allosteric modulator drugs 1–4 (benzodiazepines, referred to as ‘benzo’ or BZD), which potentiate the effect of the neurotransmitter γ-aminobutyric acid (GABA) at the ionotropic GABAA receptor, the concept of allosteric modulation for a wide range of molecular targets has gained momentum in modern drug discovery (Figure 1).4,9 Benzodiazepines, for example, possess a number of modes of pharmacology and include positive allosteric modulators (PAMs), which potentiate GABAA receptor response, negative allosteric modulators (NAMs), which decrease channel activity and modulate the ability of these GABAergic receptors to elicit sedative, hypnotic, and anxiolytic effects. In addition to PAMs and NAMs, silent allosteric modulators (SAMs, or neutral allosteric ligands) bind at allosteric sites and can block the activity of PAMs and NAMs but, importantly, have no effect on orthosteric ligand responses. In contrast to the potentially deadly effects of direct acting GABAA agonists, allosteric modulation of GABAA by the benzodiazepine class has proven clinically safe and effective.4,9 With advances in molecular pharmacology and screening technology, allosteric modulators have now been developed for other ion channels, kinases, phospholipases and 7 Transmembrane Spanning Receptors (7TMRs, also known as G protein-coupled receptors (GPCRs)).1,4–8,10–15 n n n nFigure 1 n nBenzodiazepines, the first allosteric modulators with clinical success, and marketed as GABAA allosteric modualtors. A generic benzodiazepine scaffold 1 highlighting the classical substitution patterns. 2 (Librium™) was the first benzodiazepine ... n n n n n1.2 7TMRs Structure and Ligands n7TMRs are the largest class of cell surface receptors, accounting for over 30% of currently marketed drugs and over 50% of all known drugs.4–7 7TMRs are plasma membrane proteins that receive stimuli (in the form of hormones, neurotransmitters, light, ions or odorants) on the extracellular surface to alter receptor conformation, which in turn activates signaling cascades and effector systems located within the intracellular cytosol via coupling to G proteins and other accessory proteins.4–7 Much of our understanding of the basic structure and function of 7TMRs is based on biochemical, genetic, imaging, and molecular pharmacological research, as crystal structures of 7TMRs (Rhodopsin, opsin, beta2 and beta 1 (agonist and antagonist bound), dopamine D3, Adenosine 2A (agonist and antagonist bound), chemokine CXCR4, histamine H1) have only recently been solved definitively.4–7,16–32 However, these crystal structures have powered the development of homology models for multiple 7TMRs, and afforded avenues for ligand design efforts. Structurally, all 7TMRs possess seven transmembrane helices, three extracellular and three intracellular loops, with an extracellular N-terminal tail and an intracellular C-terminal tail (Figure 2).4–7,16–32 The heptahelical transmembrane domain is largely hydrophobic whereas the extracellular (e1–e3) and intracellular (i1–i3) segments, or loops, are generally hydrophilic as would be anticipated for amino acids exposed to the phospholipid-rich membrane and the water-rich environments, respectively. The seven transmembrane helices are each approximately two-dozen amino acids long, while the C- and N-terminal tails as well as the loops can vary widely in length with up to hundreds of amino acids.4–7,16–32 Based on sequence homology and functional roles, 7TMRs commonly divided into three main Families (or classes): A (e.g., M1 mAChR), B (e.g., CRF1) and C (e.g., mGlu5) (Figure 2). The families are readily distinguished by comparing their amino acid sequences; Family B are distinguished from Family A by the presence of a larger extracellular loop and Family C have a large, bi-lobed N-terminal Venus Fly Trap (VFT) domain. A second major difference between the families concerns the location of the orthosteric binding site and the nature of the orthosteric ligand. As shown in Figure 2, the orthosteric binding site of many Family A 7TMRs is located with the 7TM domain whereas the orthosteric binding site is located in the large extracellular loop within Family B and within the VFT domain in Family C. The orthosteric ligands for Family A and C are neurotransmitters, for example, 5 (acetylcholine, for the mAChRs) and 9 (glutamate, for the mGluRs), respectively.4–7 The orthosteric ligands for Family B 7TMRs are large peptide ligands with usually >30 amino acids, such as the 41 amino acid peptide 7 (hCRF) for corticotrophin releasing factor 1 (CRF1). In contrast, allosteric ligands are structurally distinct from orthosteric ligands and bind at distinct sights, often, but not always, topologically distant from the orthosteric site.4–7 For example, the Family A M1 mAChR PAM 6 (BQCA),33 is believed to bind in a region above the TMs among the extracellular loops, whereas the Family B PAM, 8 (DMP696),34 and the Family C NAM, 10 (MPEP),35,36 bind within the TM domains. n n n nFigure 2 n nStructural topology of typical orthosteric and allosteric sites of Family A, B and C 7TMRs, highlighting representative orthosteric and allosteric ligands for each Family. n n n nAre there naturally occurring allosteric modulators? This question is invariably posed during any discussion of allosteric modulators, and one must understand the complexity of identifying such ligands within the chemical diversity of ligands within the human body.1,2,37 However, a few natural allosteric modulators have been described, including the unnatural amino acid D-serine (an allosteric modulator of the NMDA receptor),38 L-phenylalanine and L-tryptophan (allosteric modulators of the calcium receptor)39 and the tetrepeptide Leu-Ser-Ala-Leu, also known as 5-HT moduline (an allosteric modulator of the 5-HT1B receptor).40,41

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships

Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu5) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu5 to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu5-mediated Ca2+ mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities.

mGlu5 negative allosteric modulators: a patent review (2010 – 2012)

Introduction: The design and development of small molecule negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGlu5) has been an area of intense interest for over a decade. Potential roles have been established for mGlu5 NAMs in the treatment of diseases such as pain, anxiety, gastroesophageal reflux disease (GERD), Parkinsons disease levodopa-induced dyskinesia (PD-LID), fragile X syndrome (FXS), autism, addiction, and depression. Areas covered: This review begins with an update of the clinical trial efforts with mGlu5 NAMs. Following that update, the review summarizes small molecule mGlu5 NAM patent applications published between 2010 and 2012. These summaries are subdivided into three separate groups: inventions related to improvements in drug properties and/or developability, new chemical entities that contain a disubstituted alkyne, and new chemical entities that do not contain a disubstituted alkyne. Expert opinion: Given the abundant promise found within the mGlu5 NAM field, optimism remains that a drug will emerge from this therapeutic class. Still, the launch of a new drug is far from a certainty. It is encouraging to observe the ever-increasing chemical diversity among mGlu5 NAMs. Finally, in spite of the mature nature of this field, room remains for new advancements.

Metabotropic glutamate receptor 3 activation is required for long-term depression in medial prefrontal cortex and fear extinction

Significance Recent genetic studies suggest that variations in the gene encoding metabotropic glutamate receptor 3 (mGlu3) can influence aspects of cognitive function that involve the prefrontal cortex (PFC). Furthermore, mutations in this gene may predispose individuals to developing psychiatric disorders in which altered function of the PFC has been implicated. However, little is known about the precise roles of mGlu3 in regulating the function of the PFC. In the present study, we took advantage of newly identified molecular probes to show that mGlu3 can strongly influence synaptic plasticity within the PFC and that blockade of this receptor impairs specific learning abilities in mice. These results suggest that mGlu3 may be a therapeutic target for cognitive dysfunction in mental disorders. Clinical studies have revealed that genetic variations in metabotropic glutamate receptor 3 (mGlu3) affect performance on cognitive tasks dependent upon the prefrontal cortex (PFC) and may be linked to psychiatric conditions such as schizophrenia, bipolar disorder, and addiction. We have performed a series of studies aimed at understanding how mGlu3 influences PFC function and cognitive behaviors. In the present study, we found that activation of mGlu3 can induce long-term depression in the mouse medial PFC (mPFC) in vitro. Furthermore, in vivo administration of a selective mGlu3 negative allosteric modulator impaired learning in the mPFC-dependent fear extinction task. The results of these studies implicate mGlu3 as a major regulator of PFC function and cognition. Additionally, potentiators of mGlu3 may be useful in alleviating prefrontal impairments associated with several CNS disorders.

Practical Strategies and Concepts in GPCR Allosteric Modulator Discovery: Recent Advances with Metabotropic Glutamate Receptors

Allosteric modulation of GPCRs has initiated a new era of basic and translational discovery, filled with therapeutic promise yet fraught with caveats. Allosteric ligands stabilize unique conformations of the GPCR that afford fundamentally new receptors, capable of novel pharmacology, unprecedented subtype selectivity, and unique signal bias. This review provides a comprehensive overview of the basics of GPCR allosteric pharmacology, medicinal chemistry, drug metabolism, and validated approaches to address each of the major challenges and caveats. Then, the review narrows focus to highlight recent advances in the discovery of allosteric ligands for metabotropic glutamate receptor subtypes 1-5 and 7 (mGlu1-5,7) highlighting key concepts (molecular switches, signal bias, heterodimers) and practical solutions to enable the development of tool compounds and clinical candidates. The review closes with a section on late-breaking new advances with allosteric ligands for other GPCRs and emerging data for endogenous allosteric modulators.

Discovery of (R)-(2-Fluoro-4-((-4-methoxyphenyl)ethynyl)phenyl) (3-Hydroxypiperidin-1-yl)methanone (ML337), An mGlu3 Selective and CNS Penetrant Negative Allosteric Modulator (NAM)

A multidimensional, iterative parallel synthesis effort identified a series of highly selective mGlu3 NAMs with submicromolar potency and good CNS penetration. Of these, ML337 resulted (mGlu3 IC50 = 593 nM, mGlu2 IC50 >30 μM) with B:P ratios of 0.92 (mouse) to 0.3 (rat). DMPK profiling and shallow SAR led to the incorporation of deuterium atoms to address a metabolic soft spot, which subsequently lowered both in vitro and in vivo clearance by >50%.

A Novel Class of Succinimide-Derived Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 1 Provides Insight into a Disconnect in Activity between the Rat and Human Receptors

Recent progress in the discovery of mGlu₁ allosteric modulators has suggested the modulation of mGlu₁ could offer possible treatment for a number of central nervous system disorders; however, the available chemotypes are inadequate to fully investigate the therapeutic potential of mGlu₁ modulation. To address this issue, we used a fluorescence-based high-throughput screening assay to screen an allosteric modulator-biased library of compounds to generate structurally diverse mGlu₁ negative allosteric modulator hits for chemical optimization. Herein, we describe the discovery and characterization of a novel mGlu₁ chemotype. This series of succinimide negative allosteric modulators, exemplified by VU0410425, exhibited potent inhibitory activity at rat mGlu₁ but was, surprisingly, inactive at human mGlu₁. VU0410425 and a set of chemically diverse mGlu₁ negative allosteric modulators previously reported in the literature were utilized to examine this species disconnect between rat and human mGlu₁ activity. Mutation of the key transmembrane domain residue 757 and functional screening of VU0410425 and the literature compounds suggests that amino acid 757 plays a role in the activity of these compounds, but the contribution of the residue is scaffold specific, ranging from critical to minor. The operational model of allosterism was used to estimate the binding affinities of each compound to compare to functional data. This novel series of mGlu₁ negative allosteric modulators provides valuable insight into the pharmacology underlying the disconnect between rat and human mGlu₁ activity, an issue that must be understood to progress the therapeutic potential of allosteric modulators of mGlu₁.

The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.

Discovery of 2‐(2‐Benzoxazoyl amino)‐4‐Aryl‐5‐Cyanopyrimidine as Negative Allosteric Modulators (NAMs) of Metabotropic Glutamate Receptor 5 (mGlu5): From an Artificial Neural Network Virtual Screen to an In Vivo Tool Compound

Glutamate, the major excitatory neurotransmitter, functions in the brain via activation of ligand gated cation channels and also the eight subtypes of Class C G protein-coupled metabotropic glutamate receptors (mGlus).[1] Selective allosteric modulation of mGlu5 has been shown to have potential for treatment of a variety of neurological disorders[2,3] including anxiety disorders[4,5], Parkinson’s disease[6–8], Fragile X syndrome[9] and schizophrenia.[10–14] The majority of mGlu5 negative allosteric modulators (NAMs) developed to date either contain an alkyne moiety 1–4 or employ the alkyne topology as basis for ligand design,[15] as in 5–8 (Figure 1). Only recently have mGlu5 NAM chemotypes been identified, through high-throughput screening (HTS) campaigns, that are structurally unrelated to the classical acetylenic derivatives, such as 9–12 (Figure 1).[16] Due to the prevalence of ‘molecular switch‘phenomenon in MPEP related scaffolds, our interest focused on the discovery and development of novel mGlu5 NAM chemotypes, by both HTS and Artificial Neural Network (ANN) virtual screens.