Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.

We describe here a series of metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulators (NAMs) with a saturable range of activity in inhibiting responses to an orthosteric agonist in two distinct in vitro pharmacological assays. The range of inhibition among compounds in this scaffold provides highly structurally related ligands with differential degrees of receptor blockade that can be used to understand inhibitory efficacy profiles in native tissue or in vivo.

Discovery of a potent M5 antagonist with improved clearance profile. Part 2: Pyrrolidine amide-based antagonists.

This Letter describes our ongoing effort to improve the clearance of selective M5 antagonists. Herein, we report the replacement of the previously disclosed piperidine amide (4, disclosed in Part 1) with a pyrrolidine amide core. Several compounds within this series provided good potency, subtype selectivity, and low to moderate clearance profiles. Interestingly, the left-hand side SAR for this series diverged from our earlier efforts.

Discovery of a potent M5 antagonist with improved clearance profile. Part 1: Piperidine amide-based antagonists.

The lack of potent and selective tool compounds with pharmaceutically favorable properties limits the in vivo understanding of muscarinic acetylcholine receptor subtype 5 (M5) biology. Previously, we presented a highly potent and selective M5 antagonist VU6019650 with a suboptimal clearance profile as our second-generation tool compound. Herein, we disclose our ongoing efforts to generate next-generation M5 antagonists with improved clearance profiles. A mix and match approach between VU6019650 (lead) and VU0500325 (HTS hit) generated a piperidine amide-based novel M5 antagonist series. Several analogs within this series, including 29f, provided good on-target potency with improved clearance profiles, though room for improvement remains.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators.

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the "druggable genome" that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

Discovery of a novel class of heteroaryl-pyrrolidinones as positive allosteric modulators of the muscarinic acetylcholine receptor M1.

This Letter describes the synthesis and optimization of a series of heteroaryl-pyrrolidinone positive allosteric modulators (PAMs) of the muscarinic acetylcholine receptor M1 (mAChR M1). Through the continued optimization of M1 PAM tool compound VU0453595, with a focus on replacement of the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one with a wide variety of alternative 4,5-dihydropyrrolo-fused heteroaromatics, the generation of M1 PAMs with structurally novel chemotypes is disclosed. Two compounds from these subseries, 8b (VU6005610) and 20a (VU6005852), show robust selectivity for the M1 mAChR, and no M1 agonism. Both compounds have favorable preliminary PK profiles in vitro;8b additionally demonstrates high brain exposure in a rodent IV cassette model.

Discovery and optimization of a novel CNS penetrant series of mGlu4 PAMs based on a 1,4-thiazepane core with in vivo efficacy in a preclinical Parkinsonian model.

A high throughput screen (HTS) identified a novel, but weak (EC50 = 6.2 µM, 97% Glu Max) mGlu4 PAM chemotype based on a 1,4-thiazepane core, VU0544412. Reaction development and chemical optimization delivered a potent mGlu4 PAM VU6022296 (EC50 = 32.8 nM, 108% Glu Max) with good CNS penetration (Kp = 0.45, Kp,uu = 0.70) and enantiopreference. Finally, VU6022296 displayed robust, dose-dependent efficacy in reversing Haloperidol-Induced Catalepsy (HIC), a rodent preclinical Parkinson's disease model.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Synthesis and SAR of a series of mGlu7 NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core.

A High-Throughput Screening (HTS) campaign identified a fundamentally new mGlu7 NAM chemotype, based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core. The initial hit, VU0226390, was a potent mGlu7 NAM (IC50 = 647 nM, 6% L-AP4 min) with selectivity versus the other group III mGlu receptors (>30 µM vs. mGlu4 and mGlu8). A multi-dimensional optimization effort surveyed all regions of this new chemotype, and found very steep SAR, reminiscent of allosteric modulators, and unexpected piperazine mimetics (whereas classical bioisosteres failed). While mGlu7 NAM potency could be improved (IC50s ~ 350 nM), the necessity of the ethyl ester moiety and poor physiochemical and DMPK properties precluded optimization towards in vivo tool compounds or clinical candidates. Still, this hit-to-lead campaign afforded key medicinal chemistry insights and new opportunities.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain.

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Structure-Activity Relationships, Pharmacokinetics, and Pharmacodynamics of the Kir6.2/SUR1-Specific Channel Opener VU0071063.

Glucose-stimulated insulin secretion from pancreatic ß-cells is controlled by ATP-regulated potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The KATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of ß-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

Further exploration of an N-aryl phenoxyethoxy pyridinone-based series of mGlu3 NAMs: Challenging SAR, enantiospecific activity and in vivo efficacy.

This letter describes the further optimization of a series of mGlu3 NAMs based on an N-aryl phenoxyethoxy pyridinone core. A multidimensional optimization campaign, with focused matrix libraries, quickly established challenging SAR, enantiospecific activity, differences in assay read-outs (Ca2+ flux via a promiscuous G protein (Gα15) versus native coupling to GIRK channels), identified both full and partial mGlu3 NAMs and a new in vivo tool compound, VU6017587. This mGlu3 NAM showed efficacy in tail suspension, elevated zero maze and marble burying, suggesting selective inhibition of mGlu3 affords anxiolytic-like and antidepressant-like phenotypes in mice.

Surveying heterocycles as amide bioisosteres within a series of mGlu7 NAMs: Discovery of VU6019278.

This letter describes a diversity-oriented library approach to rapidly assess diverse heterocycles as bioisosteric replacements for a metabolically labile amide moiety within a series of mGlu7 negative allosteric modulators (NAMs). SAR rapidly honed in on either a 1,2,4- or 1,3,4-oxadizaole ring system as an effective bioisostere for the amide. Further optimization of the southern region of the mGlu7 NAM chemotype led to the discovery of VU6019278, a potent mGlu7 NAM (IC50 = 501 nM, 6.3% L-AP4 Min) with favorable plasma protein binding (rat fu = 0.10), low predicted hepatic clearance (rat CLhep = 27.7 mL/min/kg) and high CNS penetration (rat Kp = 4.9, Kp,uu = 0.65).

Discovery of 4-alkoxy-6-methylpicolinamide negative allosteric modulators of metabotropic glutamate receptor subtype 5.

This letter describes the further chemical optimization of VU0424238 (auglurant), an mGlu5 NAM clinical candidate that failed in non-human primate (NHP) 28 day toxicology due to accumulation of a species-specific aldehyde oxidase (AO) metabolite of the pyrimidine head group. Here, we excised the pyrimidine moiety, identified the minimum pharmacophore, and then developed a new series of saturated ether head groups that ablated any AO contribution to metabolism. Putative back-up compounds in this novel series provided increased sp3 character, uniform CYP450-mediated metabolism across species, good functional potency and high CNS penetration. Key to the optimization was a combination of matrix and iterative libraries that allowed rapid surveillance of multiple domains of the allosteric ligand.

The discovery of VU0652957 (VU2957, Valiglurax): SAR and DMPK challenges en route to an mGlu4 PAM development candidate.

This letter describes the first account of the chemical optimization (SAR and DMPK profiling) of a new series of mGlu4 positive allosteric modulators (PAMs), leading to the identification of VU0652957 (VU2957, Valiglurax), a compound profiled as a preclinical development candidate. Here, we detail the challenges faced in allosteric modulator programs (e.g., steep SAR, as well as subtle structural changes affecting overall physiochemical/DMPK properties and CNS penetration).

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Isoform selective PLD inhibition by novel, chiral 2,8-diazaspiro[4.5]decan-1-one derivatives.

This letter describes the on-going SAR efforts to develop PLD1, PLD2 and dual PLD1/2 inhibitors with improved physiochemical and disposition properties as well as securing intellectual property position. Previous PLD inhibitors, based on a triazaspiro[4.5]decanone core proved to be highly selective PLD2 inhibitors, but with low plasma free fraction (rat, human fu < 0.03), high predicted hepatic clearance (rat CLhep > 65 mL/min/kg) and very short half-lives in vivo (t1/2 < 0.15 h). Removal of a nitrogen atom from this core generated a 2,8-diazaspiro[4.5]decanone core, harboring a new chiral center, as well as increased sp3 character. This new core demonstrated enantioselective inhibition of the individual PLD isoforms, enhanced free fraction (rat, human fu < 0.13), engendered moderate predicted hepatic clearance (rat CLhep ∼ 43 mL/min/kg), improved half-lives in vivo (t1/2 > 3 h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors.

Discovery of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5.

Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu5 negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu5 NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu5 assay, and an exemplar analog 27 was >60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism.

The discovery of VU0486846: steep SAR from a series of M1 PAMs based on a novel benzomorpholine core.

This letter describes the chemical optimization of a new series of M1 positive allosteric modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 (7), devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes affecting CNS penetration and overall physiochemical and DMPK properties).

Discovery and characterization of N-(1,3-dialkyl-1H-indazol-6-yl)-1H-pyrazolo[4,3-b]pyridin-3-amine scaffold as mGlu4 positive allosteric modulators that mitigate CYP1A2 induction liability.

Previous reports from our laboratory disclosed the structure and activity of a novel 1H-pyrazolo[4,3-b]pyridine-3-amine scaffold (VU8506) which showed excellent potency, selectivity and in vivo efficacy in preclinical rodent models of Parkinson's disease. Unfortunately, this compound suffered from significant CYP1A2 induction as measured through upstream AhR activation (125-fold) and thus was precluded from further advancement in chronic studies. Herein, we report a new scaffold developed recently which was systematically studied in order to mitigate the CYP1A2 liabilities presented in the earlier scaffolds. We have identified a novel structure that maintains the potency and selectivity of other mGlu4 PAMs, leading to 9i (hmGlu4 EC50 = 43 nM; AhR activation = 2.3-fold).

Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets.

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.