Background Deficits in prefrontal cortex (PFC) inhibitory transmission are thought to underlie the cognitive symptoms of schizophrenia that remain untreated by current pharmacotherapies. The mGlu1 subtype of metabotropic glutamate receptor is a promising therapeutic target because its activation enhances inhibitory transmission in the PFC through excitation of somatostatin-expressing GABAergic interneurons (SST-INs). Methods We used a subchronic phencyclidine (PCP) N-methyl-D-aspartate receptor antagonist mouse model, mGlu1 positive allosteric modulators (PAMs), and mice with selective deletion of mGlu1 receptors from SST-INs to examine the cell type-specific role of mGlu1 signaling in cortical inhibition and cognition. Ex vivo whole-cell patch-clamp electrophysiology was used to assess inhibitory transmission in the PFC, and cognitive performance was evaluated using tests of working memory and novel object recognition. Results Subchronic PCP treatment impaired inhibitory synaptic transmission in the PFC, particularly onto layer V pyramidal neurons projecting to the nucleus accumbens. Pharmacological activation of mGlu1 restored inhibitory drive and reversed PCP-induced deficits in working memory and novel object recognition. Importantly, selective deletion of mGlu1 receptors from SST-INs abolished the ability of mGlu1 activation to restore cortical inhibition and cognitive function, demonstrating that these receptors are required for the therapeutic effects of mGlu1 PAMs. Conclusions These findings identify SST interneuron-expressed mGlu1 receptors as critical regulators of prefrontal cortical inhibition and cognition. Positive allosteric modulation of mGlu1 represents a promising therapeutic strategy for treating the cognitive deficits associated with schizophrenia by restoring inhibitory circuit function.

This work describes progress toward an M4 PAM preclinical candidate. The SAR to address potency, clearance, subtype selectivity, CNS exposure, and P-gp efflux are detailed within. A novel 1-(7,8-dimethyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-ol scaffold was identified, and optimization provided a highly potent analog VU6025733 (hM4 EC50 = 23 nM; rM4 EC50 = 55 nM). Further characterization revealed a highly selective compound across muscarinic acetylcholine receptor subtypes with exceptional DMPK properties (in vivo rat CLp = 5.9 mL/min/kg; t1/2 = 4.8 h; CYP1A2 & CYP2C9 IC50s > 30 μM, CYP2D6 IC50 > 9 μM; CYP3A4 IC50 > 25 μM). Moreover, VU6025733 demonstrated robust in vivo efficacy in a rat amphetamine-induced hyperlocomotion model in a dose-dependent manner. However, hepatotoxicity risk precluded further development.

We report the discovery of an additional selective metabotropic glutamate receptor subtype 3 (mGlu(3)) positive allosteric modulator (PAM) tool compound, VU6052959/BI'7927. A high-throughput screening (HTS) campaign identified a selective mGlu(3) PAM VU6046180/BI'3690 (7) containing a benzoxazine core (an orthogonal chemotype to previously reported thiophene tool compound VU6053371/BI'8809 (6)). Lead optimization efforts focused on improving PK parameters led to the identification of VU6052959/BI'7927 (8). Orthogonal PAM 8 demonstrated efficacy in a rat novel object recognition task with conservation of our previously determined PK/PD relationship. However, an Ames positive metabolite halted further progression of 8.

Herein, we report the structure-activity relationship (SAR) to develop novel mGlu(5) negative allosteric modulator (NAM) scaffolds devoid of the aryl/heterobiaryl acetylene moiety found in many historic mGlu(5) NAMs, which has been linked to metabolic liabilities and hepatotoxicity. This endeavor utilized a scaffold-hopping strategy from the predecessor compound VU6031545, in which we replace an ether-linked tetrahydrofuran with various carbon-linked heteroaryl motifs to generate highly potent and selective mGlu(5) NAMs. One such compound, VU6035386, displayed low nanomolar potency against human mGlu(5) and was highly brain penetrant. Moreover, VU6035386 showed a vast improvement in predicted human hepatic clearance versus predecessor compound VU6031545.

Abstract ID 95290Poster Board 322Aim: Exposure to psychostimulants, such as cocaine, during adolescence produces persistent changes in the prefrontal cortex (PFC) which parallel cognitive deficits seen in adulthood. Further, adolescent exposure to psychostimulants impairs inhibitory transmission in the PFC in adulthood, suggesting that enhancing PFC inhibitory transmission may be a promising strategy to reverse drug-induced cognitive deficits. Activation of the mGlu1 subtype of metabotropic glutamate receptor increases inhibitory transmission in the PFC and working memory by selective excitation of somatostatin-expressing GABA interneurons (SST-INs). Therefore, we hypothesize that repeated exposure to cocaine during a critical developmental period in adolescence disrupts PFC inhibition via SST-INs and drives working memory impairments in adulthood which can be mitigated by activation of mGlu1.Methods: Male and female SST- and PV-Ai9 tdTomato mice were injected once daily with cocaine (20 mg/kg, i.p.) for 7 days (postnatal day 35-42). Whole-cell patch-clamp electrophysiological recordings from interneuron populations within the PFC were conducted between 10-12 weeks of age. Additionally, touchscreen-based automated cognition testing was used to determine working memory performance in adult mice. Novel mGlu1 positive allosteric modulators (PAMs) were leveraged in behavioral and electrophysiology studies to determine their procognitive efficacy and mechanism of action within the PFC.Results: We found that repeated administration of cocaine during a critical adolescent period impaired PFC SST-IN, but not parvalbumin-expressing interneuron (PV-IN), firing compared to saline-treated mice. Adolescent cocaine exposure significantly decreased the frequency of spontaneous excitatory postsynaptic currents onto SST-INs but not PV-INs. These findings were paralleled by adolescent cocaine-induced impairments in spatial working memory in adulthood. Importantly, these physiological and behavioral effects of adolescent cocaine exposure were reversed by selective mGlu1 activation. Lastly, repeated amphetamine administration during the same adolescent critical period did not result in impaired SST-IN function or spatial working memory in adulthood.Conclusions: These studies show that: 1) cocaine, but not amphetamine, exposure during an adolescent critical period induces persistent and selective deficits in PFC SST-IN function and cognition in adulthood and 2) selective activation of mGlu1 with PAMs represents a novel strategy for reversing cocaine-induced cognitive impairments.This work is supported by MH119673, NS031373, MH062646, MH073676, MH065215

Herein, we report the discovery and development of the first-in-class (FIC), selective, and centrally active metabotropic glutamate receptor subtype 3 (mGlu3) positive allosteric modulator (PAM), VU6053371/BI03738809. A high-throughput screening campaign identified a potent and selective mGlu3 PAM VU6048261/DI013166572 based on a tetra-substituted thiophene core but with poor DMPK properties. Chemical lead optimization efforts managed to dramatically improve protein binding and in vivo rat PK to afford VU6053371/BI03738809. With an FIC in vivo tool compound, VU6053371/BI03738809 demonstrated robust efficacy in rat novel object recognition (NOR) (minimum effective dose (MED) = 3 mg/kg PO) and a clear pharmacokinetic/pharmacodynamic (PK/PD) relationship (PD efficacy observed when free brain concentrations were at, or above, the rat mGlu3 EC50). Thus, selective activation of mGlu3 represents a novel mechanism to address the cognitive impairment associated with schizophrenia (CIAS) and other neurodegenerative diseases. Moreover, the discovery of VU6053371/BI03738809 completes the group II mGlu receptor toolkit of in vivo PAM and NAM probes for both mGlu2 and mGlu3.

Herein, we report the structure-activity relationship to develop novel tricyclic M4 positive allosteric modulator scaffolds with improved pharmacological properties. This endeavor involved modifying a 5-amino-3,4-dimethylthieno[2,3-c]pyridazine-6-carboxamide core via a "tie-back" strategy to discover a novel tricyclic 3,4-dimethylpyrimido[4',5':4,5]thieno[2,3-c]pyridazine core. From this exercise, VU6008055/AF98943 was identified as a preclinical candidate, which displays low nanomolar potency against both human and rat M4. Moreover, VU6008055 is highly brain penetrant, has an overall superior pharmacological and DMPK profile to previously reported M4 PAMs, and demonstrates efficacy in preclinical models of antipsychotic-like activity.

This Letter describes the discovery of novel mGlu5 NAMs VU6031545 and VU6024945. Starting from previously reported picolinamide compounds, a structure-activity relationship study of various core isosteres was conducted, leading to the identification of thieno[3,2-b]pyridine-5-carboxamide and 2,3-difluorobenzamide as competent core replacements. These compounds are highly potent as well as brain penetrant with an IVIVC agreement and improved oral bioavailability in rats.

Standard treatment for Parkinson's disease (PD) is dopamine replacement therapy with L-DOPA. However, chronic treatment often results in abnormal involuntary movements called L-DOPA-induced dyskinesia (LID). Prior evidence indicates that heightened striatal cholinergic tone may contribute to LID. Restoring cholinergic inhibition by targeting the inhibitory M4 muscarinic acetylcholine (ACh) receptor (M4) reduces LID in preclinical models. Although intrinsic striatal sources of ACh have been considered for their role in LID, extrinsic sources of ACh such as the pedunculopontine nucleus (PPN) have not been well investigated for their role in LID. Therefore, the current study employed hemiparkinsonian Long-Evans rats with a PPN-targeted cannula ipsilateral to 6-OHDA lesion. We examined the effect of local unilateral PPN infusion of M4 PAM VU0467154 on LID, motor performance, and c-fos expression within the PPN. It was expected that PPN infusion of VU0467154 would reduce LID, reduce L-DOPA's motor benefit, and globally reduce c-fos expression in the PPN. Contrary to our expectations, PPN infusion of M4 PAM did not significantly affect LID severity. Furthermore, M4 PAM did not alter L-DOPA-mediated motor improvement, and decreased c-fos expression specifically in PPN cholinergic neurons. These results suggest that local PPN ACh dynamics differ from those of the striatum. In the context of prior work, our results suggest that PPN cholinergic modulation or global PPN modulation may be a promising strategy for altering freezing of gait without decreasing motor benefit of L-DOPA and without increasing LID severity.

While traditional antipsychotic drugs provide symptomatic relief for positive symptoms in patients with schizophrenia, many patients are refractory to traditional antipsychotics. Furthermore, these medicines are ineffective in treating negative and cognitive symptoms and have serious adverse effects that limit their utility. Traditional antipsychotics act as antagonists or partial agonists of D2 dopamine receptors, an action that is key to their antipsychotic efficacy and adverse effects. Muscarinic acetylcholine receptor (mAChR) agonists have now emerged as the first truly novel treatments for schizophrenia. This represents a fundamental breakthrough that provides a new treatment option to reduce psychotic symptoms and possibly improve negative and cognitive symptoms in patients with schizophrenia. Mechanistic studies are shedding light on the specific mAChR subtypes involved and the specific neural circuits where mAChR agonists may exert these effects. These studies may pave the way for a new generation of drugs to treat schizophrenia and other neuropsychiatric disorders.

The development of cholinergic neurotransmitter based cognitive enhancers for Alzheimer’s disease and other neuropsychiatric disorders have focused recently on allosteric modulation of specific muscarinic acetylcholine receptor (mAChR) subtypes to reduce dose-limiting side-effects that have been the hallmark of earlier orthosteric mAChR agonists. VU0467319 (VU319) is an investigational positive allosteric modulator of the M1 mAChR. A Phase 1 first-in-human study was conducted assessing safety and brain activity utilizing cognitive tasks and event-related potentials (ERPs) in single-ascending dose and food effect studies. VU319 was given orally to 52 healthy volunteers aged 18–55 years. The single ascending dose study tested 40 participants in five dose escalating cohorts (60, 120, 240, 400, 600 mg; 6 VU319/2 placebo per dose). The food effect study involved 12 participants, 10 VU319 (120 mg)/2 placebo. Exploratory cognitive and electrophysiological tasks were examined pre-dosing and at 5 h post-dose. Tolerability was good with no observed dose limiting side effects throughout the full dose range tested. In the single ascending dose study, there were 47 TEAEs reported across the 5 cohorts, 14 in the placebo group and 33 across the 5 active dose cohorts. In the food effect study, there were 20 TEAEs reported, 6 in the placebo group and 14 in the fed and fasted conditions. Drug exposure increased with dose in a less than dose-proportional manner with a half-life ranging from 30 to 55 h. Peak concentration was observed between 5 and 9.5 h across the dosage groups. Absorption was increased with food. Exploratory cognitive/ERP testing showed evidence for drug-induced CNS activity on higher doses of VU319 compared to placebo. Single dose VU319 across five ascending cohorts appeared to have a favorable safety profile and a PK profile consistent with once daily dosing. Target engagement results suggest stimulation of the cholinergic system functioning in healthy adults following a single dose of VU319. These results provide a strong foundation for further studies of positive allosteric modulators of muscarinic M1 receptors for potential cognitive or behavioral benefits.

The M1 receptor has long been investigated as a promising CNS drug target, yet further research is essential to fully elucidate compound's Pharmacodynamic (PD) as well as Toxicokinetic (TK) effects. In this context, the development of structurally diverse and high-profile M1 PAM tool compounds remains highly valuable, as existing advanced tools exhibit notable structural similarity. One approach that can be considered during scaffold hopping exercise and can improve drug-like properties is to introduce additional sp3 carbon atoms and increase Fsp3 values; the fraction of sp3 hybridized carbons. Determining the correct location to incorporate sp3 carbon atoms can be challenging, but once the right position is identified, it often leads to novel optimization opportunities. Reported herein is the discovery of a novel sp3-rich M1 positive allosteric modulator series utilizing a N-cyclopentyl pyrazole core. Also, an iterative library synthesis approach provided an enhanced understanding of the minimum pharmacophore. Several compounds within the series showed favorable on-target potencies and DMPK properties. In conclusion, the reported sp3-rich N-cyclopentyl pyrazole-based M1 PAM scaffold offers a promising structure-activity relationship starting point to discover structurally distinct M1 PAM chemotypes.

Recently, we disclosed VU0467319, an M1 positive allosteric modulator (PAM) clinical candidate that had successfully completed a phase I single ascending dose clinical trial. Pharmacokinetic assessment revealed that, in humans upon increasing dose, a circulating, inactive metabolite constituted a major portion of the total drug-related area under the curve (AUC). One approach the team employed to reduce inactive metabolite formation in the back-up program was the kinetic isotope effect, replacing the metabolically labile C-H bonds with shorter, more stable C-D bonds. The C-D dipole afforded VU6045422, a more potent M1 PAM (human EC50 = 192 nM, 80% ACh Max) than its proteocongener VU0467319 (human EC50 = 492 nM, 71% ACh Max), and retained the desired profile of minimal M1 agonism. Overall, the profile of VU6045422 supported advancement, as did greater in vitro metabolic stability in both microsomes and hepatocytes than did VU0467319. In both rat and dog in vivo, low doses proved to mirror the in vitro profile; however, at higher doses in 14-day exploratory toxicology studies, the amount of the same undesired metabolite derived from VU6045422 was equivalent to that produced from VU0467319. This unexpected IVIVC result, coupled with less than dose-proportional increases in exposure and no improvement in solubility, led to discontinuation of VU0467319/VU6045422 development.

Mutations or deletions in the SHANK3 gene have been identified in up to 1% of autism spectrum disorder cases and are considered the primary cause of neuropsychiatric symptoms in Phelan McDermid syndrome (PMS). While synaptic dysfunctions have been extensively documented in the absence of Shank3, other mechanisms through which Shank3 may regulate neuronal functions remain unclear. In this study, we report that the ribosomal protein Rpl3 and overall protein synthesis are downregulated in the cortex and striatum of Shank3 knockout (KO) mice and in neurons differentiated from human-induced pluripotent stem cells (hiPSCs) derived from a PMS patient. Moreover, restoring Rpl3 expression in the striatum of Shank3 KO mice was sufficient to rescue protein synthesis and mitigate excessive grooming, suggesting that the behavioral alterations observed in Shank3 KO mice might be, at least in part, caused by Rpl3 downregulation and consequent impaired protein synthesis. Furthermore, we demonstrated that chronic inhibition of mGlu5 is sufficient to reduce Rpl3 expression, which in turn impairs global protein synthesis. Consequently, chronic treatment with VU0409551, a potent and selective mGlu5 positive allosteric modulator, rescues Rpl3 expression and the resulting reduction in protein synthesis, leading to long-lasting improvements in behavioral deficits in Shank3 KO mice Altogether, we propose a new role for Shank3 in modulating Rpl3 protein expression, ribosomal function, and protein synthesis by downregulating mGlu5 receptor activity.

Herein, we report the further chemical optimization of the metabotropic glutamate receptor subtype 1 (mGlu(1)) positive allosteric modulator (PAM) VU6024578/BI02982816 and the discovery of VU6033685/BI1752. PAM VU6033685/BI1752 was developed through an iterative process wherein, after the furanyl moiety (a potential toxicophore) was replaced by an N-linked pyrazole, a diversity screen identified a quinoline core, which was further truncated to a pyridine scaffold. PAM VU6033685/BI1752 proved to be a potent and selective mGlu(1) PAM with efficacy in both amphetamine-induced hyperlocomotion (AHL) and novel object recognition (NOR) with a clear pharmacokinetic-pharmacodynamic (PK/PD) relationship. VU6024578/BI02982816 was efficacious and well tolerated in rats but not dogs, whereas VU6033685/BI1752 elicited adverse events (AEs) in both rats and dogs. These AEs, noted in two distinct mGlu(1) PAM chemotypes, cast a shadow on an otherwise promising molecular target to address multiple symptom clusters in schizophrenic patients.

We recently disclosed VU0467319, a muscarinic acetylcholine receptor subtype 1 (M1) Positive Allosteric Modulator (PAM) clinical candidate that had successfully completed a Phase I Single Ascending Dose (SAD) clinical trial, but the identification of an inactive metabolite constituting a major portion of the total plasma AUC detracted from the molecules' pharmacokinetic profile and contributed to clinical development discontinuation. Attempts to block metabolism with the incorporation of deuterium atoms proved successful in vitro and in vivo at low exposures; however, in high-dose nonclinical toxicology studies, the degree of oxidative metabolism and metabolite accumulation was comparable to that of the proteo-congener. Here, we describe a second-generation back-up effort based on the VU0467319 scaffold to discover VU6052254. Strategic placement of a tertiary hydroxyl moiety afforded VU6052254, a potent M1 PAM (EC50 = 59 nM, 79% ACh max), with high CNS exposure (rat Kp = 1.07; Kp,uu = 1.27; P-gp ER = 1.97, Papp = 23 × 10-6 cm/s), reduced metabolism across species, excellent pharmacodynamic responses (MED in rat NOR = 1 mg/kg PO; MED in rat CFC = 0.3 mg/kg PO), excellent multispecies PK (Clps < 10 mL/min/kg, %F > 65), and favorable human PK and dose projections. Based on these beneficial attributes, VU6052254 was nominated for further nonclinical development. However, possible CYP450 induction liability as well as uncertain projected margins for human efficacy at those systemic concentrations where dose/exposure-related clinical and anatomic pathology kidney findings were observed in a 14-day exploratory toxicity study in male rats, precluded further development.