Discovery and first-time disclosure of CVN766, an exquisitely selective orexin 1 receptor antagonist.

Modulators of orexin receptors are being developed for neurological illnesses such as sleep disorders, addictive behaviours and other psychiatric diseases. We herein describe the discovery of CVN766, a potent orexin 1 receptor antagonist that has greater than 1000-fold selectivity for the orexin 1 receptor over the orexin 2 receptor and demonstrates low off target hits in a diversity screen. In agreement with its in vitro ADME data, CVN766 demonstrated moderate in vivo clearance in rodents and displayed good brain permeability and target occupancy. This drug candidate is currently being investigated in clinical trials for schizophrenia and related psychiatric conditions.

Luvadaxistat: A Novel Potent and Selective D-Amino Acid Oxidase Inhibitor Improves Cognitive and Social Deficits in Rodent Models for Schizophrenia.

N-methyl-D-aspartate (NMDA) receptor hypofunctionality is a well-studied hypothesis for schizophrenia pathophysiology, and daily dosing of the NMDA receptor co-agonist, D-serine, in clinical trials has shown positive effects in patients. Therefore, inhibition of D-amino acid oxidase (DAAO) has the potential to be a new therapeutic approach for the treatment of schizophrenia. TAK-831 (luvadaxistat), a novel, highly potent inhibitor of DAAO, significantly increases D-serine levels in the rodent brain, plasma, and cerebrospinal fluid. This study shows luvadaxistat to be efficacious in animal tests of cognition and in a translational animal model for cognitive impairment in schizophrenia. This is demonstrated when luvadaxistat is dosed alone and in conjunction with a typical antipsychotic. When dosed chronically, there is a suggestion of change in synaptic plasticity as seen by a leftward shift in the maximum efficacious dose in several studies. This is suggestive of enhanced activation of NMDA receptors in the brain and confirmed by modulation of long-term potentiation after chronic dosing. DAAO is highly expressed in the cerebellum, an area of increasing interest for schizophrenia, and luvadaxistat was shown to be efficacious in a cerebellar-dependent associative learning task. While luvadaxistat ameliorated the deficit seen in sociability in two different negative symptom tests of social interaction, it failed to show an effect in endpoints of negative symptoms in clinical trials. These results suggest that luvadaxistat potentially could be used to improve cognitive impairment in patients with schizophrenia, which is not well addressed with current antipsychotic medications.

The oDGal Mouse: A Novel, Physiologically Relevant Rodent Model of Sporadic Alzheimer's Disease.

Sporadic Alzheimer's disease (sAD) represents a serious and growing worldwide economic and healthcare burden. Almost 95% of current AD patients are associated with sAD as opposed to patients presenting with well-characterized genetic mutations that lead to AD predisposition, i.e., familial AD (fAD). Presently, the use of transgenic (Tg) animals overexpressing human versions of these causative fAD genes represents the dominant research model for AD therapeutic development. As significant differences in etiology exist between sAD and fAD, it is perhaps more appropriate to develop novel, more sAD-reminiscent experimental models that would expedite the discovery of effective therapies for the majority of AD patients. Here we present the oDGal mouse model, a novel model of sAD that displays a range of AD-like pathologies as well as multiple cognitive deficits reminiscent of AD symptomology. Hippocampal cognitive impairment and pathology were delayed with N-acetyl-cysteine (NaC) treatment, which strongly suggests that reactive oxygen species (ROS) are the drivers of downstream pathologies such as elevated amyloid beta and hyperphosphorylated tau. These features demonstrate a desired pathophenotype that distinguishes our model from current transgenic rodent AD models. A preclinical model that presents a phenotype of non-genetic AD-like pathologies and cognitive deficits would benefit the sAD field, particularly when translating therapeutics from the preclinical to the clinical phase.

A Phase I, First-in-Human, Healthy Volunteer Study to Investigate the Safety, Tolerability, and Pharmacokinetics of CVN424, a Novel G Protein-Coupled Receptor 6 Inverse Agonist for Parkinson's Disease.

CVN424 is a novel small molecule and first-in-class candidate therapeutic to selectively modulate GPR6, an orphan G-protein coupled receptor. Expression of GPR6 is largely confined to the subset of striatal projection neurons that give rise to the indirect (striatopallidal) pathway, important in the control of movement. CVN424 improves motor function in preclinical animal models of Parkinson's disease. Here, we report results of a phase 1, first-in-human study investigating the safety, tolerability, and pharmacokinetics of CVN424 in healthy volunteers. The study (NCT03657030) was randomized, double-blind, and placebo controlled. CVN424 was orally administered in ascending doses to successive cohorts as inpatients in a clinical research unit. Single doses ranged from 1 mg to 225 mg, and repeated (7 day) daily doses were 25, 75, or 150 mg. CVN424 peak plasma concentrations were reached within 2 h post-dose in the fasted state and increased with increasing dose. Dosing after a standardized high-fat meal reduced and delayed the peak plasma concentration, but total plasma exposure was similar. Mean terminal half-life ranged from 30 to 41 h. CVN424 was generally well tolerated: no serious or severe adverse effects were observed, and there were no clinically significant changes in vital signs or laboratory parameters. We conclude that CVN424, a nondopaminergic compound that modulates a novel therapeutic target, was safe and well tolerated. A phase 2 study in patients with Parkinson's disease is underway. SIGNIFICANCE STATEMENT: This is the first-in-human clinical study of a first-in-class candidate therapeutic. CVN424 modulates a novel drug target, GPR6, which is selectively expressed in a pathway in the brain that has been implicated in the motor dysfunction of patients with Parkinson's disease. This study paves the way for investigating this novel mechanism of action in patients with Parkinson's disease.

Synthesis and SAR of novel GPR39 agonists and positive allosteric modulators.

We report a significant decrease in transcription of the G protein-coupled receptor GPR39 in striatal neurons of Parkinson's disease patients compared to healthy controls, suggesting that a positive modulator of GPR39 may beneficially impact neuroprotection. To test this notion, we developed various structurally diverse tool molecules. While we elaborated on previously reported starting points, we also performed an in silico screen which led to completely novel pharmacophores. In vitro studies indicated that GPR39 agonism does not have a profound effect on neuroprotection.

Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

Assessment of the Target Engagement and D-Serine Biomarker Profiles of the D-Amino Acid Oxidase Inhibitors Sodium Benzoate and PGM030756.

Irregular N-methyl-D-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of schizophrenia. Although direct agonism of the NMDAR has not yielded promising therapeutics, advances have been made by modulating the NMDAR co-agonist site which is activated by glycine and D-serine. One approach to activate the co-agonist site is to increase synaptic D-serine levels through inhibition of D-amino acid oxidase (DAO), the major catabolic clearance pathway for this and other D-amino acids. A number of DAO inhibitors have been developed but most have not entered clinical trials. One exception to this is sodium benzoate which has demonstrated efficacy in small trials of schizophrenia and Alzheimer's disease. Herein we provide data on the effect of sodium benzoate and an optimised Takeda compound, PGM030756 on ex vivo DAO enzyme occupancy and cerebellar D-serine levels in mice. Both compounds achieve high levels of enzyme occupancy; although lower doses of PGM030756 (1, 3 and 10 mg/kg) were required to achieve this compared to sodium benzoate (300, 1000 mg/kg). Cerebellar D-serine levels were increased by both agents with a delay of approximately 6 h after dosing before the peak effect was achieved. Our data and methods may be useful in understanding the effects of sodium benzoate that have been seen in clinical trials of schizophrenia and Alzheimer's disease and to support the potential clinical assessment of other DAO inhibitors, such as PGM030756, which demonstrate good enzyme occupancy and D-serine increases following administration of low oral doses.

Insulin-like peptide 5 is an orexigenic gastrointestinal hormone.

The gut endocrine system is emerging as a central player in the control of appetite and glucose homeostasis, and as a rich source of peptides with therapeutic potential in the field of diabetes and obesity. In this study we have explored the physiology of insulin-like peptide 5 (Insl5), which we identified as a product of colonic enteroendocrine L-cells, better known for their secretion of glucagon-like peptide-1 and peptideYY. i.p. Insl5 increased food intake in wild-type mice but not mice lacking the cognate receptor Rxfp4. Plasma Insl5 levels were elevated by fasting or prolonged calorie restriction, and declined with feeding. We conclude that Insl5 is an orexigenic hormone released from colonic L-cells, which promotes appetite during conditions of energy deprivation.

Discovery of CVN293, a Brain Permeable KCNK13 (THIK-1) Inhibitor Suitable for Clinical Assessment.

The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1ß from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.

Characterization and functional study of a cluster of four highly conserved orphan adhesion-GPCR in mouse.

BACKGROUND: Adhesion G protein-coupled receptors (aGPCR) constitute a structurally and functionally diverse class of seven-transmembrane receptor proteins. Although for some of the members important roles in immunology, neurology, as well as developmental biology have been suggested, most receptors have been poorly characterized. RESULTS: We have studied evolution, expression, and function of an entire receptor group containing four uncharacterized aGPCR: Gpr110, Gpr111, Gpr115, and Gpr116. We show that the genomic loci of these four receptors are clustered tightly together in mouse and human genomes and that this cluster likely derives from a single common ancestor gene. Using transcriptional profiling on wild-type and knockout/LacZ reporter knockin mice strains, we have obtained detailed expression maps that show ubiquitous expression of Gpr116, co-expression of Gpr111 and Gpr115 in developing skin, and expression of Gpr110 in adult kidney. Loss of Gpr110, Gpr111, or Gpr115 function did not result in detectable defects, indicating that genes of this aGPCR group might function redundantly. CONCLUSIONS: The aGPCR cluster Gpr110, Gpr111, Gpr115, and Gpr116 developed from one common ancestor in vertebrates. Expression suggests a role in epithelia, and one can speculate about a possible redundant function of GPR111 and GPR115.

Discovery of CVN417, a Novel Brain-Penetrant α6-Containing Nicotinic Receptor Antagonist for the Modulation of Motor Dysfunction.

Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.

Discovery and Characterization of Novel CNS-Penetrant GPR55 Agonists.

From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.

Discovery of CVN636: A Highly Potent, Selective, and CNS Penetrant mGluR7 Allosteric Agonist.

The low affinity metabotropic glutamate receptor mGluR7 has been implicated in numerous CNS disorders; however, a paucity of potent and selective activators has hampered full delineation of the functional role and therapeutic potential of this receptor. In this work, we present the identification, optimization, and characterization of highly potent, novel mGluR7 agonists. Of particular interest is the chromane CVN636, a potent (EC50 7 nM) allosteric agonist which demonstrates exquisite selectivity for mGluR7 compared to not only other mGluRs, but also a broad range of targets. CVN636 demonstrated CNS penetrance and efficacy in an in vivo rodent model of alcohol use disorder. CVN636 thus has potential to progress as a drug candidate in CNS disorders involving mGluR7 and glutamatergic dysfunction.

Characterisation of C101248: A novel selective THIK-1 channel inhibitor for the modulation of microglial NLRP3-inflammasome.

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1ß, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1ß from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.

Mismatch negativity as an index of target engagement for excitation/inhibition-based treatment development: a double-blind, placebo-controlled, randomized, single-dose cross-over study of the serotonin type-3 receptor antagonist CVN058.

Serotonin type-3 receptor (5-HT3R) antagonists show potential as a treatment for cognitive deficits in schizophrenia. CVN058, a brain-penetrant, potent and selective 5-HT3R antagonist, shows efficacy in rodent models of cognition and was well-tolerated in Phase-1 studies. We evaluated the target engagement of CVN058 using mismatch negativity (MMN) in a randomized, double-blind, placebo-controlled, cross-over study. Subjects were stable outpatients with schizophrenia or schizoaffective disorder treated with antipsychotics. Subjects were not permitted to use other 5-HT3R modulators or serotonin reuptake inhibitors. Each subject received a high (150 mg) and low (15 mg or 75 mg) oral dose of CVN058 and placebo in a randomized order across 3 single-day treatment visits separated by at least 1 week. The primary pre-registered outcome was amplitude of duration MMN. Amplitude of other MMN deviants (frequency, intensity, frequency modulation, and location), P50, P300 and auditory steady-state response (ASSR) were exploratory endpoints. 19 of 22 randomized subjects (86.4%) completed the study. Baseline PANSS scores indicated moderate impairment. CVN058 150 mg led to significant improvement vs. placebo on the primary outcome of duration MMN (p = 0.02, Cohen's d = 0.48). A significant treatment effect was also seen in a combined analysis across all MMN deviants (p < 0.001, d = 0.57). Effects on location MMN were independently significant (p < 0.007, d = 0.46). No other significant effects were seen for other deviants, doses or EEG measures. There were no clinically significant treatment related adverse effects. These results show MMN to be a sensitive target engagement biomarker for 5-HT3R, and support the potential utility of CVN058 in correcting the excitatory/inhibitory imbalance in schizophrenia.

Pharmacological targeting of glutamatergic neurons within the brainstem for weight reduction.

Food intake and body weight are tightly regulated by neurons within specific brain regions, including the brainstem, where acute activation of dorsal raphe nucleus (DRN) glutamatergic neurons expressing the glutamate transporter Vglut3 (DRNVglut3) drive a robust suppression of food intake and enhance locomotion. Activating Vglut3 neurons in DRN suppresses food intake and increases locomotion, suggesting that modulating the activity of these neurons might alter body weight. Here, we show that DRNVglut3 neurons project to the lateral hypothalamus (LHA), a canonical feeding center that also reduces food intake. Moreover, chronic DRNVglut3 activation reduces weight in both leptin-deficient (ob/ob) and leptin-resistant diet-induced obese (DIO) male mice. Molecular profiling revealed that the orexin 1 receptor (Hcrtr1) is highly enriched in DRN Vglut3 neurons, with limited expression elsewhere in the brain. Finally, an orally bioavailable, highly selective Hcrtr1 antagonist (CVN45502) significantly reduces feeding and body weight in DIO. Hcrtr1 is also co-expressed with Vglut3 in the human DRN, suggesting that there might be a similar effect in human. These results identify a potential therapy for obesity by targeting DRNVglut3 neurons while also establishing a general strategy for developing drugs for central nervous system disorders.

Loss of MLL5 results in pleiotropic hematopoietic defects, reduced neutrophil immune function, and extreme sensitivity to DNA demethylation.

MLL5 is a divergent member of the Drosophila Trithorax-related (SET) domain and plant homeodomain (PHD) domain-containing chromatin regulators that are involved in the regulation of transcriptional "memory" during differentiation. Human MLL5 is located on chromosome 7q22, which frequently is deleted in myeloid leukemias, suggesting a possible role in hemopoiesis. To address this question, we generated a loss-of-function allele (Mll5(tm1Apa)) in the murine Mll5 locus. Unlike other Mll genes, Mll5(tm1Apa) homozygous mice are viable but display defects in immunity and hematopoiesis. First, Mll5(tm1Apa) homozygous mice show increased susceptibility to spontaneous eye infections, associated with a cell-autonomous impairment of neutrophil function. Second, Mll5(tm1Apa/tm1Apa) mice exhibit a mild impairment of erythropoiesis. Third, Mll5(tm1Apa/tm1Apa) hematopoietic stem cells (HSCs) have impaired competitive repopulating capacity both under normal conditions and when subjected to self-renewal stimulation by NUP98-HOXA10. Fourth, Mll5(tm1Apa) homozygous HSCs show a dramatic sensitivity to DNA demethylation-induced differentiation (5-azadeoxycytidine). Taken together, our data show that MLL5 is involved in terminal myeloid differentiation and the regulation of HSC self-renewal by a mechanism that involves DNA methylation. These data warrant investigation of MLL5 expression levels as a predictive marker of demethylating-agent response in patients with myelodysplastic syndromes and leukemias and identify MLL5 as a key regulator of normal hematopoiesis.

First-Time Disclosure of CVN424, a Potent and Selective GPR6 Inverse Agonist for the Treatment of Parkinson's Disease: Discovery, Pharmacological Validation, and Identification of a Clinical Candidate.

Parkinson's disease (PD) is a chronic and progressive movement disorder with the urgent unmet need for efficient symptomatic therapies with fewer side effects. GPR6 is an orphan G-protein coupled receptor (GPCR) with highly restricted expression in dopamine receptor D2-type medium spiny neurons (MSNs) of the indirect pathway, a striatal brain circuit which shows aberrant hyperactivity in PD patients. Potent and selective GPR6 inverse agonists (IAG) were developed starting from a low-potency screening hit (EC50 = 43 µM). Herein, we describe the multiple parameter optimization that led to the discovery of multiple nanomolar potent and selective GPR6 IAG, including our clinical compound CVN424. GPR6 IAG reversed haloperidol-induced catalepsy in rats and restored mobility in the bilateral 6-OHDA-lesioned rat PD model demonstrating that inhibition of GPR6 activity in vivo normalizes activity in basal ganglia circuitry and motor behavior. CVN424 is currently in clinical development to treat motor symptoms in Parkinson's disease.

SIPA1L2 controls trafficking and local signaling of TrkB-containing amphisomes at presynaptic terminals.

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.