Discovery of MK-8189, a Highly Potent and Selective PDE10A Inhibitor for the Treatment of Schizophrenia.

PDE10A is an important regulator of striatal signaling that, when inhibited, can normalize dysfunctional activity. Given the involvement of dysfunctional striatal activity with schizophrenia, PDE10A inhibition represents a potentially novel means for its treatment. With the goal of developing PDE10A inhibitors, early optimization of a fragment hit through rational design led to a series of potent pyrimidine PDE10A inhibitors that required further improvements in physicochemical properties, off-target activities, and pharmacokinetics. Herein we describe the discovery of an isomeric pyrimidine series that addresses the liabilities seen with earlier compounds and resulted in the invention of compound 18 (MK-8189), which is currently in Phase 2b clinical development for the treatment of schizophrenia.

Stem Cell-based therapies for COVID-19-related acute respiratory distress syndrome.

As the number of confirmed cases and resulting death toll of the COVID-19 pandemic continue to increase around the globe - especially with the emergence of new mutations of the SARS-CoV-2 virus in addition to the known alpha, beta, gamma, delta and omicron variants - tremendous efforts continue to be dedicated to the development of interventive therapeutics to mitigate infective symptoms or post-viral sequelae in individuals for which vaccines are not accessible, viable or effective in the prevention of illness. Many of these investigations aim to target the associated acute respiratory distress syndrome, or ARDS, which induces damage to lung epithelia and other physiologic systems and is associated with progression in severe cases. Recently, stem cell-based therapies have demonstrated preliminary efficacy against ARDS based on a number of preclinical and preliminary human safety studies, and based on promising outcomes are now being evaluated in phase II clinical trials for ARDS. A number of candidate stem cell therapies have been found to exhibit low immunogenicity, coupled with inherent tropism to injury sites. In recent studies, these have demonstrated the ability to modulate suppression of pro-inflammatory cytokine signals such as those characterizing COVID-19-associated ARDS. Present translational studies are aiming to optimize the safety, efficacy and delivery to fully validate stem cell-based strategies targeting COVID-19 associated ARDS for viable clinical application.

Redefining the Histone Deacetylase Inhibitor Pharmacophore: High Potency with No Zinc Cofactor Interaction.

A novel series of histone deacetylase (HDAC) inhibitors lacking a zinc-binding moiety has been developed and described herein. HDAC isozyme profiling and kinetic studies indicate that these inhibitors display a selectivity preference for HDACs 1, 2, 3, 10, and 11 via a rapid equilibrium mechanism, and crystal structures with HDAC2 confirm that these inhibitors do not interact with the catalytic zinc. The compounds are nonmutagenic and devoid of electrophilic and mutagenic structural elements and exhibit off-target profiles that are promising for further optimization. The efficacy of this new class in biochemical and cell-based assays is comparable to the marketed HDAC inhibitors belinostat and vorinostat. These results demonstrate that the long-standing pharmacophore model of HDAC inhibitors requiring a metal binding motif should be revised and offers a distinct class of HDAC inhibitors.

Discovery of an Anion-Dependent Farnesyltransferase Inhibitor from a Phenotypic Screen.

By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.

In Quest of Pathognomonic/Endophenotypic Markers of Attention Deficit Hyperactivity Disorder (ADHD): Potential of EEG-Based Frequency Analysis and ERPs to Better Detect, Prevent and Manage ADHD.

Attention deficit hyperactivity disorder (ADHD) is a chronic heritable developmental delay psychiatric disorder requiring chronic management, characterized by inattention, hyperactivity, hyperkinectivity and impulsivity. Subjective clinical evaluation still remains crucial in its diagnosis. Discussed are two key aspects in the "characterizing ADHD" and on the quest for objective "pathognomonic/endophenotypic diagnostic markers of ADHD". The first aspect briefly revolves around issues related to identification of pathognomonic/endophenotypic diagnostic markers in ADHD. Issues discussed include changes in ADHD definition, remission/persistence and overlapping-symptoms cum shared-heritability with its co-morbid cross-border mental disorders. The second aspect discussed is neurobiological and EEG-based studies on ADHD. Given the neurobiological and temporal aspects of ADHD symptoms the electroencephalograph (EEG) like NeuralScan by Medeia appears as an appropriate tool. The EEGs appropriateness is further enhanced when coupled with suitable behavior/cognitive/motor/psychological tasks/paradigms yielding EEG-based markers like event-related-potential (ERPs like P3 amplitudes and latency), reaction time variability (RTV), Theta:Beta ratio (TBR) and sensorimotor rhythm (SMR). At present, these markers could potentially help in the neurobiological characterization of ADHD and either help in identifying or lay the groundwork for identifying pathognomonic and/or endophenotypic EEG-based markers enabling its diagnosis, treatment and management.

Targeted next-generation sequencing of 565 neuro-oncology patients at UCLA: A single-institution experience.

BACKGROUND: Targeted next-generation sequencing (NGS) is frequently obtained at the University of California, Los Angeles (UCLA) for clinical characterization of CNS tumors. In this study, we describe the diagnostic reliability of the Foundation Medicine (FM) targeted NGS platform and its ability to explore and identify tumor characteristics of prognostic significance in gliomas. METHODS: Neuro-oncology patients seen at UCLA who have received FM testing between August 2012 and March 2019 were included in this study, and all mutations from FM test reports were recorded. Initial tumor diagnoses and diagnostic markers found via standard clinical methods were obtained from pathology reports. With overall and progression-free survival data, elastic net regularized Cox regression and Cox proportional hazards models were used to determine whether any mutations of unknown significance detected by FM could predict patient outcome in glioblastoma (GBM). RESULTS: Six hundred and three samples tested by FM from 565 distinct patients were identified. Concordance of diagnostic markers was high between standard clinical testing methods and FM. Oligodendroglial markers detected via FM were highly correlated with 1p19q codeletion in IDH mutated gliomas. FM testing of multiple tumor samples from the same patient demonstrated temporal and spatial mutational heterogeneity. Mutations in BCORL1, ERBB4, and PALB2, which are mutations of unknown significance in GBM, were shown to be statistically significant in predicting patient outcome. CONCLUSIONS: In our large cohort, we found that targeted NGS can both reliably and efficiently detect important diagnostic markers in CNS tumors.

SPINT2 is hypermethylated in both IDH1 mutated and wild-type glioblastomas, and exerts tumor suppression via reduction of c-Met activation.

PURPOSE: Both IDH1-mutated and wild-type gliomas abundantly display aberrant CpG island hypermethylation. However, the potential role of hypermethylation in promoting gliomas, especially the most aggressive form, glioblastoma (GBM), remains poorly understood. METHODS: We analyzed RRBS-generated methylation profiles for 11 IDH1WT gliomas (including 7 GBMs), 24 IDH1MUT gliomas (including 6 GBMs), and 5 normal brain samples and employed TCGA GBM methylation profiles as a validation set. Upon classification of differentially methylated CpG islands by IDH1 status, we used integrated analysis of methylation and gene expression to identify SPINT2 as a top cancer related gene. To explore functional consequences of SPINT2 methylation in GBM, we validated SPINT2 methylation status using targeted bisulfite sequencing in a large cohort of GBM samples. We assessed DNA methylation-mediated SPINT2 gene regulation using 5-aza-2'-deoxycytidine treatment, DNMT1 knockdown and luciferase reporter assays. We conducted functional analyses of SPINT2 in GBM cell lines in vitro and in vivo. RESULTS: We identified SPINT2 as a candidate tumor-suppressor gene within a group of CpG islands (designated GT-CMG) that are hypermethylated in both IDH1MUT and IDH1WT gliomas but not in normal brain. We established that SPINT2 downregulation results from promoter hypermethylation, and that restoration of SPINT2 expression reduces c-Met activation and tumorigenic properties of GBM cells. CONCLUSIONS: We defined a previously under-recognized group of coordinately methylated CpG islands common to both IDH1WT and IDH1MUT gliomas (GT-CMG). Within GT-CMG, we identified SPINT2 as a top cancer-related candidate and demonstrated that SPINT2 suppressed GBM via down-regulation of c-Met activation.

D-2-Hydroxyglutarate Is Necessary and Sufficient for Isocitrate Dehydrogenase 1 Mutant-Induced MIR148A Promoter Methylation.

Mutant isocitrate dehydrogenase (IDH) 1/2 converts α-ketoglutarate (α-KG) to D-2 hydroxyglutarate (D-2-HG), a putative oncometabolite that can inhibit α-KG-dependent enzymes, including ten-eleven translocation methylcytosine dioxygenase (TET) DNA demethylases. We recently established that miRNAs are components of the IDH1 mutant-associated glioma CpG island methylator phenotype (G-CIMP) and specifically identified MIR148A as a tumor-suppressive miRNA within G-CIMP. However, the precise mechanism by which mutant IDH induces hypermethylation of MIR148A and other G-CIMP promoters remains to be elucidated. In this study, we demonstrate that treatment with exogenous D-2-HG induces MIR148A promoter methylation and transcriptional silencing in human embryonic kidney 293T (293T) cells and primary normal human astrocytes. Conversely, we show that the development of MIR148A promoter methylation in mutant IDH1-overexpressing 293T cells is abrogated via treatment with C227, an inhibitor of mutant IDH1 generation of D-2-HG. Using dot blot assays for global assessment of 5-hydroxymethylcytosine (5-hmC), we show that D-2-HG treatment reduces 5-hmC levels, whereas C227 treatment increases 5-hmC levels, strongly suggesting TET inhibition by D-2-HG. Moreover, we show that withdrawal of D-2-HG treatment reverses methylation with an associated increase in MIR148A transcript levels and transient generation of 5-hmC. We also demonstrate that RNA polymerase II binds endogenously to the predicted promoter region of MIR148A, validating the hypothesis that its transcription is driven by an independent promoter.Implications: Establishment of D-2-HG as a necessary and sufficient intermediate by which mutant IDH1 induces CpG island methylation of MIR148A will help with understanding the efficacy of selective mutant IDH1 inhibitors in the clinic. Mol Cancer Res; 16(6); 947-60. ©2018 AACR.

The identification of a novel lead class for phosphodiesterase 2 inhibition by fragment-based drug design.

We have identified a novel PDE2 inhibitor series using fragment-based screening. Pyrazolopyrimidine fragment 1, while possessing weak potency (Ki = 22.4 µM), exhibited good binding efficiencies (LBE = 0.49, LLE = 4.48) to serve as a start for structure-based drug design. With the assistance of molecular modeling and X-ray crystallography, this fragment was developed into a series of potent PDE2 inhibitors with good physicochemical properties. Compound 16, a PDE2 selective inhibitor, was identified that exhibited favorable rat pharmacokinetic properties.

Preclinical Characterization of the Phosphodiesterase 10A PET Tracer [(11)C]MK-8193.

PURPOSE: A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [(11)C]MK-8193 is described. PROCEDURES: In vitro binding studies with [(3)H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [(11)C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor. RESULTS: [(3)H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [(11)C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships. CONCLUSIONS: [(11)C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [(11)C]MK-8193 in humans is warranted.

Discovery of pyrazolopyrimidine phosphodiesterase 10A inhibitors for the treatment of schizophrenia.

Herein, we present the identification of a novel class of pyrazolopyrimidine phosphodiesterase 10A (PDE10A) inhibitors. Beginning with a lead molecule (1) identified through a fragment-based drug discovery (FBDD) effort, lead optimization was enabled by rational design, X-ray crystallography, metabolic and off-target profiling, and fragment scaffold-hopping. We highlight the discovery of PyP-1, a potent, highly selective, and orally bioavailable pyrazolopyrimidine inhibitor of PDE10A. PyP-1 exhibits sub-nanomolar potency (PDE10A Ki=0.23nM), excellent pharmacokinetic (PK) and physicochemical properties, and a clean off-target profile. It displays dose-dependent efficacy in numerous pharmacodynamic (PD) assays that measure potential for anti-psychotic activity and cognitive improvement. PyP-1 also has a clean preclinical profile with respect to cataleptic potential in rats, prolactin secretion, and weight gain, common adverse events associated with currently marketed therapeutics. Further, PyP-1 displays in vivo preclinical target engagement as measured by PET enzyme occupancy in concert with [(11)C]MK-8193, a novel PDE10A PET tracer.

Orexin Receptor Antagonists: New Therapeutic Agents for the Treatment of Insomnia.

Since its discovery in 1998, the orexin system, composed of two G-protein coupled receptors, orexins 1 and 2, and two neuropeptide agonists, orexins A and B, has captured the attention of the scientific community as a potential therapeutic target for the treatment of obesity, anxiety, and sleep/wake disorders. Genetic evidence in rodents, dogs, and humans was revealed between 1999 and 2000, demonstrating a causal link between dysfunction or deletion of the orexin system and narcolepsy, a disorder characterized by hypersomnolence during normal wakefulness. These findings encouraged efforts to discover agonists to treat narcolepsy and, alternatively, antagonists to treat insomnia. This perspective will focus on the discovery and development of structurally diverse orexin antagonists suitable for preclinical pharmacology studies and human clinical trials. The work described herein culminated in the 2014 FDA approval of suvorexant as a first-in-class dual orexin receptor antagonist for the treatment of insomnia.

Discovery and Optimization of a Series of Pyrimidine-Based Phosphodiesterase 10A (PDE10A) Inhibitors through Fragment Screening, Structure-Based Design, and Parallel Synthesis.

Screening of a fragment library for PDE10A inhibitors identified a low molecular weight pyrimidine hit with PDE10A Ki of 8700 nM and LE of 0.59. Initial optimization by catalog followed by iterative parallel synthesis guided by X-ray cocrystal structures resulted in rapid potency improvements with minimal loss of ligand efficiency. Compound 15 h, with PDE10A Ki of 8.2 pM, LE of 0.49, and >5000-fold selectivity over other PDEs, fully attenuates MK-801-induced hyperlocomotor activity after ip dosing.

Discovery of [¹¹C]MK-8193 as a PET tracer to measure target engagement of phosphodiesterase 10A (PDE10A) inhibitors.

Phosphodiesterase 10A (PDE10A) inhibition has recently been identified as a potential mechanism to treat multiple symptoms that manifest in schizophrenia. In order to facilitate preclinical development and support key proof-of-concept clinical trials of novel PDE10A inhibitors, it is critical to discover positron emission tomography (PET) tracers that enable plasma concentration/PDE10A occupancy relationships to be established across species with structurally diverse PDE10A inhibitors. In this Letter, we describe how a high-throughput screening hit was optimized to provide [(11)C]MK-8193 (8j), a PET tracer that supports the determination of plasma concentration/PDE10A occupancy relationships for structurally diverse series of PDE10A inhibitors in both rat and rhesus monkey.

Discovery of piperidine ethers as selective orexin receptor antagonists (SORAs) inspired by filorexant.

Highly selective orexin receptor antagonists (SORAs) of the orexin 2 receptor (OX2R) have become attractive targets both as potential therapeutics for insomnia as well as biological tools to help further elucidate the underlying pharmacology of the orexin signaling pathway. Herein, we describe the discovery of a novel piperidine ether 2-SORA class identified by systematic lead optimization beginning with filorexant, a dual orexin receptor antagonist (DORA) that recently completed Phase 2 clinical trials. Changes to the ether linkage and pendant heterocycle of filorexant were found to impart significant selectivity for OX2R, culminating in lead compound PE-6. PE-6 displays sub-nanomolar binding affinity and functional potency on OX2R while maintaining >1600-fold binding selectivity and >200-fold functional selectivity versus the orexin 1 receptor (OX1R). PE-6 bears a clean off-target profile, a good overall preclinical pharmacokinetic (PK) profile, and reduces wakefulness with increased NREM and REM sleep when evaluated in vivo in a rat sleep study. Importantly, subtle structural changes to the piperidine ether class impart dramatic changes in receptor selectivity. To this end, our laboratories have identified multiple piperidine ether 2-SORAs, 1-SORAs, and DORAs, providing access to a number of important biological tool compounds from a single structural class.

Discovery of MK-3697: a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia.

Orexin receptor antagonists have demonstrated clinical utility for the treatment of insomnia. The majority of clinical efforts to date have focused on the development of dual orexin receptor antagonists (DORAs), small molecules that antagonize both the orexin 1 and orexin 2 receptors. Our group has recently disclosed medicinal chemistry efforts to identify highly potent, orally bioavailable selective orexin 2 receptor antagonists (2-SORAs) that possess acceptable profiles for clinical development. Herein we report additional SAR studies within the 'triaryl' amide 2-SORA series focused on improvements in compound stability in acidic media and time-dependent inhibition of CYP3A4. These studies resulted in the discovery of 2,5-disubstituted isonicotinamide 2-SORAs such as compound 24 that demonstrated improved stability and TDI profiles as well as excellent sleep efficacy across species.

Shaping suvorexant: application of experimental and theoretical methods for driving synthetic designs.

Dual Orexin Receptor Antagonists (DORA) bind to both the Orexin 1 and 2 receptors. High resolution crystal structures of the Orexin 1 and 2 receptors, both class A GPCRs, were not available at the time of this study, and thus, ligand-based analyses were invoked and successfully applied to the design of DORAs. Computational analysis, ligand based superposition, unbound small-molecule X-ray crystal structures and NMR analysis were utilized to understand the conformational preferences of key DORAs and excellent agreement between these orthogonal approaches was seen in the majority of compounds examined. The predominantly face-to-face (F2F) interaction observed between the distal aromatic rings was the core 3D shape motif in our design principle and was used in the development of compounds. A notable exception, however, was seen between computation and experiment for suvorexant where the molecule exhibits an extended conformation in the unbound small-molecule X-ray structure. Even taking into account solvation effects explicitly in our calculations, we nevertheless find support that the F2F conformation is the bioactive conformation. Using a dominant states approximation for the partition function, we made a comprehensive assessment of the free energies required to adopt both an extended and a F2F conformation of a number of DORAs. Interestingly, we find that only a F2F conformation is consistent with the activities reported.

Genetic deletion and pharmacological inhibition of phosphodiesterase 10A protects mice from diet-induced obesity and insulin resistance.

Phosphodiesterase 10A (PDE10A) is a novel therapeutic target for the treatment of schizophrenia. Here we report a novel role of PDE10A in the regulation of caloric intake and energy homeostasis. PDE10A-deficient mice are resistant to diet-induced obesity (DIO) and associated metabolic disturbances. Inhibition of weight gain is due to hypophagia after mice are fed a highly palatable diet rich in fats and sugar but not a standard diet. PDE10A deficiency produces a decrease in caloric intake without affecting meal frequency, daytime versus nighttime feeding behavior, or locomotor activity. We tested THPP-6, a small molecule PDE10A inhibitor, in DIO mice. THPP-6 treatment resulted in decreased food intake, body weight loss, and reduced adiposity at doses that produced antipsychotic efficacy in behavioral models. We show that PDE10A inhibition increased whole-body energy expenditure in DIO mice fed a Western-style diet, achieving weight loss and reducing adiposity beyond the extent seen with food restriction alone. Therefore, chronic THPP-6 treatment conferred improved insulin sensitivity and reversed hyperinsulinemia. These data demonstrate that PDE10A inhibition represents a novel antipsychotic target that may have additional metabolic benefits over current medications for schizophrenia by suppressing food intake, alleviating weight gain, and reducing the risk for the development of diabetes.

Discovery of 5''-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2':5',3''-terpyridine-3'-carboxamide (MK-1064): a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia.

The field of small-molecule orexin antagonist research has evolved rapidly in the last 15 years from the discovery of the orexin peptides to clinical proof-of-concept for the treatment of insomnia. Clinical programs have focused on the development of antagonists that reversibly block the action of endogenous peptides at both the orexin 1 and orexin 2 receptors (OX1 R and OX2 R), termed dual orexin receptor antagonists (DORAs), affording late-stage development candidates including Merck's suvorexant (new drug application filed 2012). Full characterization of the pharmacology associated with antagonism of either OX1 R or OX2 R alone has been hampered by the dearth of suitable subtype-selective, orally bioavailable ligands. Herein, we report the development of a selective orexin 2 antagonist (2-SORA) series to afford a potent, orally bioavailable 2-SORA ligand. Several challenging medicinal chemistry issues were identified and overcome during the development of these 2,5-disubstituted nicotinamides, including reversible CYP inhibition, physiochemical properties, P-glycoprotein efflux and bioactivation. This article highlights structural modifications the team utilized to drive compound design, as well as in vivo characterization of our 2-SORA clinical candidate, 5''-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2':5',3''-terpyridine-3'-carboxamide (MK-1064), in mouse, rat, dog, and rhesus sleep models.

The novel phosphodiesterase 10A inhibitor THPP-1 has antipsychotic-like effects in rat and improves cognition in rat and rhesus monkey.

Phosphodiesterase 10A (PDE10A) is a novel target for the treatment of schizophrenia that may address multiple symptomatic domains associated with this disorder. PDE10A is highly expressed in the brain and functions to metabolically inactivate the important second messengers cAMP and cGMP. Here we describe effects of a potent and orally bioavailable PDE10A inhibitor [2-(6-chloropyridin-3-yl)-4-(2-methoxyethoxy)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl](imidazo[1,5-a]pyridin-1-yl)methanone] (THPP-1) on striatal signaling pathways, in behavioral tests that predict antipsychotic potential, and assays that measure episodic-like memory in rat and executive function in rhesus monkey. THPP-1 exhibits nanomolar potency on the PDE10A enzyme, demonstrates excellent pharmacokinetic properties in multiple preclinical animal species, and is selective for PDE10A over other PDE families of enzymes. THPP-1 significantly increased phosphorylation of proteins in the striatum involved in synaptic plasticity, including the a-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor (AMPA) GluR1 subunit, extracellular receptor kinase (ERK), and cAMP-response element binding protein (CREB). THPP-1 produced dose-dependent effects in preclinical assays predictive of antipsychotic activity including attenuation of MK-801-induced psychomotor activation and condition avoidance responding in rats. At similar plasma exposures, THPP-1 significantly increased object recognition memory in rat and attenuated a ketamine-induced deficit in the object retrieval detour task in rhesus monkey. These findings suggest that PDE10A inhibitors have the potential to impact multiple symptomatic domains of schizophrenia including positive symptoms and cognitive impairment. This article is part of a Special Issue entitled 'Cognitive Enhancers'.