Fragment-Based Screening Identifies New Quinazolinone-Based Inositol Hexakisphosphate Kinase (IP6K) Inhibitors.

A high-throughput fragment-based screen has been employed to discover a series of quinazolinone inositol hexakisphosphate kinase (IP6K) inhibitors. IP6Ks have been studied for their role in glucose homeostasis, metabolic disease, fatty liver disease, chronic kidney disease, blood coagulation, neurological development, and psychiatric disease. IP6Ks phosphorylate inositol hexakisphosphate (IP6) to form pyrophosphate 5-diphospho-1,2,3,4,6-pentakisphosphate (IP7). Molecular docking studies and investigation of structure-activity relationships around the quinazolinone core resulted in compounds with submicromolar potency and interesting selectivity for IP6K1 versus the closely related IP6K2 and IP6K3 isoforms.

Promyelinating drugs promote functional recovery in an autism spectrum disorder mouse model of Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is an autism spectrum disorder caused by autosomal dominant mutations in the human transcription factor 4 gene (TCF4). One pathobiological process caused by murine Tcf4 mutation is a cell autonomous reduction in oligodendrocytes and myelination. In this study, we show that the promyelinating compounds, clemastine, sobetirome and Sob-AM2 are effective at restoring myelination defects in a Pitt-Hopkins syndrome mouse model. In vitro, clemastine treatment reduced excess oligodendrocyte precursor cells and normalized oligodendrocyte density. In vivo, 2-week intraperitoneal administration of clemastine also normalized oligodendrocyte precursor cell and oligodendrocyte density in the cortex of Tcf4 mutant mice and appeared to increase the number of axons undergoing myelination, as EM imaging of the corpus callosum showed a significant increase in the proportion of uncompacted myelin and an overall reduction in the g-ratio. Importantly, this treatment paradigm resulted in functional rescue by improving electrophysiology and behaviour. To confirm behavioural rescue was achieved via enhancing myelination, we show that treatment with the thyroid hormone receptor agonist sobetirome or its brain penetrating prodrug Sob-AM2, was also effective at normalizing oligodendrocyte precursor cell and oligodendrocyte densities and behaviour in the Pitt-Hopkins syndrome mouse model. Together, these results provide preclinical evidence that promyelinating therapies may be beneficial in Pitt-Hopkins syndrome and potentially other neurodevelopmental disorders characterized by dysmyelination.

Membrane bound catechol-O-methytransferase is the dominant isoform for dopamine metabolism in PC12 cells and rat brain.

Impaired dopamine activity in the dorsolateral prefrontal cortex (DLPFC) is thought to contribute to cognitive deficits in diseases such as schizophrenia, attention deficit hyperactivity disorder (ADHD) and traumatic brain injury. Catechol-O-methyltransfease (COMT) metabolizes dopamine and is an important regulator of dopamine signaling in the DLPFC. In mammalian species, two isoforms of COMT protein, membrane-bound COMT (MB-COMT) and soluble COMT (S-COMT), are encoded by one COMT gene and expressed widely. While S-COMT is thought to play a dominant role in the peripheral tissues, MB-COMT is suggested to have a greater role in dopamine metabolism in the brain. However, whether a selective inhibitor for MB-COMT may effectively block dopamine metabolism remains unknown. We generated a knockout of MB-COMT in PC12 cells using CRISPR-cas9 technology to evaluate the effect of both MB and S-COMT on dopamine metabolism. Deletion of MB-COMT in PC12 cells significantly decreased homovanillic acid (HVA), completely depleted 3-methyoxytyramine (3-MT), and significantly increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels. Comparison of the effect of a MB-COMT selective inhibitor LI-1141 on dopamine metabolism in wild type and MB-COMT knockout PC12 cells allowed us to confirm the selectivity of LI-1141 with respect to MB-COMT in cells. Under conditions in which LI-1141 was shown to inhibit only MB-COMT but not S-COMT, it effectively changed dopamine metabolites similar to the effect induced by tolcapone, a non-selective COMT inhibitor, suggesting that selective inhibition of MB-COMT will be effective in blocking dopamine metabolism, providing an attractive therapeutic approach in improving cognition for patients.

Novel, non-nitrocatechol catechol-O-methyltransferase inhibitors modulate dopamine neurotransmission in the frontal cortex and improve cognitive flexibility.

RATIONALE: Cognitive impairment is a primary feature of many neuropsychiatric disorders and there is a need for new therapeutic options. Catechol-O-methyltransferase (COMT) inhibitors modulate cortical dopaminergic function and have been proposed as potential cognitive enhancers. Unfortunately, currently available COMT inhibitors are not good candidates due to either poor blood-brain barrier penetration or severe toxicity. OBJECTIVES: To address the need for safe, brain-penetrant COMT inhibitors, we tested multiple novel compounds in a set of preclinical in vivo efficacy assays in rats to determine their ability to inhibit COMT function and viability as potential clinical candidates. METHODS: We measured the change in concentration of dopamine (DA) metabolites in cerebrospinal fluid (CSF) from the cisterna magna and extracellular fluid (ECF) from the frontal cortex produced by our novel compounds. Additionally, we tested the effects of our brain-penetrant COMT inhibitors in an attentional set-shifting assay (ASST). We benchmarked the performance of the novel COMT inhibitors to the effects produced by the known COMT inhibitor tolcapone. RESULTS: We found that multiple COMT inhibitors, exemplified by LIBD-1 and LIBD-3, significantly modulated dopaminergic function measured as decreases in homovanillic acid (HVA) and increases in 3,4-Dihydroxyphenylacetic acid (DOPAC), two DA metabolites, in CSF and the frontal cortex. Additionally, we found that LIBD-1 significantly improved cognitive flexibility in the ASST, an effect previously reported following tolcapone administration. CONCLUSIONS: These results demonstrate that LIBD-1 is a novel COMT inhibitor with promising in vivo activity and the potential to serve as a new therapy for cognitive impairment.

Development of a PC12 Cell Based Assay for Screening Catechol-O-methyltransferase Inhibitors.

The male rat adrenal pheochromocytoma cell-derived PC12 cell line can synthesize and release catecholamine neurotransmitters, and it has been widely used as a model system in cell biology and toxicology research. Catechol-O-methyltransferase (COMT) is involved in the inactivation of the catecholamine neurotransmitters, and it is particularly important for the regulation of dopamine. In this study, we explored the feasibility of using PC12 cells as an in vitro drug screening platform to compare the activity of multiple COMT inhibitors. Incubation of PC12 cells with tolcapone, a highly potent and selective COMT inhibitor, increased the concentrations of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) while reducing the metabolites 3-methoxytyramine (3-MT) and homovanillic acid (HVA) in the cell culture medium. LIBD-3, a novel, non-nitrocatechol COMT inhibitor, produced similar effects compared to tolcapone. LIBD-4, a less potent inhibitor, exhibited the expected right-shift in functional inhibition in the assay. These results match the known in vivo effects of COMT inhibition in rodents. Together, these data support the continued use of PC12 cells as an in vitro screen that bridges cell-free enzyme assays and more costly in vivo assays.

Aminopyrazole Carboxamide Bruton's Tyrosine Kinase Inhibitors. Irreversible to Reversible Covalent Reactive Group Tuning.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors of Catechol O-Methyltransferase.

A series of bicyclic pyridones were identified as potent inhibitors of catechol O-methyltransferase (COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series, but with high free fraction due to remarkably low levels of protein and tissue binding. In rat biomarker studies, levels of unbound drug exposure are seen in the brain, which exceed their respective IC50s, leading to changes in the levels of dopamine metabolites in a manner consistent with COMT inhibition.

Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase.

A series of 8-hydroxy quinolines were identified as potent inhibitors of catechol O-methyltransferase (COMT) with selectivity for the membrane-bound form of the enzyme. Small substituents at the 7-position of the quinoline were found to increase metabolic stability without sacrificing potency. Compounds with good pharmacokinetics and brain penetration were identified and demonstrated in vivo modulation of dopamine metabolites in the brain. An X-ray cocrystal structure of compound 21 in the S-COMT active site shows chelation of the active site magnesium similar to catechol-based inhibitors. These compounds should prove useful for treatment of many neurological and psychiatric conditions associated with compromised cortical dopamine signaling.

Novel tetrahydro-beta-carboline-1-carboxylic acids as inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2).

A structure-activity relationship study was conducted on a series of tetrahydro-beta-carboline-1-carboxylic acid analogs in order to identify the key functionality responsible for activity against the mitogen-activated protein kinase-activated protein kinase 2 enzyme (MK-2). The compounds were further evaluated for their ability to inhibit TNFalpha production in U937 cells and in vivo. These compounds represent a novel structural class of compounds capable of inhibiting MK-2 with remarkable selectivity.

Evidence for separate binding and scaffolding domains in the immunosuppressive and antitumor marine natural product, pateamine a: design, synthesis, and activity studies leading to a potent simplified derivative.

Pateamine A (PatA), a marine metabolite from Mycale sp., is a potent inhibitor of the intracellular signal transduction pathway emanating from the T-cell receptor leading to the transcription of cytokines such as interleukin-2 (IL-2). On the basis of the structure of PatA and initial biological results, a hypothesis was developed regarding the presence of distinct binding and scaffolding domains in the PatA structure with respect to interactions with its putative cellular receptor(s). Employing a highly convergent approach involving a Hantzsch coupling strategy, we probed this hypothesis by preparing a simplified PatA derivative (desmethyl, desamino PatA, DMDAPatA, 3). This derivative was prepared in 10 fewer synthetic steps relative to PatA and was indeed found to exhibit equal to greater potency (IC50 0.81 +/- 0.27 nM) in inhibition of IL-2 production relative to PatA (IC50 4.01 +/- 0.94 nM) thus providing support for the binding/scaffolding domain hypothesis. In addition, as a means to find more stable derivatives and gain further insights into structure-activity relationships, several PatA derivatives were synthesized and assayed in the IL-2 reporter gene assay. Several of these derivatives displayed lower potency but marked stability relative to the natural product and provide further insights into the nature of the binding domain required for activity.