The IP6K Inhibitor LI-2242 Ameliorates Diet-Induced Obesity, Hyperglycemia, and Hepatic Steatosis in Mice by Improving Cell Metabolism and Insulin Signaling.

Obesity and nonalcoholic fatty liver disease (NAFLD) are global health concerns, and thus, drugs for the long-term treatment of these diseases are urgently needed. We previously discovered that the inositol pyrophosphate biosynthetic enzyme IP6K1 is a target in diet-induced obesity (DIO), insulin resistance, and NAFLD. Moreover, high-throughput screening (HTS) assays and structure-activity relationship (SAR) studies identified LI-2242 as a potent IP6K inhibitor compound. Here, we tested the efficacy of LI-2242 in DIO WT C57/BL6J mice. LI-2242 (20 mg/kg/BW daily, i.p.) reduced body weight in DIO mice by specifically reducing the accumulation of body fat. It also improved glycemic parameters and reduced hyperinsulinemia. LI-2242-treated mice displayed reduced the weight of various adipose tissue depots and an increased expression of metabolism- and mitochondrial-energy-oxidation-inducing genes in these tissues. LI-2242 also ameliorated hepatic steatosis by reducing the expression of genes that enhance lipid uptake, lipid stabilization, and lipogenesis. Furthermore, LI-2242 enhances the mitochondrial oxygen consumption rate (OCR) and insulin signaling in adipocytes and hepatocytes in vitro. In conclusion, the pharmacologic inhibition of the inositol pyrophosphate pathway by LI-2242 has therapeutic potential in obesity and NAFLD.

Desmoglein compensation hypothesis fidelity assessment in Pemphigus.

The pemphigus group of autoimmune blistering diseases encompasses pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Lesion location in pemphigus has been elegantly postulated by the Desmoglein Compensation Hypothesis (DCH), which references the distribution of desmoglein (Dsg) proteins in the epidermis along with a patient's autoantibody profile to describe three different lesion phenotypes: PF is characterized by subcorneal lesions in the presence of anti-Dsg1 antibodies only, while lesions in PV are suprabasilar and accompanied by anti-Dsg3 antibodies only in mucosal PV, or both anti-Dsg3 and anti-Dsg1 in the case of mucocutaneous PV. While the validity of this hypothesis has been supported by several studies and is prominently featured in textbooks of dermatology, a number of logical inconsistencies have been noted and exceptions have been published in several small-scale studies. We sought to comprehensively assess the extent to which patient clinical and autoantibody profiles contradict the DCH, and characterize these contradictions in a large sample size of 266 pemphigus patients. Remarkably, we find that roughly half of active PV and PF patients surveyed present with a combination of lesion morphology and anti-Dsg3/1 levels that contradict the DCH, including: patients with a cutaneous only PV presentation, mucocutaneous disease in the absence of either Dsg3, Dsg1, or both, and mucosal disease in the absence of Dsg3 or presence of Dsg1. We also find stark differences in fidelity to the DCH based on ethnicity and HLA-association, with the lowest proportion of adherence in previously understudied populations. These findings underscore the need to expand our understanding of pemphigus morphology beyond the DCH, in particular for populations that have not been a focus in previous investigation.

Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil.

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.

Identification of Small-Molecule Inhibitors of Human Inositol Hexakisphosphate Kinases by High-Throughput Screening.

Inositol hexakisphosphate kinases (IP6Ks) catalyze pyrophosphorylation of inositol hexakisphosphate (IP6) into inositol 5-diphospho-1,2,3,4,6-pentakisphosphate (IP7), which is involved in numerous areas of cell physiology including glucose homeostasis, blood coagulation, and neurological development. Inhibition of IP6Ks may be effective for the treatment of Type II diabetes, obesity, metabolic complications, thrombosis, and psychiatric disorders. We performed a high-throughput screen (HTS) of 158 410 compounds for IP6K1 inhibitors using a previously developed ADP-Glo Max assay. Of these, 1206 compounds were found to inhibit IP6K1 kinase activity by more than 25%, representing a 0.8% hit rate. Structural clustering analysis of HTS-active compounds, which were confirmed in the dose-response testing using the same kinase assay, revealed diverse clusters that were feasible for future structure-activity relationship (SAR) optimization to potent IP6K inhibitors. Medicinal chemistry SAR efforts in three chemical series identified potent IP6K1 inhibitors which were further validated in an orthogonal LC-MS IP7 analysis. The effects of IP6K1 inhibitors on cellular IP7 levels were further confirmed and were found to correlate with cellular IP6K1 binding measured by a high-throughput cellular thermal shift assay (CETSA).

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.

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.

Design, synthesis, and structure-activity relationships of pyridoquinazolinecarboxamides as RNA polymerase I inhibitors.

RNA polymerase I (Pol I) is a dedicated polymerase that transcribes the 45S ribosomal (r) RNA precursor. The 45S rRNA precursor is subsequently processed into the mature 5.8S, 18S, and 28S rRNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. On the basis of the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides have been evaluated as inhibitors of Pol I and activators of the destruction of RPA194, the Pol I large catalytic subunit protein. Structure-activity relationships in assays of nucleolar stress and cell viability demonstrate key pharmacophores and their physicochemical properties required for potent activation of Pol I stress and cytotoxicity. This work identifies a set of bioactive compounds that potently cause RPA194 degradation that function in a tightly constrained chemical space. This work has yielded novel derivatives that contribute to the development of Pol I inhibitory cancer therapeutic strategies.

The relationship of clinical QT prolongation to outcome in the conscious dog using a beat-to-beat QT-RR interval assessment.

QT interval prolongation of the electrocardiogram has been associated with the occurrence of life-threatening fatal ventricular arrhythmias. To understand the relationship between preclinical cardiac conduction assessment to clinical outcome, comparisons of free (unbound)-plasma drug concentrations and their associated effects in the conscious mongrel dog were made to the free plasma concentrations in humans reported to produce QT prolongation. E-4031 (an experimental class III antiarrhythmic), cisapride, terfenadine, terodiline, and verapamil all affect cardiac repolarization and can produce QT prolongation in humans. In the conscious dog, the QT interval was assessed on a beat-to-beat basis in relation to each preceding RR interval at concentrations approximating the same unbound human concentrations. E-4031, cisapride and terodiline statistically increased the QT(RR1000) interval [the QT interval at a 60 beats/min (bpm) heart rate] 23, 8, and 9 ms, respectively, at concentrations 0.3 to 15.8 times their relevant clinical level. Increases were not observed for terfenadine or verapamil (p > 0.05 at all doses). Inspection of individual dog QT versus RR interval relationships showed clear QT interval responses specific to each treatment but not readily apparent when data are averaged at a heart rate of 60 bpm. For specific rectifier K(+) current (IKr) blockers, robust effects on mean QT prolongation can be detected. However, for drugs that affect repolarization through multiple channels, the effect on the mean QT interval may be more difficult to detect. Inspection of the beat-to-beat QT-RR interval relationship in an individual animal can increase the sensitivity for more accurate clinical prediction.