REV-ERB activation as a novel pharmacological approach for treating inflammatory pain

Pain is a complex problem affecting millions of people worldwide. The current therapies to reduce pain are limited as many treatment options inadequately address the causes of pain, lead to tolerance of the drug, or have adverse effects including abuse potential. While there are many causes of pain, one underlying mechanism to the pathogenesis and maintenance of pain conditions is chronic inflammation driven by the NLRP3 inflammasome. Several inflammasome inhibitors are currently under investigation however have the potential to suppress the functioning of the innate immune system, which may cause unwanted affects in patients. Here, we show that the nuclear receptor REV-ERB can suppress the activation of the inflammasome when pharmacologically activated with small molecule agonists. Additionally, REV-ERB activation appears to have analgesic potential in a model of acute inflammatory pain, likely as a result of inflammasome suppression

REV-ERB agonism improves liver pathology in a mouse model of NASH

Non-alcoholic fatty liver disease (NAFLD) affects a significant number of people worldwide and currently there are no pharmacological treatments. NAFLD often presents with obesity, insulin resistance, and in some cases cardiovascular diseases. There is a clear need for treatment options to alleviate this disease since it often progresses to much more the much more severe non-alcoholic steatohepatitis (NASH). The REV-ERB nuclear receptor is a transcriptional repressor that regulates physiological processes involved in the development of NAFLD including lipogenesis and inflammation. We hypothesized that pharmacologically activating REV-ERB would suppress the progression of fatty liver in a mouse model of NASH. Using REV-ERB agonist SR9009 in a mouse NASH model, we demonstrate the beneficial effects of REV-ERB activation that led to an overall improvement of hepatic health by suppressing hepatic fibrosis and inflammatory response

REV-ERB activation as a novel pharmacological approach for treating inflammatory pain

Pain is a complex problem affecting millions of people worldwide. The current therapies to reduce pain are limited as many treatment options inadequately address the causes of pain, lead to tolerance of the drug, or have adverse effects including abuse potential. While there are many causes of pain, one underlying mechanism to the pathogenesis and maintenance of pain conditions is chronic inflammation driven by the NLRP3 inflammasome. Several inflammasome inhibitors are currently under investigation however have the potential to suppress the functioning of the innate immune system, which may cause unwanted affects in patients. Here, we show that the nuclear receptor REV-ERB can suppress the activation of the inflammasome when pharmacologically activated with small molecule agonists. Additionally, REV-ERB activation appears to have analgesic potential in a model of acute inflammatory pain, likely as a result of inflammasome suppression

A two-hit model of alcoholic liver disease that exhibits rapid, severe fibrosis

Alcoholic liver disease (ALD) is responsible for an average of 50.4% and 44.2%of liver disease deaths among males and females respectively. Driven by alcohol misuse, ALD is often reversible by cessation of consumption. However, abstinence programs can have limited success at curtailing abuse, and the loss of life. ALD, therefore, remains a significant clinical challenge. There is a need for effective treatments that prevent or reverse alcohol-induced liver damage to complement or supplant behavioral interventions. Metabolic syndrome, which is disproportionally prevalent in ALD patients, accelerates the progression of ALD and increases liver disease mortality. Current rodent models of ALD unfortunately do not account for the contribution of the western diet to ALD pathology. To address this, we have developed a rodent model of ALD that integrates the impact of the western diet and alcohol; the WASH-diet model. We show here that the WASH diet, either chronically or in small time-restricted bouts, accelerated ALD pathology with severe steatohepatitis, elevated inflammation and increased fibrosis compared to mice receiving chronic alcohol alone. We also validated our WASH-diet model as an in vivo system for testing the efficacy of experimental ALD treatments. The efficacy of the inverse-agonist SR9238, previously shown to inhibit both non-alcohol and alcohol-induced steatohepatitis progression, was conserved in our WASH-diet model. These findings suggested that the WASH-diet may be useful for in vivo pre-clinical assessment of novel therapies

A two-hit model of alcoholic liver disease that exhibits rapid, severe fibrosis

Alcoholic liver disease (ALD) is responsible for an average of 50.4% and 44.2%of liver disease deaths among males and females respectively. Driven by alcohol misuse, ALD is often reversible by cessation of consumption. However, abstinence programs can have limited success at curtailing abuse, and the loss of life. ALD, therefore, remains a significant clinical challenge. There is a need for effective treatments that prevent or reverse alcohol-induced liver damage to complement or supplant behavioral interventions. Metabolic syndrome, which is disproportionally prevalent in ALD patients, accelerates the progression of ALD and increases liver disease mortality. Current rodent models of ALD unfortunately do not account for the contribution of the western diet to ALD pathology. To address this, we have developed a rodent model of ALD that integrates the impact of the western diet and alcohol; the WASH-diet model. We show here that the WASH diet, either chronically or in small time-restricted bouts, accelerated ALD pathology with severe steatohepatitis, elevated inflammation and increased fibrosis compared to mice receiving chronic alcohol alone. We also validated our WASH-diet model as an in vivo system for testing the efficacy of experimental ALD treatments. The efficacy of the inverse-agonist SR9238, previously shown to inhibit both non-alcohol and alcohol-induced steatohepatitis progression, was conserved in our WASH-diet model. These findings suggested that the WASH-diet may be useful for in vivo pre-clinical assessment of novel therapies

Mitochondrial pyruvate carrier inhibitors improve metabolic parameters in diet-induced obese mice

The mitochondrial pyruvate carrier (MPC) is an inner mitochondrial membrane complex that plays a critical role in intermediary metabolism. Inhibition of the MPC, especially in liver, may have efficacy for treating type 2 diabetes mellitus. Herein, we examined the antidiabetic effects of zaprinast and 7ACC2, small molecules which have been reported to act as MPC inhibitors. Both compounds activated a bioluminescence resonance energy transfer-based MPC reporter assay (reporter sensitive to pyruvate) and potently inhibited pyruvate-mediated respiration in isolated mitochondria. Furthermore, zaprinast and 7ACC2 acutely improved glucose tolerance in diet-induced obese mice in vivo. Although some findings were suggestive of improved insulin sensitivity, hyperinsulinemic-euglycemic clamp studies did not detect enhanced insulin action in response to 7ACC2 treatment. Rather, our data suggest acute glucose-lowering effects of MPC inhibition may be due to suppressed hepatic gluconeogenesis. Finally, we used reporter sensitive to pyruvate to screen a chemical library of drugs and identified 35 potentially novel MPC modulators. Using available evidence, we generated a pharmacophore model to prioritize which hits to pursue. Our analysis revealed carsalam and six quinolone antibiotics, as well as 7ACC1, share a common pharmacophore with 7ACC2. We validated that these compounds are novel inhibitors of the MPC and suppress hepatocyte glucose production and demonstrated that one quinolone (nalidixic acid) improved glucose tolerance in obese mice. In conclusion, these data demonstrate the feasibility of therapeutic targeting of the MPC for treating diabetes and provide scaffolds that can be used to develop potent and novel classes of MPC inhibitors

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. In cancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. In cancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach

From DNA to Protein: a study of genomic instability candidate genes during zebrafish development

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation during the early stages of embryogenesis, that can be detected with the mosaic eye assay at 48-72 hpf. Using published genome and mapping data, several candidate genes for two of the gin mutations were identified and studied during early zebrafish development. The gin mutations are heritable, ENU-induced, and have both maternal and zygotic effects during zebrafish development. The first aim of this project was to study the normal gene characteristics of the gin-10 candidate genes, synbl, rfx4, and sir2 that are located on chromosome 18. Semi-quantitative RT-PCR, whole-mount in situ hybridization, and gene knockdown (using morpholino oligonucleotides) techniques were utilized in both wildtype and transgenic (Tg-synbl) zebrafish lines to gain an understanding of the function of each of these genes during zebrafish embryogenesis. Additionally, the synbl paralog, ric8a, was also explored, as it has been implicated in the control of asymmetric cell division in C. elegans. Single gene knockdowns were performed for each candidate in the golden heterozygous (pigment mutant) zebrafish background to test for genomic instability activity. Genomic instability activity was not observed, however the results showed that these genes are expressed throughout zebrafish embryogenesis, and are necessary for the proper development of the central nervous system, notochord and tail, as well as metabolic functions in the early embryo. Moreover, the transgenic line used for the paralog studies of synbl and ric8a was incorrectly genotyped. Using PCR analysis and sequencing, it was found that the viral insert for the Tg-synbl fish was disrupting the cry1b gene on an adjacent contig. The second aim focused on the gin-12 region on chromosome 4, where the mdm1 gene is located. Originally cloned from a transformed mouse cell line with mdm2, the function of the mdm1 gene in these cells or during development had not yet been identified. To allow the Mdm1 protein to be evaluated, custom antibodies targeting Mdm1 were produced and the detection of Mdm1 optimized in zebrafish embryos. This would allow us to then determine whether Mdm1 was a possible regulator of the p53-Mdm2/Mdm4 pathway. Additionally, the mdm1 gene was studied in situ and in vivo to determine the normal gene expression patterns and developmental role in the embryonic zebrafish. Moreover, this gene was also studied in the golden heterozygous zebrafish line to assess whether it had a role in modulating genomic instability activity using the mosaic eye assay. Collectively, morpholino oligonucleotides, RNA rescue, whole-mount antibody staining, and overexpression studies suggest that the mdm1 gene is involved in the development of the eye and portions of the central nervous system, but did not appear to be the gin-12 mutant. While the genes in this study did not appear to have genomic instability activity in the embryonic zebrafish based on the mosaic eye assay in the golden heterozygotes, normal developmental gene expression patterns were identified for synbl, ric8a, rfx4, sir2, and mdm1 in wildtype zebrafish embryos. Additional information was gained by the reverse genetic studies using gene knockdowns, which identified the functional roles of these genes at various stages of embryogenesis. Notably, it was determined that the mdm1 gene may be involved in retinal degenerative diseases based on our studies and recently published data. Future research of the Mdm1 protein should identify protein interactions and the specific role during eye development and retinal diseases

Inhibition of Hepatotoxicity by a LXR Inverse Agonist in a Model of Alcoholic Liver Disease

Alcohol abuse is a major cause of liver disease and mortality worldwide and is a significant public health issue. Patients with alcoholic liver disease (ALD) have severe hepatic lipid accumulation, inflammation, and fibrosis. Therapies for ALD are very limited and even abstinence from alcohol consumption does not necessarily protect patients from progression of the disease. We sought to evaluate the efficacy of a liver X receptor (LXR) inverse agonist, SR9238, in an animal model of ALD. SR9238 suppresses hepatic lipogenesis, a pathological hallmark of ALD, and we hypothesized that targeting suppression of hepatic metabolic pathways that are activated in ALD may be an effective treatment for the disease. A chronic ethanol diet with or without a final ethanol binge treatment was used to induce ALD in mice. Mice were administered the liver specific LXR inverse agonist SR9238 for 4 weeks after the mice had been maintained on the ethanol diet for 14 days. Mice developed all the hallmarks of advanced ALD demonstrating significant pathophysiology and hepatotoxicity. SR9238 significantly attenuated liver injury and hepatic steatosis and fibrosis was nearly eliminated in SR9238 treated mice. SR9238 treatment reversed the damage associated with chronic ethanol use returning the liver to near normal morphology. These results indicate that inhibiting LXR activity using the inverse agonist has a hepatoprotective effect in rodent models of ALD; thus, this pharmacological approach may be efficacious for treatment of ALD in humans