Michael D. Cameron

Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation

PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5 (ref. 2). Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.

Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists

Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-β have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.

A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a ‘co-clinical’ trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

Insular hypocretin transmission regulates nicotine reward

Damage to the insular cortex can profoundly disrupt tobacco addiction in human smokers, reflected in spontaneous cessation of the tobacco habit and persistently decreased urge to smoke. Little is known concerning the neurobiological mechanisms through which the insula may control the maintenance of the tobacco habit. Emerging evidence suggests that hypocretin (orexin) transmission may play an important role in drug reinforcement processes, but its role in the rewarding actions of nicotine, considered the key addictive component of tobacco smoke, remains largely unexplored. Here we show that blockade of hypocretin transmission at hypocretin-1 (Hcrt-1; orexin-1) receptors decreases i.v. nicotine self-administration in rats and the motivation to obtain the drug. Blockade of Hcrt-1 receptors also abolished the stimulatory effects of nicotine on brain reward circuitries, as measured by reversal of nicotine-induced lowering of intracranial self-stimulation thresholds. In addition, we show that hypocretin-containing fibers innervate the insula, Hcrt-1 receptors are located on insular cells, and blockade of Hcrt-1 receptors in the insula but not in the adjacent somatosensory cortex decreases nicotine self-administration. These data demonstrate that insular hypocretin transmission plays a permissive role in the motivational properties of nicotine, and therefore may be a key neurobiological substrate necessary for maintaining tobacco addiction in human smokers.

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics.

Abstract The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals.

Blocking Lactate Export by Inhibiting the Myc Target MCT1 Disables Glycolysis and Glutathione Synthesis

Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1 and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP, NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide, mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies.

Pharmacological Inhibition of BMK1 Suppresses Tumor Growth through Promyelocytic Leukemia Protein

BMK1 is activated by mitogens and oncogenic signals and, thus, is strongly implicated in tumorigenesis. We found that BMK1 interacted with promyelocytic leukemia protein (PML), and inhibited its tumor-suppressor function through phosphorylation. Furthermore, activated BMK1 notably inhibited PML-dependent activation of p21. To further investigate the BMK-mediated inhibition of the tumor suppressor activity of PML in tumor cells, we developed a small-molecule inhibitor of the kinase activity of BMK1, XMD8-92. Inhibition of BMK1 by XMD8-92 blocked tumor cell proliferation in vitro and significantly inhibited tumor growth in vivo by 95%, demonstrating the efficacy and tolerability of BMK1-targeted cancer treatment in animals.

Full Pharmacological Efficacy of a Novel S1P1 Agonist That Does Not Require S1P-Like Headgroup Interactions

Strong evidence exists for interactions of zwitterionic phosphate and amine groups in sphingosine-1 phosphate (S1P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S1P receptors. The contribution of Arg120 and Glu121 for high-affinity ligand-receptor interactions is essential, because single-point R120A or E121A S1P1 mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly selective S1P1 agonist [2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino) ethanol (CYM-5442)] that does not require Arg120 or Glu121 residues for activating S1P1-dependent p42/p44 mitogen-activated protein kinase phosphorylation, which defines a new hydrophobic pocket in S1P1. CYM-5442 is a full agonist in vitro for S1P1 internalization, phosphorylation, and ubiquitination. It is noteworthy that CYM-5442 was a full agonist for induction and maintenance of S1P1-dependent blood lymphopenia, decreasing B lymphocytes by 65% and T lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose- and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S1P1 activation by CYM-5442 were noncompetitively inhibited by a specific S1P1 antagonist [(R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146)], competitive for S1P, 2-amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720-P), and 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2, 4-oxadiazole (SEW2871). In addition, lymphopenia induced by CYM-5442 was reversed by W146 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S1P1-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket separate from the orthosteric site of S1P binding that is headgroup-dependent.

Cellobiose dehydrogenase–an extracellular fungal flavocytochrome

Wood-degrading fungi, including white-rot and soft-rot fungi as well as at least one brown-rot fungus, produce cellobiose dehydrogenase (CDH). CDH has generated recent interest because of its ability to facilitate the formation of free radicals and because it makes a nice model to study intraprotein electron transfer. While the physiological function of CDH is not known, a considerable portion of this review discusses the strength of the data dealing with individual hypotheses. New evidence dealing with proteolysis of CDH in relationship to the interaction of CDH with lignin and manganese peroxidases are discussed. Additionally, recent information dealing with the catalytic mechanism and reactivity of the individual domains of CDH is detailed.

Genetic regulation of behavioral and neuronal responses to fluoxetine.

Despite widespread use of antidepressants, the factors underlying the behavioral response to antidepressants are unknown. It has been shown that antidepressant treatment promotes the proliferation and survival of neurons in the adult hippocampus via enhanced serotonergic signaling, but it is unclear whether hippocampal neurogenesis is responsible for the behavioral response to antidepressants. Furthermore, a large subpopulation of patients fails to respond to antidepressant treatment due to presumed underlying genetic factors. In the present study, we have used the phenotypic and genotypic variability of inbred mouse strains to show that there is a genetic component to both the behavioral and neuronal effects of chronic fluoxetine treatment, and that this antidepressant induces an increase in hippocampal cell proliferation only in the strains that also show a positive behavioral response to treatment. Furthermore, the behavioral and neuronal responses are associated with an upregulation of genes known to promote neuronal proliferation and survival. These results suggest that inherent genetic predisposition to increased serotonin-induced neurogenesis may be a determinant of antidepressant efficacy.