A pharmacological screening approach for discovery of neuroprotective compounds in ischemic stroke.
ABSTRACT: With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-?-hydroxysteroid dehydrogenase-1 (11?-HSD1), gave rise to similarly potent in vivo neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11?-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.
Project description:Insulin resistance and associated metabolic sequelae are common in chronic kidney disease (CKD) and are positively and independently associated with increased cardiovascular mortality. However, the pathogenesis has yet to be fully elucidated. 11?-Hydroxysteroid dehydrogenase type 1 (11?HSD1) catalyzes intracellular regeneration of active glucocorticoids, promoting insulin resistance in liver and other metabolic tissues. Using two experimental rat models of CKD (subtotal nephrectomy and adenine diet) which show early insulin resistance, we found that 11?HSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11?HSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11?HSD inhibitor carbenoxolone to uremic rats for 2 wk improved glucose tolerance and insulin sensitivity, improved insulin signaling, and reduced hepatic expression of gluconeogenic and lipogenic genes. Furthermore, 11?HSD1(-/-) mice and rats treated with a specific 11?HSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11?HSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11?HSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.
Project description:<h4>Background</h4>Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11?-hydroxysteroid dehydrogenase 1 (11?-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11?-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11?-HSD1 inhibitors.<h4>Results</h4>The yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11?-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11?-HSD1 inhibitory activity. As proof of concept the correlation between 11?-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11?-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11?-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11?-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times.<h4>Conclusions</h4>Our yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11?-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11?-HSD1 inhibitors at intracellular level.
Project description:Stress slows cutaneous wound healing (WH) in an endogenous glucocorticoid (GC)-dependent fashion. We investigated whether stress/GC-induced delays in WH require further intracutaneous activation of endogenous GC; and whether blockade or down-regulation of peripheral activation normalizes WH in the face of stress. Delayed WH in our motion-restricted murine model of stress could be attributed to elevated systemic GC, because blockade of GC production (using corticotropin-releasing factor inhibitor, antalarmin), or of peripheral binding to the GC receptor [GCr], with an antagonist, Ru-486, normalized WH. We next investigated whether local blockade or down-regulation of the peripheral GC-activating enzyme, 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1), accelerates cutaneous WH. Topical applications of nonspecific (carbenoxolone) as well as an isoform-specific 11?-HSD1 inhibitor overcame stress and exogenous GC-induced delays in WH. Moreover, two liver X receptor ligands, TO901317 and GW3695, down-regulated expression of 11?-HSD1, attenuating stress-induced delays in WH. Combined inhibitor and liver X receptor ligand applications accelerated WH in the face of stress/systemic GC. Thus: (1) intracutaneous conversion of inactive-to-active GC accounts for stress (GC)-induced delays in WH; and (2) blockade or down-regulation of 11?-HSD1 and/or GCr normalize cutaneous WH in the face of stress/GC. Local blockade or down-regulation of cutaneous GC activation could help enhance WH in various clinical settings.
Project description:Activity and selectivity assessment of new bi-aryl amide 11?-hydroxysteroid dehydrogenase 1 (11?-HSD1) inhibitors, prepared in a modular manner via Suzuki cross-coupling, are described. Several compounds inhibiting 11?-HSD1 at nanomolar concentrations were identified. Compounds 2b, 3e, 7b and 12e were shown to selectively inhibit 11?-HSD1 over 11?-HSD2, 17?-HSD1 and 17?-HSD2. These inhibitors also potently inhibited 11?-HSD1 activity in intact HEK-293 cells expressing the recombinant enzyme and in intact primary human keratinocytes expressing endogenous 11?-HSD1. Moreover, compounds 2b, 3e and 12e were tested for their activity in human skin biopsies. They were able to prevent, at least in part, both the cortisone- and the UV-mediated decreases in collagen content. Thus, inhibition of 11?-HSD1 by these compounds can be further investigated to delay or prevent UV-mediated skin damage and skin aging.
Project description:Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO2) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1?mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40?min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO2. We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.
Project description:Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11β-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11β-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11β-HSD1 inhibition by carbenoxolone (CBX) in mice treated with corticosterone (CORT) or vehicle for 6 weeks. Mice were treated bidaily with topical CBX or vehicle (VEH) 7 days before wounding and during wound healing. CORT mice displayed skin thinning and impaired wound healing but also increased epidermal integrity. 11β-HSD1 activity was elevated in unwounded CORT skin and was inhibited by CBX. CORT mice treated with CBX displayed 51%, 59%, and 100% normalization of wound healing, epidermal thickness, and epidermal integrity, respectively. Gene expression studies revealed normalization of interleukin 6, keratinocyte growth factor, collagen 1, collagen 3, matrix metalloproteinase 9, and tissue inhibitor of matrix metalloproteinase 4 by CBX during wound healing. Importantly, proinflammatory cytokine expression and resolution of inflammation were unaffected by 11β-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11β-HSD1 inhibition can limit the cutaneous effects of GC excess, which may improve the safety profile of systemic steroids and the prognosis of chronic wounds.
Project description:Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage.We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures.Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons.Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences.
Project description:The microsomal enzyme 11?-hydroxysteroid deydrogenase type 1 (11?-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11?-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11?-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11?-HSD1. Indeed we found that tea extracts inhibited 11?-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (-)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11?-HSD1 activity (reduction: IC50?=?57.99 µM; oxidation: IC50?=?131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki?=?22.68 µM for microsomes and Ki?=?18.74 µM for purified 11?-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11?-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11?-HSD1.
Project description:11?-hydroxysteroid dehydrogenase type?1 (11?-HSD1) plays a key role in converting intracellular cortisone to physiologically active cortisol, which is implicated in the development of several phenotypes of metabolic syndrome. Inhibition of 11?-HSD1 activity with selective inhibitors has beneficial effects on various conditions, including diabetes, dyslipidemia and obesity, and therefore constitutes a promising strategy to discover novel therapies for metabolic and cardiovascular diseases. A series of novel adamantyl heterocyclic ketones provides potent and selective inhibitors of human 11?-HSD1. Lead compounds display low nanomolar inhibition against human and mouse 11?-HSD1 and are selective with no activity against 11?-HSD2 and 17?-HSD1. Selected potent 11?-HSD1 inhibitors show moderate metabolic stability upon incubation with human liver microsomes and weak inhibition of human CYP450 enzymes.
Project description:Reduced adenosine triphosphate (ATP) levels in ischemic stroke constitute an upstream contributor to neuronal cell death. We have recently created a small chemical, named Kyoto University Substance 121 (KUS121), which can reduce cellular ATP consumption. In this study, we examined whether KUS121 has neuroprotective effects in rodent cerebral ischemia models. We evaluated cell viability and ATP levels in vitro after oxygen glucose deprivation (OGD) in rat cortical primary neuronal cultures incubated with or without KUS121. We found that KUS121 protected neurons from cell death under OGD by preventing ATP depletion. We also used in vivo ischemic stroke models of transient distal middle cerebral artery occlusion in C57BL/6 and B-17 mice. Administration of KUS121 in these models improved functional deficits and reduced brain infarction volume after transient focal cerebral ischemia in both C57BL/6 and B-17 mice. These results indicate that KUS121 could be a novel type of neuroprotective drug for ischemic stroke.