Memory deficits in a juvenile rat model of type 1 diabetes are due to excess 11ß-HSD1 activity, which is upregulated by high glucose concentrations rather than insulin deficiency.

AIMS/HYPOTHESIS: Children with diabetes may display cognitive alterations although vascular disorders have not yet appeared. Variations in glucose levels together with relative insulin deficiency in treated type 1 diabetes have been reported to impact brain function indirectly through dysregulation of the hypothalamus-pituitary-adrenal axis. We have recently shown that enhancement of glucocorticoid levels in children with type 1 diabetes is dependent not only on glucocorticoid secretion but also on glucocorticoid tissue concentrations, which is linked to 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity. Hypothalamus-pituitary-adrenal axis dysfunction and memory alteration were further dissected in a juvenile rat model of diabetes showing that excess 11ß-HSD1 activity within the hippocampus is associated with hippocampal-dependent memory deficits. Here, to investigate the causal relationships between diabetes, 11ß-HSD1 activity and hippocampus-dependent memory deficits, we evaluated the beneficial effect of 11ß-HSD1 inhibition on hippocampal-related memory in juvenile diabetic rats. We also examined whether diabetes-associated enhancement of hippocampal 11ß-HSD1 activity is due to an increase in brain glucose concentrations and/or a decrease in insulin signalling. METHODS: Diabetes was induced in juvenile rats by daily i.p. injection of streptozotocin for 2 consecutive days. Inhibition of 11ß-HSD1 was obtained by administrating the compound UE2316 twice daily by gavage for 3 weeks, after which hippocampal-dependent object location memory was assessed. Hippocampal 11ß-HSD1 activity was estimated by the ratio of corticosterone/dehydrocorticosterone measured by LC/MS. Regulation of 11ß-HSD1 activity in response to changes in glucose or insulin levels was determined ex vivo on acute brain hippocampal slices. The insulin regulation of 11ß-HSD1 was further examined in vivo using virally mediated knockdown of insulin receptor expression specifically in the hippocampus. RESULTS: Our data show that inhibiting 11ß-HSD1 activity prevents hippocampal-related memory deficits in diabetic juvenile rats. A significant increase (53.0±9.9%) in hippocampal 11ß-HSD1 activity was found in hippocampal slices incubated in high glucose conditions (13.9 mmol/l) vs normal glucose conditions (2.8 mmol/l) without insulin. However, 11ß-HSD1 activity was not affected by variations in insulin concentration either in the hippocampal slices or after a decrease in hippocampal insulin receptor expression. CONCLUSIONS/INTERPRETATION: Together, these data demonstrate that an increase in 11ß-HSD1 activity contributes to memory deficits observed in juvenile diabetic rats and that an excess of hippocampal 11ß-HSD1 activity stems from high glucose levels rather than insulin deficiency. 11ß-HSD1 might be a therapeutic target for treating cognitive impairments associated with diabetes.

11Beta-hydroxysteroid dehydrogenase-1 deficiency or inhibition enhances hepatic myofibroblast activation in murine liver fibrosis.

A hallmark of chronic liver injury is fibrosis, with accumulation of extracellular matrix orchestrated by activated hepatic stellate cells (HSCs). Glucocorticoids limit HSC activation in vitro, and tissue glucocorticoid levels are amplified by 11beta-hydroxysteroid dehydrogenase-1 (11ßHSD1). Although 11ßHSD1 inhibitors have been developed for type 2 diabetes mellitus and improve diet-induced fatty liver in various mouse models, effects on the progression and/or resolution of liver injury and consequent fibrosis have not been characterized. We have used the reversible carbon tetrachloride-induced model of hepatocyte injury and liver fibrosis to show that in two models of genetic 11ßHSD1 deficiency (global, Hsd11b1-/- , and hepatic myofibroblast-specific, Hsd11b1fl/fl /Pdgfrb-cre) 11ßHSD1 pharmacological inhibition in vivo exacerbates hepatic myofibroblast activation and liver fibrosis. In contrast, liver injury and fibrosis in hepatocyte-specific Hsd11b1fl/fl /albumin-cre mice did not differ from that of controls, ruling out 11ßHSD1 deficiency in hepatocytes as the cause of the increased fibrosis. In primary HSC culture, glucocorticoids inhibited expression of the key profibrotic genes Acta2 and Col1α1, an effect attenuated by the 11ßHSD1 inhibitor [4-(2-chlorophenyl-4-fluoro-1-piperidinyl][5-(1H-pyrazol-4-yl)-3-thienyl]-methanone. HSCs from Hsd11b1-/- and Hsd11b1fl/fl /Pdgfrb-cre mice expressed higher levels of Acta2 and Col1α1 and were correspondingly more potently activated. In vivo [4-(2-chlorophenyl-4-fluoro-1-piperidinyl][5-(1H-pyrazol-4-yl)-3-thienyl]-methanone administration prior to chemical injury recapitulated findings in Hsd11b1-/- mice, including greater fibrosis. CONCLUSION: 11ßHSD1 deficiency enhances myofibroblast activation and promotes initial fibrosis following chemical liver injury; hence, the effects of 11ßHSD1 inhibitors on liver injury and repair are likely to be context-dependent and deserve careful scrutiny as these compounds are developed for chronic diseases including metabolic syndrome and dementia. (Hepatology 2018;67:2167-2181).

Exploring N-acyl-4-azatetracyclo[5.3.2.02,6.08,10]dodec-11-enes as 11ß-HSD1 Inhibitors.

We recently found that a cyclohexanecarboxamide derived from 4-azatetracyclo[5.3.2.02,6.08,10]dodec-11-ene displayed low nanomolar inhibition of 11ß-HSD1. In continuation of our efforts to discover potent and selective 11ß-HSD1 inhibitors, herein we explored several replacements for the cyclohexane ring. Some derivatives exhibited potent inhibitory activity against human 11ß-HSD1, although with low selectivity over the isoenzyme 11ß-HSD2, and poor microsomal stability.

The discovery of potent and selective kynurenine 3-monooxygenase inhibitors for the treatment of acute pancreatitis.

A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.

Elevated hepatic 11ß-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia.

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. 11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) 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 11ßHSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11ßHSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11ßHSD 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, 11ßHSD1(-/-) mice and rats treated with a specific 11ßHSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11ßHSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11ßHSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.

Novel 11ß-HSD1 inhibitors: C-1 versus C-2 substitution and effect of the introduction of an oxygen atom in the adamantane scaffold.

The adamantane scaffold is found in several marketed drugs and in many investigational 11ß-HSD1 inhibitors. Interestingly, all the clinically approved adamantane derivatives are C-1 substituted. We demonstrate that, in a series of paired adamantane isomers, substitution of the adamantane in C-2 is preferred over the substitution at C-1 and is necessary for potency at human 11ß-HSD1. Furthermore, the introduction of an oxygen atom in the hydrocarbon scaffold of adamantane is deleterious to 11ß-HSD1 inhibition. Molecular modeling studies provide a basis to rationalize these features.

Searching for novel applications of the benzohomoadamantane scaffold in medicinal chemistry: Synthesis of novel 11ß-HSD1 inhibitors.

The structural and physicochemical properties of the adamantane nucleus account for its use as a chemical scaffold in multiple drugs. In the last years, we have developed new polycyclic scaffolds as surrogates of the adamantane group with encouraging results in multiple targets. As adamantane is a common structural feature in several 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors, we have explored the ability of the 6,7,8,9,10,11-hexahydro-5H-5,9:7,11-dimethanobenzo[9]annulen-7-yl scaffold to act as a surrogate of the adamantane nucleus in a novel series of 11ß-HSD1 inhibitors. Of note, within this family of compounds one derivative is endowed with submicromolar 11ß-HSD1 inhibitory activity. Molecular modeling studies support the binding of the compounds to the active site of the enzyme. However, a fine tuning of the hydrophobicity of the size-expanded nucleus may be beneficial for the inhibitory potency.

Mass spectrometry imaging for dissecting steroid intracrinology within target tissues.

Steroid concentrations within tissues are modulated by intracellular enzymes. Such "steroid intracrinology" influences hormone-dependent cancers and obesity and provides targets for pharmacological inhibition. However, no high resolution methods exist to quantify steroids within target tissues. We developed mass spectrometry imaging (MSI), combining matrix assisted laser desorption ionization with on-tissue derivatization with Girard T and Fourier transform ion cyclotron resonance mass spectrometry, to quantify substrate and product (11-dehydrocorticosterone and corticosterone) of the glucocorticoid-amplifying enzyme 11ß-HSD1. Regional steroid distribution was imaged at 150-200 µm resolution in rat adrenal gland and mouse brain sections and confirmed with collision induced dissociation/liquid extraction surface analysis. In brains of mice with 11ß-HSD1 deficiency or inhibition, MSI quantified changes in subregional corticosterone/11-dehydrocorticosterone ratio, distribution of inhibitor, and accumulation of the alternative 11ß-HSD1 substrate, 7-ketocholesterol. MSI data correlated well with LC-MS/MS in whole brain homogenates. MSI with derivatization is a powerful new tool to investigate steroid biology within tissues.

Discovery, synthesis and in combo studies of a tetrazole analogue of clofibric acid as a potent hypoglycemic agent.

A tetrazole isosteric analogue of clofibric acid (1) was prepared using a short synthetic route and was characterized by elemental analysis, NMR ((1)H, (13)C) spectroscopy, and single-crystal X-ray diffraction. The in vitro inhibitory activity of 1 against 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) was evaluated, showing a moderate inhibitory enzyme activity (51.17% of inhibition at 10 µM), being more active than clofibrate and clofibric acid. The antidiabetic activity of compound 1 was determined at 50 mg/Kg single dose using a non insulin dependent diabetes mellitus rat model. The results indicated a significant decrease of plasma glucose levels, during the 7h post-administration. Additionally, we performed a molecular docking of 1 into the ligand binding pocket of one subunit of human 11ß-HSD1. In this model, compound 1 binds into the catalytic site of 11ß-HSD1 in two different orientations. Both of them, show important short contacts with the catalytic residues Ser 170, Tyr 183, Asp 259 and also with the nicotinamide ring of NADP(+).

11ß-HSD1 inhibition does not affect murine tumour angiogenesis but may exert a selective effect on tumour growth by modulating inflammation and fibrosis.

Glucocorticoids inhibit angiogenesis by activating the glucocorticoid receptor. Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) reduces tissue-specific glucocorticoid action and promotes angiogenesis in murine models of myocardial infarction. Angiogenesis is important in the growth of some solid tumours. This study used murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC) to test the hypothesis that 11ß-HSD1 inhibition promotes angiogenesis and subsequent tumour growth. SCC or PDAC cells were injected into female FVB/N or C57BL6/J mice fed either standard diet, or diet containing the 11ß-HSD1 inhibitor UE2316. SCC tumours grew more rapidly in UE2316-treated mice, reaching a larger (P<0.01) final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). However, PDAC tumour growth was unaffected. Immunofluorescent analysis of SCC tumours did not show differences in vessel density (CD31/alpha-smooth muscle actin) or cell proliferation (Ki67) after 11ß-HSD1 inhibition, and immunohistochemistry of SCC tumours did not show changes in inflammatory cell (CD3- or F4/80-positive) infiltration. In culture, the growth/viability (assessed by live cell imaging) of SCC cells was not affected by UE2316 or corticosterone. Second Harmonic Generation microscopy showed that UE2316 reduced Type I collagen (P<0.001), whilst RNA-sequencing revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated SCC tumours. 11ß-HSD1 inhibition increases SCC tumour growth, likely via suppression of inflammatory/immune cell signalling and extracellular matrix deposition, but does not promote tumour angiogenesis or growth of all solid tumours.

Kynurenine monooxygenase regulates inflammation during critical illness and recovery in experimental acute pancreatitis.

Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but the link between KMO and systemic inflammation has eluded discovery until now. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signaling to exacerbate systemic inflammation during experimental AP. We find a tissue-specific role for KMO, where mice lacking Kmo solely in hepatocytes have elevated plasma 3-hydroxykynurenine levels that prime inflammatory gene transcription. 3-Hydroxykynurenine synergizes with interleukin-1ß to cause cellular apoptosis. Critically, mice with elevated 3-hydroxykynurenine succumb fatally earlier and more readily to experimental AP. Therapeutically, blockade with the highly selective KMO inhibitor GSK898 rescues the phenotype, reducing 3-hydroxykynurenine and protecting against critical illness and death. Together, our findings establish KMO and 3-hydroxykynurenine as regulators of inflammation and the innate immune response to sterile inflammation. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.

Partial deficiency or short-term inhibition of 11beta-hydroxysteroid dehydrogenase type 1 improves cognitive function in aging mice.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) regenerates active glucocorticoids (GCs) from intrinsically inert 11-keto substrates inside cells, including neurons, thus amplifying steroid action. Excess GC action exerts deleterious effects on the hippocampus and causes impaired spatial memory, a key feature of age-related cognitive dysfunction. Mice with complete deficiency of 11ß-HSD1 are protected from spatial memory impairments with aging. Here, we tested whether lifelong or short-term decreases in 11ß-HSD1 activity are sufficient to alter cognitive function in aged mice. Aged (24 months old) heterozygous male 11ß-HSD1 knock-out mice, with ∼60% reduction in hippocampal 11ß-reductase activity throughout life, were protected against spatial memory impairments in the Y-maze compared to age-matched congenic C57BL/6J controls. Pharmacological treatment of aged C57BL/6J mice with a selective 11ß-HSD1 inhibitor (UE1961) for 10 d improved spatial memory performance in the Y-maze (59% greater time in novel arm than vehicle control). These data support the use of selective 11ß-HSD1 inhibitors in the treatment of age-related cognitive impairments.

Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Modulation of 11beta-hydroxysteroid dehydrogenase type 1 activity by 1,5-substituted 1H-tetrazoles.

Inhibitors of 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) show promise as drugs to treat metabolic disease and CNS disorders such as cognitive impairment. A series of 1,5-substituted 1H-tetrazole 11beta-HSD1 inhibitors has been discovered and chemically modified. Compounds are selective for 11beta-HSD1 over 11beta-HSD2 and possess good cellular potency in human and murine 11beta-HSD1 assays. A range of in vitro stabilities are observed in human liver microsome assays.

Vitamin A deficiency impairs contextual fear memory in rats: Abnormalities in the glucocorticoid pathway.

Vitamin A and its active metabolite, retinoic acid (RA), play a key role in the maintenance of cognitive functions in the adult brain. Depletion of RA using the vitamin A deficiency (VAD) model in Wistar rats leads to spatial memory deficits in relation to elevated intrahippocampal basal corticosterone (CORT) levels and increased hippocampal 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity. All of these effects are normalised by vitamin A supplementation. However, it is unknown whether vitamin A status also modulates contextual fear conditioning (CFC) in a glucocorticoid-associated fear memory task dependent on the functional integrity of the hippocampus. In the present study, we investigated the impact of VAD and vitamin A supplementation in adult male rats on fear memory processing, plasma CORT levels, hippocampal retinoid receptors and 11ß-HSD1 expression following a novelty-induced stress. We also examined whether vitamin A supplementation or a single injection of UE2316, a selective 11ß-HSD1 inhibitor, known to modulate local glucocorticoid levels, had any beneficial effects on contextual fear memory and biochemical parameters in VAD rats. We provide evidence that VAD rats exhibit a decreased fear conditioning response during training with a poor contextual fear memory 24 hours later. These VAD-induced cognitive impairments are associated with elevated plasma CORT levels under basal conditions, as well as following a stressful event, with saturated CORT release, altered hippocampal retinoid receptors and 11ß-HSD1 expression. Vitamin A supplementation normalises VAD-induced fear conditioning training deficits and all biochemical effects, although it cannot prevent fear memory deficits. Moreover, a single injection of UE2316 not only impairs contextual fear memory, but also reduces plasma CORT levels, regardless of the vitamin A status and decreases slightly hippocampal 11ß-HSD1 activity in VAD rats following stress. The present study highlights the importance of vitamin A status with respect to modulating fear memory conditioning in relation to plasma CORT levels and hippocampal 11ß-HSD1.

Kynurenine 3-monooxygenase is a critical regulator of renal ischemia-reperfusion injury.

Acute kidney injury (AKI) following ischemia-reperfusion injury (IRI) has a high mortality and lacks specific therapies. Here, we report that mice lacking kynurenine 3-monooxygenase (KMO) activity (Kmonull mice) are protected against AKI after renal IRI. We show that KMO is highly expressed in the kidney and exerts major metabolic control over the biologically active kynurenine metabolites 3-hydroxykynurenine, kynurenic acid, and downstream metabolites. In experimental AKI induced by kidney IRI, Kmonull mice had preserved renal function, reduced renal tubular cell injury, and fewer infiltrating neutrophils compared with wild-type (Kmowt) control mice. Together, these data confirm that flux through KMO contributes to AKI after IRI, and supports the rationale for KMO inhibition as a therapeutic strategy to protect against AKI during critical illness.

Antidiabetic activity of N-(6-substituted-1,3-benzothiazol-2-yl)benzenesulfonamides.

N-(6-Substituted-1,3-benzothiazol-2-yl)benzenesulfonamide derivatives 1-8 were synthesized and evaluated for their in vivo antidiabetic activity in a non-insulin-dependent diabetes mellitus rat model. Several compounds synthesized showed significant lowering of plasma glucose level in this model. As a possible mode of action, the compounds were in vitro evaluated as 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibitors. The most active compounds (3 and 4) were docked into the crystal structure of 11beta-HSD1. Docking results indicate potential hydrogen bond interactions with catalytic amino acid residues.

Diclofenac Identified as a Kynurenine 3-Monooxygenase Binder and Inhibitor by Molecular Similarity Techniques.

In this study, we apply a battery of molecular similarity techniques to known inhibitors of kynurenine 3-monooxygenase (KMO), querying each against a repository of approved, experimental, nutraceutical, and illicit drugs. Four compounds are assayed against KMO. Subsequently, diclofenac (also known by the trade names Voltaren, Voltarol, Aclonac, and Cataflam) has been confirmed as a human KMO protein binder and inhibitor in cell lysate with low micromolar KD and IC50, respectively, and low millimolar cellular IC50. Hit to drug hopping, as exemplified here for one of the most successful anti-inflammatory medicines ever invented, holds great promise for expansion into new disease areas and highlights the not-yet-fully-exploited potential of drug repurposing.

Corrigendum: Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness.

This corrects the article DOI: 10.1038/nm.4115.

Design, synthesis and in vivo study of novel pyrrolidine-based 11ß-HSD1 inhibitors for age-related cognitive dysfunction.

Recent findings suggest that treatment with 11ß-HSD1 inhibitors provides a novel approach to deal with age-related cognitive dysfunctions, including Alzheimer's disease. In this work we report potent 11ß-HSD1 inhibitors featuring unexplored pyrrolidine-based polycyclic substituents. A selected candidate administered to 12-month-old SAMP8 mice for four weeks prevented memory deficits and displayed a neuroprotective action. This is the first time that 11ß-HSD1 inhibitors have been studied in this broadly-used mouse model of accelerated senescence and late-onset Alzheimer's disease.