Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure–activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.     

Design and synthesis of aryl sulfonamide-based nonsteroidal mineralocorticoid receptor antagonists

Hit-to-lead medicinal chemistry efforts are described starting from a screening hit 1, leading to a new class of aryl sulfonamide-based MR antagonist, exemplified by 17, that possesses favourable MR binding affinity, selectivity profile against closely related NHRs, physicochemical properties and metabolic stability.     

The mineralocorticoid signaling pathway throughout development: Expression,regulation and pathophysiological implications

The mineralocorticoid signaling pathway has gained interest over the past few years, considering not only its implication in numerous pathologies but also its emerging role in physiological processes during kidney, brain, heart and lung development. This review aims at describing the setting and regulation of aldosterone biosynthesis and the expression of the mineralocorticoid receptor (MR), a nuclear receptor mediating aldosterone action in target tissues, during the perinatal period. Specificities concerning MR expression and regulation during the development of several major organs are highlighted. We provide evidence that MR expression is tightly controlled in a tissue-specific manner during development, which could have major pathophysiological implications in the neonatal period.     

A colonic mineralocorticoid receptor cell model expressing epithelial Na channels

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of β- and γ-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.     

Differential response to ischemia in adjacent hippocampalsectors: neuronal death in CA1 versus neurogenesis in dentate gyrus

Two hippocampal sectors show distinct responses to transient ischemia: the cornu Ammonis (CA)1 sector undergoes a delayed neuronal death followed by a lack of neuronal generation, while the dentate gyrus (DG) shows slight postischemic damage followed by an increased neurogenesis. Using the monkey experimental paradigm of transient whole brain global ischemia, the 'calpain-cathepsin hypothesis' was formulated in 1998. This hypothesis proposes that following ischemia calpain compromises the integrity of lysosomal membrane, causing a leakage of degrading hydrolytic enzymes--cathepsins--into the cytoplasm. Ischemia induces Ca(2+) mobilization, calpain activation, lysosomal membrane disruption, and cathepsin release, which all occur specifically in the CA1 sector and cause neuronal death. In the postischemic DG, a vascular niche has been implicated in adult neurogenesis, in that adventitial cells of the DG microvascular environment provoke postischemic up-reguation of neurogenesis with the aid of brain-derived neurotrophic factor and polysialylated form of the neural cell adhesion molecule. In parallel, Down's syndrome cell adhesion molecule has recently been shown to be expressed specifically in the neural progenitor cells of DG. In this review, we focus on the monkey experimental paradigm to reveal the remarkable contrasts between CA1 and DG in response to the ischemic insult.     

Identification of spirooxindole and dibenzoxazepine motifs as potent mineralocorticoid receptor antagonists

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.     

The human mineralocorticoid receptor only partially differentiates between different ligands after expression in fission yeast

Cardiac failure is a major health problem with increasing incidence due to aging of the population. Studies in both experimental animals and humans have suggested that aldosterone excess may have deleterious effects on cardiac function. In order to generate a novel screening system for the identification of aldosterone antagonists, we expressed the human mineralocorticoid receptor (MR) and the human glucocorticoid receptor (GR), respectively, in the fission yeast Schizosaccharomyces pombe. Reporter plasmids containing two hormone-responsive elements upstream of a fission yeast minimal promotor and either a lacZ gene (for quantification) or a neomycin gene (for survival screening) were constructed and cotransformed into fission yeast strains with expression plasmids for MR or GR. The functionality of the reporter systems was then tested using physiological ligands of both receptors as well as known inhibitors. Transactivating activity of MR could be stimulated by aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, cortisone, and spironolactone, but not by progesterone, while GR activity was stimulated by cortisol and cortisone, but also not by progesterone. Taken together, we have succeeded in establishing fission yeast-based screening systems that allow the identification of MR- or GR-interacting compounds. Moreover, our data show that after expression in fission yeast both receptors did not differentiate between steroids with different configurations at positions 11beta, 17 and 18. This finding suggests that only recognition of C-21 substituents may be accomplished by the receptor proteins alone, while the physiologically important selectivity towards other positions of the steroid ligand depends on other factors which are not conserved from fission yeast to man.     

Development of indazole mineralocorticoid receptor antagonists and investigation into their selective late-stage functionalization

The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern CH functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharmaceutical intermediates. We report herein a case study in late-stage functionalization towards the development of a new class of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the electronics of the core indazole heterocycle inspired the use of modern CH borylation chemistry. New reactivity patterns were revealed and studied computationally. Ultimately, a de novo synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic stability.     

神经元再生:抑郁症治疗的新策略

成年哺乳动物一生中,海马等脑区神经元是可以再生的,而海马脑区神经元再生的减少和增多分别是抑郁症发生和恢复的重要因素。如果神经元再生过程被抑制,在抑郁症的动物模型上抗抑郁剂将会失去其行为学效应。长期给予不同种类的抗抑郁剂可以显著地促进动物海马神经元再生。随着对神经元再生调节机制研究的不断深入,为进一步探讨抑郁症的发生机制,以及发展新型抗抑郁治疗药物提供了新的思路与视角。     

Regulation of cardiac and renal mineralocorticoid receptor expression by captopril following myocardial infarction in rats

Myocardial infarction (MI) activates the renin-angiotensin system in the heart and increases local production of aldosterone. This hormone may increase reactive fibrosis in the myocardium favoring heart failure development. To elucidate the potential contribution of aldosterone to cardiac remodeling following MI, we evaluated the expression of mineralocorticoid receptors (MCR) in the left ventricle (LV) and kidney of rats after MI and captopril treatment. MI was induced by ligation of the coronary artery in Wistar rats, which were separated into (1) sham-operated group, (2) MI group, (3) MI-captopril treated group (cap, 50 mg kg(-1) day(-1)). One month later angiotensin converting enzyme (ACE) activity was assayed in the plasma, LV and kidney. Cardiac and renal angiotensin II (Ang II) levels were determined by ELISA and MCR mRNA expression and protein were measured by Taqman RT-PCR and Western blot, respectively. Cardiac MCR mRNA and protein levels increased nearly by 80% after MI and Cap treatment normalized cardiac MCR protein and mRNA expression. Kidney MCR expression was not affected. ACE activity increased 34% in the plasma and 83% in the LV after MI. This increase was prevented by Cap. Ang II concentration increased 225% in the LV and 193% in kidney, which was partially attenuated by Cap. Our data demonstrate upregulation of MCR in the heart following MI what may facilitate the effects of aldosterone in the ventricular remodeling process. ACE inhibitors may reduce reactive fibrosis not only by decreasing Ang II production but also by attenuating the aldosterone-signaling pathway by decreasing the expression of MCR receptors.     

Nifedipine, a calcium channel blocker, inhibits inflammatory and fibrogenic gene expressions in advanced glycation end product (AGE)-exposed fibroblasts via mineralocorticoid receptor antagonistic activity

Advanced glycation end products (AGE) are involved in tissue damage and remodeling. This study investigated whether AGE could elicit inflammatory and fibrogenic reactions in fibroblast cell line MRC-5 cells via autocrine production of aldosterone and if nifedipine could block the AGE actions through mineralocorticoid receptor (MR) antagonistic activity. AGE significantly up-regulated monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β (TGF-β), type III collagen and receptor for AGE (RAGE) mRNA levels in MRC-5 cells, all of which were completely blocked by nifedipine or an MR antagonist spironolactone. Aldosterone also dose-dependently increased MCP-1, TGF-β and type III collagen mRNA levels in MRC-5 cells, which were suppressed by nifedipine, but not amlodipine, a control calcium channel blocker. Further, AGE significantly stimulated aldosterone generation in MRC-5 cells, which was partially blocked by nifedipine or spironolactone. In this study, we demonstrated for the first time that AGE could evoke inflammatory and fibrogenic reactions in MRC-5 cells via aldosterone production, which were blocked by the MR antagonistic activity of nifedipine. Our present study provides a unique beneficial aspect of nifedipine on tissue damage and remodeling; it could work as an anti-inflammatory and anti-fibrogenic agent against AGE via MR antagonistic activity.     

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.     

Loss-of-function of KMT5B leads to neurodevelopmental disorder and impairs neuronal development and neurogenesis

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that cause severe social, communication, and behavioral problems. Recent studies show that the variants of a histone methyltransferase gene KMT5B cause neurodevelopmental disorders (NDDs), including ASD, and the knockout of Kmt5b in mice is embryonic lethal. However, the detailed genotype-phenotype correlations and functional effects of KMT5B in neurodevelopment are unclear. By targeted sequencing of a large Chinese ASD cohort, analyzing published genome-wide sequencing data, and mining literature, we curated 39 KMT5B variants identified from NDD individuals. A genotype-phenotype correlation analysis for 10 individuals with KMT5B pathogenic variants reveals common symptoms, including ASD, intellectual disability, languages problem, and macrocephaly. In vitro knockdown of the expression of Kmt5b in cultured mouse primary cortical neurons leads to a decrease in neuronal dendritic complexity and an increase in dendritic spine density, which can be rescued by expression of human KMT5B but not that of pathogenic de novo missense mutants. In vivo knockdown of the Kmt5b expression in the mouse embryonic cerebral cortex by in utero electroporation results in decreased proliferation and accelerated migration of neural progenitor cells. Our findings reveal essential roles of histone methyltransferase KMT5B in neuronal development, prenatal neurogenesis, and neuronal migration.     

Neuroglobin and neuronal cell survival

The balance between neuronal apoptosis and survival sculpts the developing brain and has an important role in neurodegenerative diseases. Thus, the individuation of signals that could modulate the cell death machinery as well as enhance survival in neurons promises to provide multiple points of therapeutic intervention in neurodegenerative diseases. Neuroglobin (NGB), the first nerve globin identified in neuronal tissues of humans, seems to possess a protective role in the brain only after up-regulation. Here, the NGB physiological role in the control of neuronal survival is reviewed. In vitro studies suggested that cytosolic NGB could react very rapidly with cytochrome c released from mitochondria, thus interfering with the intrinsic pathway of apoptosis. Although very suggestive, these data do not explain either the role of NGB up-regulation in neuroprotection or the recently reported NGB localization into mitochondria. Recently, we identified the steroid hormone 17β-estradiol (E2) as an endogenous modulator of NGB levels in neuroblastoma SK-N-BE cell line. Upon E2 stimulation, NGB reallocates mainly into mitochondria where the association with the mitochondrial cytochrome c occurs. Remarkably, E2 treatment before an apoptotic stimulus strongly enhances the NGB:cytochrome c association reducing cytochrome c release into the cytosol. As a consequence, a decrease of caspase-3 activation and, in turn, of the apoptotic cascade activation take place. Besides E2, other compounds have been reported to up-regulate the NGB expression highlighting the possibility to develop NGB-mediated therapeutic strategies against stroke damage and neurodegenerative diseases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Increased cell proliferation in the adult mouse hippocampus following chronic administration of group II metabotropic glutamate receptor antagonist, MGS0039

We have previously reported that MGS0039, a novel antagonist of group II metabotropic glutamate receptors (mGluRs), exerts antidepressant-like effects in experimental animal models. Recent studies suggest that the behavioral effects of chronic antidepressant treatment are mediated by the stimulation of neurogenesis in the hippocampus. In the present study, we examined the effects of MGS0039 on cell proliferation in the adult mouse hippocampus. MGS0039 (5 or 10mg/kg) or fluvoxamine was administered chronically to male ICR mice over a period of 14 days. Multiple bromodeoxyuridine (BrdU) administrations were performed after the last drug injection to label dividing cells. Immunohistochemical analyses after BrdU injections revealed that chronic MGS0039 treatment enhanced BrdU-positive cells in the dentate gyrus ( approximately 62% increase) in the same manner as chronic fluvoxamine treatment. This is the first in vivo study to demonstrate an increase in cell proliferation following a blockade of group II mGluRs. These findings raise the possibility that MGS0039 may exert antidepressant-like effects by modulating cell proliferation in the hippocampus.