Piperazine sulfonamide BACE1 inhibitors: Design,synthesis, and in vivo characterization

With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2′ sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral Aβ₄₀ in transgenic mice with a single subcutaneous dose.     

Discovery of novel,orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.     

Optimization of orally bioavailable alkyl amine renin inhibitors

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC₅₀ of 0.83 nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10 mg/kg resulted in >20 h reduction of blood pressure in a double transgenic rat model of hypertension.     

The habenula prevents helpless behavior in larval zebrafish

Animals quickly learn to avoid predictable danger. However, if pre-exposed to a strong stressor, they do not display avoidance even if this causes continued contact with painful stimuli [1, 2]. In rodents, lesioning the habenula, an epithalamic structure that regulates the monoaminergic system, has been reported to reduce avoidance deficits caused by inescapable shock [3]. This is consistent with findings that inability to overcome a stressor is accompanied by an increase in serotonin levels [4]. However, other studies conclude that habenula lesions cause avoidance deficits [5, 6]. These contradictory results may be caused by lesions affecting unintended regions [6]. To clarify the role of the habenula, we used larval zebrafish, whose transparency and amenability to genetic manipulation enables more precise disruption of cells. We show that larval zebrafish learn to avoid a light that has been paired with a mild shock but fail to do so when pre-exposed to inescapable shock. Photobleaching of habenula afferents expressing the photosensitizer KillerRed causes a similar failure in avoidance. Expression of tetanus toxin in dorsal habenula neurons is sufficient to prevent avoidance. We suggest that this region may signal the ability to control a stressor, and that its disruption could contribute to anxiety disorders.     

Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety: Synthesis,biological evaluation and nitric oxide release studies

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a–b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a–b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4–5.8% range). In comparison, the percentage NO released was higher (3.1–8.4% range) when these nitrate prodrugs were incubated in the presence of l-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure–activity relationship data acquired showed that compounds having a MeSO₂ COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H₂NSO₂ substituent. Compounds having a MeSO₂ COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED₅₀ = 132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED₅₀ = 118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED₅₀ = 128.7 mg/kg po) but lower than ibuprofen (ED₅₀ = 67.4 mg/kg po).     

Bioactivity-guided isolation of mosquitocidal constituents from the rhizomes of Plumbago capensis Thunb

A bioassay-guided fractionation and chemical examination of chloroform extract of Plumbago capensis roots resulted in isolation and characterization of two new napthaquinone derivatives (4, 8) along with six known compounds (1–3, 5–7). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds were tested for their mosquito larvicidal activity against fourth instar larvae of Aedes aegypti, and compared with that of rotenone. Among the tested compounds, isoshinanolone (3) and plumbagin (1) showed excellent toxicity with LC₅₀ values of 1.26 and 5.43 μg/mL. New compound (8) displayed moderate toxicity against the tested mosquito species.     

<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> (Fenugreek) Protects Against Selenite-Induced Oxidative Stress in Experimental Cataractogenesis

Cataract is the opacification in eye lens and leads to 50% of blindness worldwide. The present study was undertaken to evaluate the anticataract potential of Trigonella foenum-graecum Linn seeds (fenugreek) in selenite-induced in vitro and in vivo cataract. In vitro enucleated rat lenses were maintained in organ culture containing Dulbecco’s modified Eagles medium (DMEM) alone or in addition with 100 μM selenite and served as the normal and control groups, respectively. For the test group, the medium was supplemented with selenite and T. foenum-graecum aqueous extract. The lenses were incubated for 24 h at 37°C. After incubation, the lenses were processed for the estimation of reduced glutathione (GSH), lipid peroxidation product (malondialdehyde), and the antioxidant enzymes. In vivo selenite cataract was induced in 9-day-old rats by subcutaneous injection of sodium selenite (25 μmol/kg body weight). Animals in the test group were injected with different doses of aqueous extract of T. foenum-graecum 4 h before the selenite challenge. A fall in GSH and a rise in malondialdehyde levels were observed in control as compared to normal lenses. T. foenum-graecum significantly (P < 0.01) restored glutathione and decreased malondialdehyde levels. A significant restoration in the activities of antioxidant enzymes such as superoxide dismutase (P < 0.01), catalase, (P < 0.01), glutathione peroxidase (P < 0.01), and glutathione-S-transferase (P < 0.01) was observed in the T. foenum-graecum supplemented group as compared to control. In vivo, none of the eyes was found with nuclear cataract in treated group as opposed to 72.5% in the control group. T. foenum-graecum protects against experimental cataract by virtue of its antioxidant properties. Further studies are warranted to explore its role in human cataract.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Candida albicans PEP12 Is Required for Biofilm Integrity and In Vivo Virulence

To investigate the role of the prevacuolar secretion pathway in biofilm formation and virulence in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae prevacuolar trafficking gene PEP12. C. albicans PEP12 encodes a deduced t-SNARE that is 28% identical to S. cerevisiae Pep12p, and plasmids bearing C. albicans PEP12 complemented the abnormal vacuolar morphology and temperature-sensitive growth of an S. cerevisiae pep12 null mutant. The C. albicans pep12 Δ null mutant was defective in endocytosis and vacuolar acidification and accumulated 40- to 60-nm cytoplasmic vesicles near the plasma membrane. Secretory defects included increased extracellular proteolytic activity and absent lipolytic activity. The pep12Δ null mutant was more sensitive to cell wall stresses and antifungal agents than the isogenic complemented strain or the control strain DAY185. Notably, the biofilm formed by the pep12Δ mutant was reduced in overall mass and fragmented completely upon the slightest disturbance. The pep12Δ mutant was markedly reduced in virulence in an in vitro macrophage infection model and an in vivo mouse model of disseminated candidiasis. These results suggest that C. albicans PEP12 plays a key role in biofilm integrity and in vivo virulence.In Saccharomyces cerevisiae, distinct secreted marker proteins are trafficked differentially through a prevacuolar compartment (PVC) prior to exocytosis (14). Furthermore, prevacuolar protein sorting genes play an important role in cargo transport in the prevacuolar branch of the exocytic pathway in S. cerevisiae (13, 15). By isolating dense- and light-vesicle populations in S. cerevisiae vps1 sec6-4, vps4 sec6-4, and pep12 sec6-4 mutants, it was observed that mutants blocked in this prevacuolar pathway missort marker proteins that are normally found in high-density post-Golgi compartment vesicles into low-density vesicles (15). Gurunathan et al. (13) also demonstrated these findings for vps1 and pep12 mutants with a late secretory mutant (snc1) background similar to that of the sec6-4 strains. These results indicate that some exocytic cargo, including the conditionally regulated soluble secretory proteins invertase and acid phosphatase, are differentially sorted through a PVC prior to exocytosis in the model yeast S. cerevisiae.To study the prevacuolar branch of exocytosis in Candida albicans and its role in virulence, we have previously cloned and analyzed the C. albicans prevacuolar trafficking genes VPS1 and VPS4. We demonstrated that C. albicans VPS4 is required for extracellular secretion of Sap2p and Sap4-6p and for virulence in an in vivo model of disseminated candidiasis (19, 20). C. albicans VPS1 is required for Sap2p secretion and biofilm formation (4). Interestingly, although the C. albicans null mutant lacking VPS4 forms a biofilm that is denser than that formed by the isogenic reintegrant strain, the conditional mutant lacking VPS1 expression forms a patchy biofilm of reduced density (4, 34). Thus, it appears that interference with normal prevacuolar trafficking affects both the secretion of virulence-associated proteins and biofilm formation.S. cerevisiae PEP12 encodes a 288-amino-acid syntaxin which regulates docking of Golgi compartment-derived transport vesicles at the PVC (3). Pep12p interacts with the v-SNARE Vti1p, and overexpression of Pep12p suppresses extracellular missorting of carboxypeptidase in the vti1 mutant (37). The S. cerevisiae pep12 null mutant displays a temperature-sensitive growth defect and is characterized by an enlarged vacuole with morphology defined as class D (3). A search of the C. albicans genome database identified a structural homolog of S. cerevisiae PEP12. Thus, the experiments described below were designed to determine whether the C. albicans PEP12 homolog is functionally homologous to S. cerevisiae PEP12 and to investigate its role in secretion, biofilm formation, and virulence.     

Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.