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-ß 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.

Role of inflammatory cells and adenosine in lung ischemia reoxygenation injury using a model of lung donation after cardiac death.

AIM: The objective of this study is to analyze the role of inflammation in the lung ischemia reperfusion (IR) injury and determine the protective role of adenosine in an in vitro lung transplantation model. MATERIALS AND METHODS: We used a hybrid model of lung donor after cardiac death, with warm ischemia in corpo of varying duration (2 h, 4 h) followed by in vitro lung slices culture for reoxygenation (1 h, 4 h and 24 h), in the presence or not of lymphocytes and of adenosine. To quantify the inflammatory lesions, we performed TNFα, IL2 assays, and histological analysis. RESULTS: In this model of a nonblood perfused system, the addition of lymphocytes during reoxygenation lead to higher rates of TNFα and IL2 after 4 h than after 2 h of warm ischemia (P < .05). These levels increased with the duration of reoxygenation and were maximum at 24 h (P < .05). In the presence of adenosine TNFα and IL2 decreased. After 2 h of warm ischemia, we observed a significant inflammatory infiltration, alveolar thickening and a necrosis of the bronchiolar cells. After 4 h of warm ischemia, alveolar cells necrosis was associated. CONCLUSION: This model showed that lymphocytes increased the inflammatory response and the histological lesions after 4 h of warm ischemia and that adenosine could have an anti-inflammatory role with potential reconditioning action when used in the pneumoplegia solution.

Organotypic lung culture: A new model for studying ischemia and ex vivo perfusion in lung transplantation.

AIM: Donors after cardiac death (DCD) in lung transplantation is considered as a solution for organ shortage. However, it is characterized by warm ischemic period, which could be involved in severe Ischemia-Reperfusion lesion (IR) with early graft dysfunction. We describe a new hybrid model combining in vivo ischemia followed by in vitro reoxygenation using organ-specific culture. MATERIAL AND METHODS: A hybrid model using in vivo ischemic period followed by in vitro lung slice reoxygenation was set up in rat to mimic DCD in lung transplantation with in vitro perfusion. Different markers (bioenergetics, oxidant stress assays, and histology) were measured to evaluate the viability of lung tissue after different ischemic times (I-0, I-1, I-2, I-4, I-15 hours) and reoxygenation times (R-0, R-1, R-4, R-24 hours). RESULTS: No differences were found in cell viability, ATP concentrations, extracellular LDH assays or histology, demonstrating extensive viability of up to 4 hours in lung tissue warm ischemia. We found oxidative stress mainly during the ischemic period with no burst at reoxygenation. Cytosolic anti-oxidant system was involved first (I-0,I-1,I-2) followed by mitochondrial anti-oxidant system for extensive ischemia (I-4). Histological features showed differences in this model of ischemia-reoxygenation between bronchial epithelium and lung parenchymal cells, with epithelium regeneration after 2 hours of warm ischemia and 24 hours of perfusion. CONCLUSION: The results of our hybrid model experiment suggest extensive lung viability of up to 4 hours ischemia. Our model could be an interesting tool to evaluate ex vivo reconditioning techniques after different in vivo lung insults.

The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides.

The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.

Small molecule tertiary amines as agonists of the nuclear hormone receptor Rev-erbα.

The structure-activity relationship study of a small molecule Rev-erbα agonist is reported. The potency and efficacy of the agonists in a cell-based assay were optimized as compared to the initial lead. Modest mouse pharmacokinetics coupled with an improved in vitro profile make 12e a suitable in vivo probe to interrogate the functions of Rev-erbα in animal models of disease.

Synthesis and SAR of tetrahydroisoquinolines as Rev-erbα agonists.

The design and synthesis of a novel series of Rev-erbα agonists is described. The development and optimization of the tetrahydroisoquinoline series was carried out from an earlier acyclic series of Rev-erbα agonists. Through the optimization of the scaffold 1, several potent compounds with good in vivo profiles were discovered.

Simulation of the FDA nozzle benchmark: A lattice Boltzmann study.

BACKGROUND AND OBJECTIVE: Contrary to flows in small intracranial vessels, many blood flow configurations such as those found in aortic vessels and aneurysms involve larger Reynolds numbers and, therefore, transitional or turbulent conditions. Dealing with such systems require both robust and efficient numerical methods. METHODS: We assess here the performance of a lattice Boltzmann solver with full Hermite expansion of the equilibrium and central Hermite moments collision operator at higher Reynolds numbers, especially for under-resolved simulations. To that end the food and drug administration's benchmark nozzle is considered at three different Reynolds numbers covering all regimes: (1) laminar at a Reynolds number of 500, (2) transitional at a Reynolds number of 3500, and (3) low-level turbulence at a Reynolds number of 6500. RESULTS: The lattice Boltzmann results are compared with previously published inter-laboratory experimental data obtained by particle image velocimetry. Our results show good agreement with the experimental measurements throughout the nozzle, demonstrating the good performance of the solver even in under-resolved simulations. CONCLUSION: In this manner, fast but sufficiently accurate numerical predictions can be achieved for flow configurations of practical interest regarding medical applications.

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) inhibitors.

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC(50)=40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.

Synthesis and SAR of 4-(pyrazol-3-yl)-pyridines as novel c-jun N-terminal kinase inhibitors.

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK) inhibitors is described. The development of the 4-(pyrazol-3-yl)-pyridine series was discovered from an earlier pyrimidine series of JNK inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.

Efficient methodology for the synthesis of 3-amino-1,2,4-triazoles.

A general and efficient method for the preparation of 3-amino-1,2,4-triazoles has been developed. The desired 3-amino-1,2,4-triazoles (1) were prepared in good overall yield via two convergent routes. The key intermediate within both routes is substituted hydrazinecarboximidamide derivative 2.

DABMA: A Derivative of ABMA with Improved Broad-Spectrum Inhibitory Activity of Toxins and Viruses.

The small molecule ABMA has been previously shown to protect cells against multiple toxins and pathogens including virus, intracellular bacteria, and parasite. Its mechanism of action is directly associated with host endolysosomal pathway rather than targeting toxin or pathogen itself. However, the relationship of its broad-spectrum anti-infection activity and chemical structure is not yet resolved. Here, we synthesized a series of derivatives and compared their activities against diphtheria toxin (DT). Dimethyl-ABMA (DABMA), one of the most potent analogs with about 20-fold improvement in protection efficacy against DT, was identified with a similar mechanism of action to ABMA. Moreover, DABMA exhibited enhanced efficacy against Clostridium difficile toxin B (TcdB), Clostridium sordellii lethal toxin (TcsL), Pseudomonas Exotoxin A (PE) as well as Rabies and Ebola viruses. The results revealed a structure-activity relationship of ABMA, which is a starting point for its clinical development as broad-spectrum drug against existing and emerging infectious diseases.

Retro-2 and its dihydroquinazolinone derivatives inhibit filovirus infection.

Members of the family Filoviridae cause severe, often fatal disease in humans, for which there are no approved vaccines and only a few experimental drugs tested in animal models. Retro-2, a small molecule that inhibits retrograde trafficking of bacterial and plant toxins inside host cells, has been demonstrated to be effective against a range of bacterial and virus pathogens, both in vitro and in animal models. Here, we demonstrated that Retro-2 and its derivatives, Retro-2.1 and compound 25, blocked infection by Ebola virus and Marburg virus in vitro. We show that the derivatives were more potent inhibitors of infection as compared to the parent compound. Pseudotyped virus assays indicated that the compounds affected virus entry into cells while virus particle localization to Niemann-Pick C1-positive compartments showed that they acted at a late step in virus entry. Our work demonstrates a potential for Retro-type drugs to be developed into anti-filoviral therapeutics.

Four-Year Evolution of a Thrombophylaxis Protocol in an Enhanced Recovery After Surgery (ERAS) Program: Recent Results in 485 Patients.

"Enhanced recovery after surgery" (ERAS) protocols may reduce morbidity, length of hospital stay (LOS), and costs. During the 4-year evolution of a bariatric ERAS protocol, we found that administration of thrombophylaxis selectively to high-risk morbidly obese patients (assessed postoperatively by Caprini score ≥ 3) undergoing omega loop gastric bypass ("mini" gastric bypass) or sleeve gastrectomy resulted in safe outcomes. Both procedures proved equally effective with this protocol. The vast majority of rapidly mobilized, low-risk patients did not appear to require antithrombotic heparin. Similar to other reported ERAS outcomes, our recent year's results in 485 patients included a mean LOS of 1.08 ± 0.64 days (range 1-14), with 460 (95.0%) discharged on day 1 and 99.6% by day 2. There were 13 30-day complications (2.7%), two reinterventions (0.4%), and no hemorrhages.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

Organocatalysed multicomponent synthesis of pyrazolidinones: Meldrum's acid approach.

We discovered a novel organocatalysed multicomponent domino Knoevenagel-aza-Michael-cyclocondensation reaction leading to an unprecedented straightforward synthesis of 1,5-diazabicyclo[3.3.0]octane-2,6-diones. The specific capability of the (DHQ)2PHAL organocatalyst in this process was also highlighted to eventually furnish the corresponding bicyclopyrazolidinones with up to 96 : 4 er.

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins.

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin.

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.

Synthesis of new chiral 6-carbonyl 2,3,8,8a-tetrahydro-7H-oxazolo[3,2-a]pyridines.

[reaction: see text] The preparation of new chiral 6-carbonyl 2,3,8,8a-tetrahydro-7H-oxazolo[3,2-a]pyridines by an efficient two-step procedure is described.