Discovery of benzoheterocyclic-substituted amide derivatives as apoptosis signal-regulating kinase 1 (ASK1) inhibitors.

Three series of benzoheterocyclic-substituted amide derivatives were designed and synthesized as potent ASK1 inhibitors in this work. After undergoing continuous structural optimization, compound 17a was discovered to be a novel inhibitor of ASK1 with good potency (kinase, IC50 = 26 nM), noteworthy liver microsomal stability (human, T1/2 = 340.4 min), good pharmacokinetic parameters (rat, T1/2 p.o. = 2.11 h, AUClast p.o. = 10 900 h ng mL-1) and high oral bioavailability (rat, F = 97.9%), while also being inactive towards hERG (IC50 > 10 µM).

Effectively removing the homodimer in bispecific antibodies by weak partitioning mode of anion exchange chromatography.

Small amounts of by-products are nevertheless created during the recombinant production of IgG-like bispecific antibodies due to imbalanced chain expression and improper chain pairing, despite the employment of molecular strategy techniques to promote accurate pairing. Among them, homodimers represent the species that are more difficult to remove due to their physical and chemical properties being similar to the target antibody. Homodimer by-products are always produced even though various technologies can significantly increase the expression of heterodimers, so a robust purification process to recover high-purity heterodimers is required. Most of the chromatography methods commonly adopt the bind-and-elute mode or two-step to separate homodimers, which has numerous drawbacks such as prolonged process times and limited dynamic binding capacity. Flow-through mode of anion exchange is a frequently-used polishing step for antibodies, but it is typically regarded as being more effective for host-cell protein or host-cell DNA removal rather than other product-related impurities such as homodimers and aggregates. This paper demonstrated that single-step anion exchange chromatography allows high capacity and effective clearance of the homodimer byproduct to be simultaneously achieved, suggesting that weak partitioning was a better polishing strategy for achieving a high level of heterodimer purity. And robust operation range of anion exchange chromatography steps for homodimer removal was also developed by leveraging the design of experiments.

A Comprehensive Study of High Cholesterol Diet-Induced Larval Zebrafish Model: A Short-Time In Vivo Screening Method for Non-Alcoholic Fatty Liver Disease Drugs.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease around the world. However, there is still no drug for NAFLD in the market, the study of potential therapeutic drugs on NAFLD is extraordinarily pressing and urgent. The rodent models for NAFLD drugs' study are always with a long time cost. Therefore, we aim to establish a short-time NAFLD drug screening model. A laboratory-made high cholesterol diet was used on larval zebrafish for 3 weeks to establish the NAFLD screen model. Lipid metabolism, oxidant stress, and pathology were studied to comprehensively demonstrate the whole spectrum of NAFLD on this model. Bezafibrate and pioglitazone were used to evaluate the model. Moreover, mechanism research was performed on this model.The NAFLD larval zebrafish model was established with the comprehensive process of NAFLD. Moreover, multiple index on lipid metabolism, oxidant stress, hepatic steatosis, and hepatic inflammation can be easily tested for drug screening. Furthermore, this model can be used to perform the mechanism research by testing mRNA expression. The NAFLD larval zebrafish model is a comprehensive short-time screening method for NAFLD drugs.

Simvastatin therapy in adolescent mice attenuates HFD-induced depression-like behavior by reducing hippocampal neuroinflammation.

BACKGROUND: A high-fat diet (HFD)-induced obesity/hyperlipidemia is accompanied by hormonal and neurochemical changes that can be associated with depression. Emerging studies indicate that simvastatin (SMV, decreasing cholesterol levels) has therapeutic effects on neurological and neuropsychiatric diseases through hippocampal-dependent function. However, the studies on the HFD exposure in adolescent animals, which investigate the neuroprotective effects of SMV on the hippocampal morphology, serotonin (5-HT) system and inflammation, are limited. Hence, the aim of this study was to determine whether SMV attenuates HFD-induced major depressive disorders in adolescent animals and, more specifically, acts as an anti-neuroinflammatory response. METHODS: Twenty-four male C57BL/6 mice were fed a control (n = 8), HFD (n = 8) and HFD + SMV (n = 8) for 14 weeks. In HFD + SMV group, SMV (10 mg/kg) was administrated from the 10th week of HFD feeding. The open field test (OFT) and the tail suspension test (TST) were used to examine the effect of SMV on behavioral performance. HE and Nissl staining were conducted to detect hippocampal morphology and neural survival. Expression of the inflammatory cytokine genes was assayed by quantitative polymerase chain reaction (Q-PCR). RESULTS: Firstly, alterations in lipid parameters were minimized after SMV treatment. HFD-induced depression-like behavior, which was evidenced by an increase in immobility time in TST along with considerable decrease in locomotion activity, was significantly attenuated by SMV therapy for 4 weeks. Additionally, SMV could reduce HFD-induced structural abnormality, neuronal injury, serotonergic system disturbance and pro-inflammatory cytokine over-expression in the hippocampus. Neuroimmunological changes in central hippocampus displayed a similar characteristic (only IL-1ß, IL-6, TNF-α) with that in periphery spleen, whereas they appeared in an entirely opposite trend with that in cerebral cortex. CONCLUSION: Our results suggest that SMV may be a promising treatment for HFD-induced depression-like behavior during adolescent period through brain region-specific neuroninflammatory mechanisms.

Isoquercetin Improves Hepatic Lipid Accumulation by Activating AMPK Pathway and Suppressing TGF-ß Signaling on an HFD-Induced Nonalcoholic Fatty Liver Disease Rat Model.

Isoquercetin (IQ), a glucoside derivative of quercetin, has been reported to have beneficial effects in nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the potential improvement of IQ in liver lipid accumulation, inflammation, oxidative condition, and activation in Kupffer cells (KCs) on a high-fat diet (HFD) induced NAFLD models. Male Sprague-Dawley (SD) rats were induced by HFD, lipopolysaccharides/free fatty acids (LPS/FFA) induced co-culture cells model between primary hepatocytes and Kupffer cells was used to test the effects and the underlying mechanism of IQ. Molecular docking was performed to predict the potential target of IQ. Significant effects of IQ were found on reduced lipid accumulation, inflammation, and oxidative stress. In addition, AMP-activated protein kinase (AMPK) pathway was activated by IQ, and is plays an important role in lipid regulation. Meanwhile, IQ reversed the increase of activated KCs which caused by lipid overload, and also suppression of Transforming growth factor beta (TGF-ß) signaling by TGF-ß Recptor-1 and SMAD2/3 signaling. Finally, TGF-ßR1 and TGF-ßR2 were both found may involve in the mechanism of IQ. IQ can improve hepatic lipid accumulation and decrease inflammation and oxidative stress by its activating AMPK pathway and suppressing TGF-ß signaling to alleviate NAFLD.

Adipose tissue regulates hepatic cholesterol metabolism via adiponectin.

AIMS: Lipid metabolic disorder involves multiple tissues and organs. Hepatic cholesterol metabolism is an important physiological process, which is tightly related to obesity and lipid metabolic disorders. In this study, we examined the direct effects of adipocytes on hepatic cholesterol metabolic factors and investigated the role of potential adipocytokines in it. MAIN METHODS: Male SD rats were induced by a high-fat diet (HFD) and hepatic cholesterol metabolic factors, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) and ATP-binding cassette transporter A1 (ABCA1) were measured by immunoblotting. Then the effects of adipocytes on the expressions of hepatic cholesterol metabolism proteins were examined in the co-culture system. Finally, the concentrations of several adipocytokines were detected by ELISA and the effect of adiponectin (APN) on hepatic cholesterol metabolism was confirmed by short interference RNA (siRNA) in vitro. KEY FINDINGS: Our results showed that adipocytes significantly increased ABCA1 and decreased HMGR in hepatocytes after co-culture. Lipopolysaccharide (LPS) treatment in this co-culture system reversed cholesterol metabolism compared with the untreated group. APN, which also decreased in obese rats, had a significant positive correlation with ABCA1 and inversed correlation with HMGR in vitro. Co-culturing with APN-silenced adipocytes partially restored ABCA1 and HMGR levels. SIGNIFICANCE: The present study demonstrates that adipocytes regulate hepatic cholesterol metabolism partly via APN.

A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example.

A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water-acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays.