Applications of genetic code expansion in the study of lysine acylation / 中国药科大学学报

@#Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function. However, it can be challenging to decipher the biological function of site-specific acylation modifications in living cells.The recently developed genetic code expansion (GCE) technology has enabled site-specific incorporation of unnatural amino acids (UAAs) that are structurally consistent with the natural acylation modifications in vivo through orthogonal aminoacyl-tRNA synthetase/tRNA pairs, thus facilitating the study of physicochemical properties and biological behaviors of homogeneously acylated proteins.Besides, GCE technology allows for the targeted introduction of UAAs that mimic acylation modifications but cannot be recognized by deacylases, which improves the stability of lysine acylation modification products.Moreover, the insertion of photo-crosslinked UAAs at specific sites of the target protein has been used to elucidate the reciprocal proteome of acylated modified proteins.Based on the introduction of different structural and functional acylation modifications, we described the novel design of GCE technology combined with three types of UAAs, and their application in studying the functional effects of protein acylation modifications on the enzyme activity, protein stability, cellular localization, protein-DNA interactions and protein-protein interactions of target proteins, with a description of the limitations and prospects of GCE technology in studying protein acylation modification.

SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

Farnesoid X receptor (FXR) is widely accepted as a promising target for various liver diseases; however, panels of ligands in drug development show limited clinical benefits, without a clear mechanism. Here, we reveal that acetylation initiates and orchestrates FXR nucleocytoplasmic shuttling and then enhances degradation by the cytosolic E3 ligase CHIP under conditions of liver injury, which represents the major culprit that limits the clinical benefits of FXR agonists against liver diseases. Upon inflammatory and apoptotic stimulation, enhanced FXR acetylation at K217, closed to the nuclear location signal, blocks its recognition by importin KPNA3, thereby preventing its nuclear import. Concomitantly, reduced phosphorylation at T442 within the nuclear export signals promotes its recognition by exportin CRM1, and thereby facilitating FXR export to the cytosol. Acetylation governs nucleocytoplasmic shuttling of FXR, resulting in enhanced cytosolic retention of FXR that is amenable to degradation by CHIP. SIRT1 activators reduce FXR acetylation and prevent its cytosolic degradation. More importantly, SIRT1 activators synergize with FXR agonists in combating acute and chronic liver injuries. In conclusion, these findings innovate a promising strategy to develop therapeutics against liver diseases by combining SIRT1 activators and FXR agonists.

Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1α-dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2, rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia.

Genetic incorporation of unnatural amino acids into proteins and its translational application in biomedicine / 中国药科大学学报

@#Proteins in the human body are usually made of 20 natural amino acids.Through different amino acid combinations and isomerization, proteins of diverse functions are built.An emerging genetic code expansion technology can introduce unnatural amino acids into specific sites of target protein, endowing the protein with new biological characteristics including covalently binding with proximal proteins, carrying fluorescence, and mimicking specific protein post-translational modifications.In this paper, based on the structure and function of unnatural amino acids, the applications of different types of unnatural amino acids in regulating protein''s stability, studying protein''s conformation, expression level, and localization, and uncovering heretofore unknown protein-protein interactions were reviewed.Besides, genetic code expansion of unnatural amino acids is anticipated to find broad utilities in biomedicine by bringing new ideas and methods to the design and optimization of biologics.

Research on the development of bio-pharmaceutical industry integration and collaborative innovation in the Yangtze River Delta under the mode of Industry-University-Research cooperation / 中国药科大学学报

@#After the integration of Yangtze River Delta was elevated and developed into a national strategy, few domestic studies have been conducted to explore the integrated and collaborative development of biopharmaceutical industry in the Yangtze River Delta region, which, as an important region of biopharmaceutical industry in China, has initially formed the development mode of industrial clusters. In this study, based on the industry-university-research model of patent and fund project cooperation, the social network analysis (SNA) method is used to analyze and study the cooperation of patent and Major Projects of National Natural Science Foundation of China in the biopharmaceutical industry in the Yangtze River Delta region. The study shows that the industry-university-research cooperation in the biomedical field in the Yangtze River Delta region is still in the development stage and the degree of integration needs to be improved. Universities and research institutes are more inclined to cooperate with enterprises for invention patents and with other universities for fund project research.It is suggested that the Yangtze River Delta region should adopt the deep integration mode of industry-university-research, relevant leading scientific research institutions should establish open innovation platforms, and pharmaceutical universities should cultivate innovative talents with the advantages of their disciplines, and adopt "Revealing the List and Taking Command" or other ways to tackle key core technologies.

Bile acid coordinates microbiota homeostasis and systemic immunometabolism in cardiometabolic diseases

Cardiometabolic disease (CMD), characterized with metabolic disorder triggered cardiovascular events, is a leading cause of death and disability. Metabolic disorders trigger chronic low-grade inflammation, and actually, a new concept of metaflammation has been proposed to define the state of metabolism connected with immunological adaptations. Amongst the continuously increased list of systemic metabolites in regulation of immune system, bile acids (BAs) represent a distinct class of metabolites implicated in the whole process of CMD development because of its multifaceted roles in shaping systemic immunometabolism. BAs can directly modulate the immune system by either boosting or inhibiting inflammatory responses via diverse mechanisms. Moreover, BAs are key determinants in maintaining the dynamic communication between the host and microbiota. Importantly, BAs via targeting Farnesoid X receptor (FXR) and diverse other nuclear receptors play key roles in regulating metabolic homeostasis of lipids, glucose, and amino acids. Moreover, BAs axis per se is susceptible to inflammatory and metabolic intervention, and thereby BAs axis may constitute a reciprocal regulatory loop in metaflammation. We thus propose that BAs axis represents a core coordinator in integrating systemic immunometabolism implicated in the process of CMD. We provide an updated summary and an intensive discussion about how BAs shape both the innate and adaptive immune system, and how BAs axis function as a core coordinator in integrating metabolic disorder to chronic inflammation in conditions of CMD.

Super-sensitive bifunctional nanoprobe: Self-assembly of peptide-driven nanoparticles demonstrating tumor fluorescence imaging and therapy

The development of nanomedicine has recently achieved several breakthroughs in the field of cancer treatment; however, biocompatibility and targeted penetration of these nanomaterials remain as limitations, which lead to serious side effects and significantly narrow the scope of their application. The self-assembly of intermediate filaments with arginine-glycine-aspartate (RGD) peptide (RGD-IFP) was triggered by the hydrophobic cationic molecule 7-amino actinomycin D (7-AAD) to synthesize a bifunctional nanoparticle that could serve as a fluorescent imaging probe to visualize tumor treatment. The designed RGD-IFP peptide possessed the ability to encapsulate 7-AAD molecules through the formation of hydrogen bonds and hydrophobic interactions by a one-step method. This fluorescent nanoprobe with RGD peptide could be targeted for delivery into tumor cells and released in acidic environments such as endosomes/lysosomes, ultimately inducing cytotoxicity by arresting tumor cell cycling with inserted DNA. It is noteworthy that the RGD-IFP/7-AAD nanoprobe tail-vein injection approach demonstrated not only high tumor-targeted imaging potential, but also potent antitumor therapeutic effects in vivo. The proposed strategy may be used in peptide-driven bifunctional nanoparticles for precise imaging and cancer therapy.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.

Evaluation of representative research works in pharmaceutical field based on Z-index model / 中国药科大学学报

@#This paper aims to explore the feasibility of establishing a representative work screening method based on the academic recognition of the paper. Through comparative analysis of the strong and weak points of representative method, h-index core method, the subjective selection results by faculty applying for higher professional titles, and the academic recognition method established in this research (Q1 Journal-Percentile in Subject Area-Category Normalized Citation Impact, Q1-PSA-CNCI), representative works were introduced into the Z-index to form the Zh, Zr and Zq-index, and the correlations between the indicators were analyzed. Compared with other methods, the number of representative works obtained by the Q1-PSA-CNCI method is more reasonable. There was significant correlation among the indicators (P < 0.05), with no significant difference in the Zq-index among faculty evaluated for different professional titles (P > 0.05). Studies have shown that the Q1-PSA-CNCI method could help to screen representative works and was more reasonable than the number of representative works selected based on the other two methods, so Zq-index could be used as an indicator for representative work evaluation to provide reference for the qualitative evaluation of papers. It is more reasonable to improve the review process with the participation of "small peers".

Protocatechuic aldehyde protects cardiomycoytes against ischemic injury

Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction. Protocatechuic aldehyde (PCA) is a major phenolic acid in Chinese herb Danshen (

Selective inhibition of CDK4/6: A safe and effective strategy for developing anticancer drugs

The sustained cell proliferation resulting from dysregulation of the cell cycle and activation of cyclin-dependent kinases (CDKs) is a hallmark of cancer. The inhibition of CDKs is a highly promising and attractive strategy for the development of anticancer drugs. In particular, third-generation CDK inhibitors can selectively inhibit CDK4/6 and regulate the cell cycle by suppressing the G1 to S phase transition, exhibiting a perfect balance between anticancer efficacy and general toxicity. To date, three selective CDK4/6 inhibitors have received approval from the U.S. Food and Drug Administration (FDA), and 15 CDK4/6 inhibitors are in clinical trials for the treatment of cancers. In this perspective, we discuss the crucial roles of CDK4/6 in regulating the cell cycle and cancer cells, analyze the rationale for selectively inhibiting CDK4/6 for cancer treatment, review the latest advances in highly selective CDK4/6 inhibitors with different chemical scaffolds, explain the mechanisms associated with CDK4/6 inhibitor resistance and describe solutions to overcome this issue, and briefly introduce proteolysis targeting chimera (PROTAC), a new and revolutionary technique used to degrade CDK4/6.

Herbal drug discovery for the treatment of nonalcoholic fatty liver disease

Few medications are available for meeting the increasing disease burden of nonalcoholic fatty liver disease (NAFLD) and its progressive stage, nonalcoholic steatohepatitis (NASH). Traditional herbal medicines (THM) have been used for centuries to treat indigenous people with various symptoms but without clarified modern-defined disease types and mechanisms. In modern times, NAFLD was defined as a common chronic disease leading to more studies to understand NAFLD/NASH pathology and progression. THM have garnered increased attention for providing therapeutic candidates for treating NAFLD. In this review, a new model called "multiple organs-multiple hits" is proposed to explain mechanisms of NASH progression. Against this proposed model, the effects and mechanisms of the frequently-studied THM-yielded single anti-NAFLD drug candidates and multiple herb medicines are reviewed, among which silymarin and berberine are already under U.S. FDA-sanctioned phase 4 clinical studies. Furthermore, experimental designs for anti-NAFLD drug discovery from THM in treating NAFLD are discussed. The opportunities and challenges of reverse pharmacology and reverse pharmacokinetic concepts-guided strategies for THM modernization and its global recognition to treat NAFLD are highlighted. Increasing mechanistic evidence is being generated to support the beneficial role of THM in treating NAFLD and anti-NAFLD drug discovery.

Prediction of fibril formation by early-stage amyloid peptide aggregation / 药物分析学报

Amyloid fibrils are found in systemic amyloidosis diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes. Currently, these diseases are diagnosed by observation of fibrils or plaques, which is an ineffective method for early diagnosis and treatment of disease. The goal of this study was to develop a simple and quick method to predict the possibility and speed of fibril formation before its occurrence. Oligomers generated from seven representative peptide segments were first isolated and detected by ion-mobility mass spectrometry (IM-MS). Then, their assemblies were disrupted using formic acid (FA). Interestingly, oligomers that showed small ion intensity changes upon FA addition had rapid fibril formation. By contrast, oligomers that had large ion intensity changes generated fibrils slowly. Two control peptides (aggregation/no fibrils and no aggregation/no fibrils) did not show changes in their ion intensities, which confirmed the ability of this method to predict amyloid formation. In summary, the developed method correlated MS intensity ratio changes of peptide oligomers on FA addition with their amyloid propensities. This method will be useful for monitoring peptide/protein aggregation behavior and essential for their mechanism studies.

Combined obeticholic acid and apoptosis inhibitor treatment alleviates liver fibrosis

Obeticholic acid (OCA), the first FXR-targeting drug, has been claimed effective in the therapy of liver fibrosis. However, recent clinical trials indicated that OCA might not be effective against liver fibrosis, possibly due to the lower dosage to reduce the incidence of the side-effect of pruritus. Here we propose a combinatory therapeutic strategy of OCA and apoptosis inhibitor for combating against liver fibrosis. CCl-injured mice, d-galactosamine/LPS (GalN/LPS)-treated mice and cycloheximide/TNF (CHX/TNF)-treated HepG2 cells were employed to assess the effects of OCA, or together with IDN-6556, an apoptosis inhibitor. OCA treatment significantly inhibited hepatic stellate cell (HSC) activation/proliferation and prevented fibrosis. Elevated bile acid (BA) levels and hepatocyte apoptosis triggered the activation and proliferation of HSCs. OCA treatment reduced BA levels but could not inhibit hepatocellular apoptosis. An enhanced anti-fibrotic effect was observed when OCA was co-administrated with IDN-6556. Our study demonstrated that OCA inhibits HSCs activation/proliferation partially by regulating BA homeostasis and thereby inhibiting activation of HSCs. The findings in this study suggest that combined use of apoptosis inhibitor and OCA at lower dosage represents a novel therapeutic strategy for liver fibrosis.

Rapid identification of constituents from different Ginkgo biloba preparations by high resolution mass spectrometry and metabolomics technology / 中国药科大学学报

@#A qualitative analysis was developed to identify different ingredients from three Ginkgo biloba preparations(including Yinxingye Diwan, extract of Ginkgo biloba leaves tablets and Yinxingtongzhi Diwan)based on high resolution mass spectrometry and metabolomics technology. An XSELECT HSS T3(4. 6 mm×150 mm, 3. 5 μm)column was used for separation, with the mobile phases consisting of acetonitrile and water containing 0. 1% formic acid. The column temperature was set at 40 °C. Negative ion mode was used for mass spectrometric data acquisition. Through partial least squares projection to latent structure-discriminant analysis(PLS-DA), we firstly found the different compounds among different Ginkgo biloba preparations. Subsequently, the compounds displaying different abundance levels via database searching and literature matching were identified. Finally, we identified 21 different compounds between the extract of Ginkgo biloba leaves tablets and Yinxingye Diwan, which mostly belong to the organic acids and flavonols families. Quantitative analysis showed that the ingredients which had higher abundance in extract of Ginkgo biloba leaves tablets were mainly organic acids, whereas those exhibiting higher levels in Yinxingye Diwan are flavonols. We also identified 12 different ingredients between the Yinxingye Diwan and Yinxingtongzhi Diwan, which were flavonols, sciadopitysin and organic acids. The results of this study are useful for studying different chemical constituents from distinct Ginkgo biloba preparations.

Shenmai injection inhibits infIammatory response in IipopoIysaccharides-in-duced septic mice / 中国药科大学学报

This study was to investigate the regulation of lipopolysaccharides(LPS)-induced sepsis in mice by preadministration of Shenmai injection (SMI)and the therapeutic differences between male and female.The females and males were randomly grouped by weight,including control group,LPS-induced sepsis model group and SMI administration group.After preadministration of SMI for 14 days,10 mg/kg LPS were intraperitoneally injected subsequently to induce sepsis.The survival rate of mice,level of serum cytokines and the mRNA expres-sion of proinflammatory cytokines in main tissues were detected to evaluate the impact of SMI in LPS-induced sepsis mice.From the survival rate,which is considered as a gold standard of improvement in sepsis,significant protective effect can be observed after SMI pretreatment in LPS-induced sepsis mice,a more significant effect was shown in the females.Consisting with the serum cytokines levels,SMI significantly inhibited proinflammatory cyto-kines including IL-6,IL-1βand TNF-αmRNA expression in tissues and the regulation of IL-6 was most signifi-cant,which was consistent with the results of ELISA in serum.Moreover,the liver tissue acquired a more evident impact than any other tissues,which fits with the ratio of dry/wet weight.SMI can significantly inhibit inflammato-ry response by delivery in advance in LPS-induced septic mice,providing strong evidence for elaborating mecha-nism in the treatment of cardiovascular disease related inflammation and shock.

Mass spectrometry-based protein quantification and its application in pharmacokinetic research / 中国药科大学学报

@#Quantitative proteomics is a mass spectrometry-based toolkit used to analyze and quantify entire proteins contained in whole cells, tissues or organisms. It has become an increasingly important element in exploring the mechanism of various biological processes such as discovering novel biomarkers and unknown drug targets. Emerging advances in biological mass spectrometry instrumentation and data acquisition methodologies have provided a state-of-the-art platform for protein quantification, prompting the research of proteomics evolving from the simple qualitative to the accurate quantitative approach. This review aims to introduce the most recent advancements in mass spectrometry instrumentation and methodologies of data acquisition, focusing on their characteristics and applying fields. It also highlights several significant applications of biological mass spectrometry in pharmaceutical research such as quantifitation of drug transporters and metabolizing enzymes, and pharmacokinetic study of therapeutic peptides and proteins.

Metabonomic phenotype of "formula corresponding to pattern types" based on "qi and yin deficiency pattern" of myocardial ischemia rat model / 药学学报

In order to explore the scientific connotation of "Fangzhengduiying (formula corresponding to pattern types)", "Qiyinliangxuzheng (Qi and Yin deficiency pattern)" of myocardial ischemia rat model and GC-TOF/MS based metabonomic method were used for comparing the effects of Sheng-mai injection, Salvia injection and propranolol in the present study. After data processing and pattern recognition, Sheng-mai injection showed better efficacy than the other two drugs in accordance with not only visual observation from PLS-DA scores plots but also the number of abnormal endogenous compounds restored to the normal level. Further studies showed that Sheng-mai injection could normalize the level of plasma endothelin-1, the index related to cardiovascular diseases and sleep disorders, which verified the results of metabonomics. Finally, the regulated metabolites and related metabolic pathways were analyzed, and it was supposed that the effects of Sheng-mai injection involved in the alternation of energy metabolism, lipid metabolism, amino acids metabolism, and so on. These findings provided scientific evidence to Shengmai "Fang" used for "Qi and Yin deficiency pattern" correspondingly, indicating that metabonomics has great potential in traditional Chinese medical research, which provides a novel approach and way to modernization of traditional Chinese medicine.

Pharmacokinetics and excretion of phenolicacids from mailuoning injection in rats / 中国临床药理学与治疗学

AIM: To study the pharmacokinetics of phenolic acids from Mailuoning injection in rats. METHODS: SD rats were given a single i.v. administration dose of Mailuoning injection 10 mL/kg, plasma and urine were collected before and after injection. Phenolic acid components in plasma and urine were measured by LC/MS. Pharmacokinetic parameters were determined from the plasma concentration-time data and urinary excretion-time data with the DAS software package. RESULTS: After i.v. of Mailuoning injection, chlorogenic acid (CGA), 1, 5-dicaffeylquinic acid (1,5-DCQA), 3, 4-dicaffeylquinic acid (3,4-DCQA), 3, 5-dicaffeylquinic acid (3,5-DCQA) and caffeic acid (CA) were quickly excrectioned. The t1/2 of CGA, 1,5-DCQA, 3,4-DCQA, 3,5-DCQA and CA were 0.649, 0.334, 0.479, 0.486 and 0.330 h, respectively. AUC0-∞ were (22.522±2.716), (0.353±0.062), (3.620±1.246), (5.287±1.627) and (2.257±0.360) mg·L-1·h, respectively. After i.v. of Mailuoning injection, CGA, 1,5-DCQA, 3,4-DCQA, 3,5-DCQA and CA can all be detected in the urine. The amounts of CGA, 1,5-DCQA, 3,4-DCQA, 3,5-DCQA and CA excreted from urine during 0-24 h were (122.22±26.49)%, (3.30±1.26)%, (0.24±0.11)%, (1.93±0.77)% and (18.61±4.99)% of dose given in rats, respectively. CONCLUSION: After i.v. of Mailuoning injection, phenolic acids can be excreted quickly. Only a small quantity of 1,5-DCQA, 3,4-DCQA, 3,5-DCQA and CA were excreted from urine. 3,4-DCQA and 3,5-DCQA may be metabolized into CGA in the rat plasma.