The success rate of mechanism-based drug discovery depends on the drug targets. With the rapid development of genomics and proteomics, a lot of nonenzymic proteins have been identified as potential drug targets. However, these nonenzymic proteins cannot be regulated by occupying the active site, which were recognized as undruggable targets. Direct regulation of the concentration of these proteins in cells by the innate ubiquitin-proteasome is a potential approach to target these proteins. The ubiquitination of target protein by E3 ligase is the key step for ubiquitin-proteasome mediated protein degradation. Proteolysis targeting chimeras (PROTACs) can facilitate the assembly of complex that consists of the target protein and E3 ligase. The target protein will be ubiquitinated, leading to the degradation by proteasome. This type of regulation mechanism can expand the scope of potential drug targets, and the development of PROTACs may be an innovative strategy in drug discovery.

Ferroptosis is a novel type of cell death which induced by iron-dependent lipid peroxidation accumulation. This type of cell death is significantly different from other cell death in terms of morphology, genetics and biochemistry. It has been reported that ferroptosis is involved in a variety of human diseases, particularly in liver diseases. Therefore, screening and studying of inhibitors or activators of ferroptosis may provide novel strategies for prevention and treatment of liver diseases. This review provides the biological characteristics and regulatory signaling pathways of ferroptosis, as well as the relationship between ferroptosis and liver diseases, which will contribute to new insight into the pathogenesis of liver diseases.

Depression is a complex heterogeneous psychiatric disorder with a potential to cause patient disability worldwide. In the past decades, a number of the first-line drugs (tricyclics, monoamine oxidase inhibitors, serotonin selective reuptake inhibitors, noradrenaline reuptake inhibitors, serotonin and noradrenaline reuptake inhibitors, etc.) had been discovered for the treatment of depression. CYP450 superfamily is involved in the biotransformation of antidepressants mainly through the CYP2D6, CYP2C19, CYP3A4 isoenzymes. Their genetic polymorphisms are closely related to clinical use and the interactions have been already identified between enzymes and antidepressants. This review focuses on drug metabolism and the interaction for CYP450 enzymes. The content may contribute to the development of antidepressant, improvement of the safety of antidepressants and the efficacy of treatment.

Proteolysis-targeting chimeras (PROTACs) are small-molecule protein degraders based on the ubiquitin-proteasome system. Recently, the development of specific small-molecule ligands for several E3 ligases (CRL4^CRBN, CRL2^VHL and cIAP) have significantly advanced the PROTACs technology. Several PROTACs against various oncogenic proteins including bromodomain-containing protein 4 (BRD4), estrogen receptor (ER) and androgen receptor (AR) have been developed and considered a novel approach for therapy of cancers. There are advantages of the new technology over the traditional small-molecule strategies. This review article provides a summary on the recent progress in the small-molecule-based PROTACs as antitumor drugs, and the challenges of this technology.

With the development of antibody manufacturing technology and improvement in new drug research and development (R&D) capabilities in domestic industry, more and more innovative antibody-based drugs were registered at the Investigational New Drug (IND). This type of drugs could be divided into three categories:new sequence antibodies (biobetter or new target antibodies), bispecific antibodies (or antibody cocktails), and antibody drug conjugates. Comparing with biosimilar antibodies, the innovative antibodies R&D was characterized by some significant features including "innovation", "clinical phase-appropriate" and "progressing". The minimum requirements of Chemical, Manufacturing and Control (CMC) content for innovative antibodies were obviously different from biosimilar antibodies. Here, the recent progress of antibody engineering and IND date of innovative antibodies in domestic are summarized. The general regulatory requirement and special considerations for representative innovative antibodies were proposed. Some common problems concerning innovative antibodies R&D are discussed.

Traditional Chinese medicinal materials (TCMMs) are the material basis of the traditional Chinese medicines (TCM) in the treatment of disease, and the inherent quality of the TCMMs are of crucial importance for the efficacy and safety of TCM. Most of the TCMMs are originated from the plants with complex chemical compositions, and the chemical diversity is inevitable. How to clarify the relationship between the chemical identity and the biological activity is the key point for quality control of TCMMs in the establishment of the quality standards. The components with great impact on the biological activities should be used as index compounds in the quality evaluation. In this study, based on the results in our previous studies, a research idea on the evaluation of quality difference of TCMMs is proposed. Its application and existing problems are also discussed.

Cell membrane chromatography (CMC) was first proposed by Professor He in 1996. As one of bio-affinity chromatography technique, CMC was a simple and convenient technology in the study of interactions of active components in traditional Chinese medicines (TCMs) with membrane receptors in vitro, and screen active components from complicated TCMs. Recently, the CMC technology was developed rapidly, and widely applied in the discovery of lead compounds from nature product. This review article is focused on the application of cell membrane chromatography in the identification of active components in traditional Chinese medicine, together with the recent development of CMC methodology. Combining with our previous works in the analysis of the composition of complex substances, biochromatography and TLC bioautography for quality evaluation of TCM, we proposed a new holistic quality evaluation strategy of TCM related with bioactivity, which could be summarized as the integration of screen (screening of quality control marker by CMC), macroscopic characterization (characterizing the chemical material basis by multi-dimension and multi data fingerprinting) and microscopic description (multi-component quantification). The proposed strategy would provide a new idea for the holistic quality evaluation of TCM in the composition and concentration of bioactive components as quality evaluation indicators.

In the past 20 years, tumor immunotherapy has made a significant progress in tumor inhibition effects in both laboratory studies and clinical trails. In the immune response to tumor, effective antitumor immunity is induced during the tumor progress; long-term monitoring of the tumor would be achieved through the immune memory, reducing the possibility of tumor recurrence. In the immune treatment strategies, a focus is delivery of therapeutic immune regulators with nanocarriers. It has been demonstrated that due to the special physical and chemical properties, nanocarriers are easily internalized by immune cells, which regulate the immune responses and effectively induce anti-tumor immune cascade to achieve the tumor inhibition effect. In this paper, we will discuss the progress of nanocarrier-mediated antitumor immunotherapy in recent years.

Selenium (molecular weight 78.96), is a necessary non-metallic trace elements. In recent years, more and more studies have found that selenium is closely related to human disease and could not be ignored. In this paper, we elucidate the in vivo absorption and metabolism of selenium; the active substance-selenoprotein P and so on; the correlation between selenium and pathogenesis of various diseases such as cardiovascular disease, AD/PD neurodegenerative diseases and cancer, etc. The aim is to achieve a more comprehensive understanding of selenium, and to research the in vivo process and the biological effects of selenium. At the same time, selenoproteins and related mechanisms might also be a new target for drug research and discovery.

This study was conducted to establish an in vitro 3D liver model and apply it to the drug liver toxicity evaluation. The 3D multicellular sphere model of HepaRG cells was established by hanging-drop technique for evaluation of liver function. The 3D liver model was used to test the hepatotoxicity of isoniazid and amiodarone hydrochloride compared to the 2D cell culture model. Our results showed that HepaRG cells formed a compact spheriod, and the level of cell albumin, urea and the CYP3A4 activity were significantly higher than that of 2D model. With the treatment of amiodarone hydrochloride in 2D and 3D model, the IC₅₀ were 50 and 100 μmol·L^-1, respectively. When the dose was less than 1 000 μmol·L^-1, isoniazid had no hepatocyte toxicity in 2D model, while the IC₅₀ in 3D model was 700 μmol·L^-1. The LDH activities of both drugs in 3D model showed time-and dose-dependent correlation. The results suggest that this in vitro 3D hanging-drop liver model is good for testing liver functions with a high hepatic drug-metabolizing enzyme activity. Compared with the 2D model, the 3D liver model can accurately evaluate the liver toxicity of drugs. Our results demonstrated the importance of in vitro cell culture models for detection of in vivo-relevant adverse effects of drugs.

This study was designed to investigate the effect of Yinxing Mihuan oral solution on rats with myocardial ischemia injury. Left anterior descending (LAD) coronary artery was occluded in rats to establish the model. Yinxing Mihuan oral solution was given by intragastric administration daily for one week at dosage of 309 and 618 mg·kg^-1. Cardiac ultrasound function, pathologic change and serum myocardial enzymes were determined to evaluate the effect of Yinxing Mihuan oral solution. The heart function was significantly reduced in the model group compared with sham operation group, and the pathologic damage was clear. The changes were significantly improved by Diltiazem hydrochloride tablets in heart function and ejection fraction (P < 0.01). The ejection fraction and stroke volume was improved by Yinxing Mihuan oral solution (618 mg·kg^-1) (P < 0.05, P < 0.01). In addition, Yinxing Mihuan oral solution decreased the myocardial damage and inhibited inflammatory reaction, and inhibited platelet activation factor. Yinxing Mihuan oral solution can protect against myocardial ischemia injury, inflammation and platelet activation in the rat model.

This study was designed to investigate the therapeutic effect and mechanisms of action of novel compound N-(Z)-9-octadecenyl-2-propanesulfonamide (N15) on type 2 diabetes (T2DM). A mouse model of T2DM was established with multiple injection of streptozotocin (STZ) at a low dose. N15 at different doses (50, 100 and 200 mg·kg^-1·d^-1) and pioglitazone (6 mg·kg^-1·d^-1) were administrated orally for 6 weeks. The level of fasting blood glucose (FBG) and fasting insulin (FIns) were measured in the course of the experiment for insulin resistance index (HOMA-IR). Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) were determined in the treated mice. The expression of Akt, AMPK and Glut4 in liver were analyzed by Western blot. N15 was found to reduce the level of FBG, FIns and HOMA-IR (P < 0.01) and ameliorate the glucose and insulin tolerance (P < 0.01, P < 0.001). Simultaneously the protein expression of p-Akt, p-AMPK and Glut4 was significantly increased in liver by N15 (P < 0.01). These effects were similar to those of pioglitazone (P > 0.05). These results suggested that the novel compound N15 can ameliorate insulin resistance and the potential mechanism may be associated with increased insulin signaling in liver and promotion of phosphatidyl inositol 3 phosphate phosphorylation.

The aim of the experiments is to screen human single-chain variable fragment (scFv) targeting glypican 3 from phage display library, and analyze its biological activity. After several rounds of panning, the binders with high affinity were obtained through phage ELISA and IMGT analysis. The desired scFv gene was then ligated with pET-22b vector yielding recombinant plasmids, which was then introduced into E. coli Rosetta (DE3). Soluble scFv protein was expressed and further purified using Ni²⁺ affinity chromatography. The purified proteins were identified by SDS-PAGE and Western blot. Subsequently, the affinity and cell based binding activity were measured using Surface Plasmon Resonance (SPR) and flow cytometry assay, separately. Four enriched sequences with relatively high binding affinity were found (1F7, 1D7, 1D4 and 1B10). We also found that 1F7 scFv showed better targeting ability and higher affinity. The scFv could pave the way for new immunotherapies, such as bispecific anitbody, antibody-drug conjugate and chimeric antigen receptor T-cell immunotherapy cell, etc.

A droplet microfluidic chip system was developed for drug screening against Candida albicans. The microfluidic chip was designed and prepared for the formation of droplets. Alamar blue was selected as an indicator for its characteristic of fluorescence mission in live cells. Four antifungal drugs (amphotericin B, caspofungin, 5-fluorocytosine, terbinafine) and a new drug (iodiconazole) were selected as model drugs to test the microfluidic chip approach. At the same time, 96-well microplate method was performed to verify the applicability of the chip method. The results showed that the developed droplet microfluidic chip platform was able to complete the antifungal susceptibility test within 2 h. In comparison with the 96-well microplate method, the microfluidic chip method showed a consistence of 100% with regard to the minimum inhibition concentrations and less reagent consumption. The new droplet microfluidic chip method is simple, rapid and suitable for rapid screening of antifungal drugs.

A series of ursolic acid derivatives were synthesized by the introduction of 4-chloroindole compounds at A ring and esterification and amidation at C-28 position, which structures were characterized by ¹H NMR, MS and etc. The cytotoxic activity of derivatives was evaluated against HepG2 and SGC7901 cells in vitro by MTT assay, in which paclitaxel and adriamycin were used as a positive control. The results indicated that all of derivatives can inhibit cell proliferation in HepG2 and SGC7901 cells with a better activity than ursolic acid. Especially, the compounds 6 and 12 showed significant antitumor activity comparable to the paclitaxel. The compounds are worthy to be studied further.

Interleukin-6 (IL-6)/signal transducers and activators of transcription (STAT) signaling pathway is closely related to the development and progression of atherosclerosis (AS). Taking Chinese natural product berberine (BBR) as the leading compound, a series of novel BBR analogues defined on different types of substituents on position 3 or/and 9 were designed, synthesized and evaluated for their inhibitory activities on phosphorylation of STAT-1 and STAT-3 induced by IL-6. The structure-activity relationship indicated that introduction of rigid fragment on position 3 or 9 was beneficial for enhancing their activities. Among them, compounds 2b and 9 exhibited the most satisfactory potency. The study revealed that the compounds 2b and 9 exhibit anti-inflammatory potencies via activating AMPK, and down-regulation of phosphorylation of STAT1 and STAT3 induced by IL-6 in HUVEC cells. These results suggest that BBR derivatives may inhibit the inflammatory response mediated by the IL-6/STAT signaling pathway through regulation of AMPK, which provides useful insight into the development of BBR derivatives for treatment of atherosclerosis.

The quality of Scutellaria baicalensis Georgi has a close relationship to the harvest time. With the annual and biennial Scutellaria baicalensis Georgi in different harvest periods as samples, we detected the dynamic changes of chemical compositions in their contents by UHPLC-MS/MS metabolomics technology, then identified the biomarkers of different harvest periods and analyzed their changes in content, finally analyzed the correlation among different metabolites. The results showed that chemical compositions of different harvest periods were obviously different, and showed regular changes. According to the results, if the flavonoid aglycones are chosen as the target components, the proposed harvest time could be May. If flavonoid glycosides, the time is from July to August. Plant metabolomics methodology based on UHPLC-MS/MS can be used to detect the chemical compositions of Scutellaria baicalensis Georgi in different harvest periods, which provide a reference to the optimum harvest period of Scutellaria baicalensis Georgi, and also provide a new method of quality evaluation of traditional Chinese medicinal materials.

The purpose of this article was to study the pharmacokinetic characteristics of YZG-331, plasma protein binding and metabolic stability in vivo and in vitro. Plasma and tissue concentrations of YZG-331 were determined in mice and rats after administration by LC-MS/MS analysis orally or intravenously. The plasma protein binding of YZG-331 with human, dog, monkey, rat and mouse were measured by ultrafiltration method. The stability of YZG-331 in animal and human plasma, liver microsomes, intestinal bacteria and artificial gastrointestinal fluid was also investigated in vitro. The results show that YZG-331 was absorbed rapidly in both mice and rats after oral administration, while the absorption and elimination saturation YZG-331 were also observed. The bioavailability of YZG-331 was much higher in male mice (51.2%) than that in female mice (27.7%), however, the bioavailability in male rats (27.1%) was lower than that in female rats (78.7%). YZG-331 was widely distributed in different tissues of mice, especially in certain regains of brain, including thalamus, hippocampi, cortical and striatal. YZG-331 was found to bind to human, dog, monkey, rat and mouse plasma protein in vitro (93.3%-98.9%) without significant concentration dependences and species differences. YZG-331 was stable in animal and human plasma, simulated gastric/intestinal fluid and liver microsomal incubations, except rat liver microsomes and intestinal flora. Therefore, we concluded that:the pharmacokinetics of YZG-331 in mice and rats have gender and species differences; YZG-331 was widely distributed in vivo including brain, the targets of the agent; YZG-331 had a high affinity to plasma protein and was metabolized by rat liver microsomes and intestinal flora.

Nimodipine is a selective calcium channel antagonist of cerebral vessels smooth muscle and also has polymorphs. It hasn't been reported that different crystal forms influence the metabolism process in huge animals like rhesus monkeys in vivo. This article may provide reference in the control of the quality of nimodipine and quality consistency evaluation. The powder X-ray diffraction (PXRD) method was used to identify different crystal forms and the dissolution test in vitro was used to detect the dissolution. The LC-MS method of assay nimodipine in rhesus monkey plasm was established to determine pharmacokinetics characters of different tablets from different crystal forms in rhesus monkey in vivo. As a result, the tablets inherit difference crystal forms and the dissolution of reference tablets is 1.3% higher than crystal tablets. However, the maximal blood concentration (C_max) of crystal tablet was 37.3% higher than reference tablet and AUC of crystal tablet was 29.8% higher than reference tablet. After administrated 2.5 mg·kg^-1 orally, calculated pharmacokinetics characters were observed as following:C_max was 381.4 ±327.3 and 178.0 ±214.8 μg·L^-1; AUC_0-t was 853.1 ±500.7 and 646.5 ±430.3 μg·L^-1·h respectively. The serum concentration result of different nimodipine tablets in rhesus monkeys in vivo suggests that polymorphs has a significantly distinction, which points out that controlling the crystal forms of nimodipine is essential to ensure the therapeutic efficacy. It is essential to execute quality consistency evaluation.

The aim of this study is to establish the in vitro methods for the study of induction and inhibition on CYP450 by drugs, and to validate the analytical method and incubation system. A method for the simultaneous determination of eight metabolites of seven subtypes of CYP450 enzymes probe substrates in human liver microsomes (HLM) was established and validated. The incubation system was optimized to confirm the incubation time and protein concentration of HLM, the enzyme activity of seven subtypes of CYP450 enzymes in HLM was determined, and the inhibition effects on each CYP450s were checked by positive controls. The method for the simultaneous determination of three metabolites of subtypes of CYP450 enzymes was established and validated in human primary cultured hepatocytes (HPCH) using the incubation medium. The enzyme activity of three subtypes of CYP450 enzymes in HPCH was determined, and the total RNA was extracted from HPCH after incubation. The expression of CYP450 enzymes were measured by Taqman fluorescence probe method. The induction effects on each CYP450s were examined using the positive controls. The established methods for the determination of metabolites of probe substrates were fully validated, and the results were conformed to the requirements of bioanalytical method validation. The induction and inhibition effects on each CYP450s were checked by positive controls. The established in vitro methods for the study of drug induction and inhibition on CYP450 were simple and reliable, which could be used in the investigation of enzyme induction or inhibition properties of new drug candidates and to evaluation the metabolic interactions of concomitant medication in clinical.

In this study, we used low molecular weight heparin (LH) as hydrophilic sides, doxorubicin (DOX) as hydrophobic sides, and indocyanine green (ICG) as a photosensitive drug to prepare pH-sensitive self-assembled polymeric micelles (LH-DOX/ICG). The micelles were prepared by dialysis, and evaluated for stability, pH sensibility in vitro, hemolytic test and photo-thermal effect. Wound healing test was used to evaluate the anti-metastatic effects. MTT assay and apoptosis detection kit were used to evaluate the cytotoxicity of micelles against melanoma B16F10 cells. The size of the micelles was (148.7 ±2.1) nm and the zeta potential was (-30.7 ±1.1) mV. The drug-loading content of DOX and ICG were 11.82% and 5.59%, respectively. The micelles exhibited spherical in shape, fairly uniform size. The micelles were stable within 48 h in 50% fetal bovine serum phosphate buffer. The release of DOX was pH-sensitive, while the release of ICG was sustained. The micelles were biocompatible and safe as indicated by the hemolytic test. In vitro photo-thermal effect indicated LH-DOX/ICG micelles had similar photo-thermal effect to the free ICG. Wound healing test showed that the micelles had a good ability to inhibit tumor migration with laser irradiation. MTT assay and cell apoptosis assay indicated that LH-DOX/ICG micelles displayed more efficient anti-tumor effect after near infrared laser compared to LH-DOX micelles. LH-DOX/ICG micelles are promising for the therapeutic effect of phototherapy and chemotherapy in combination for melanoma.

Photo-sensitive reactive oxygen species (ROS) responsive liposomes loaded with 1, 4, 8, 11, 15, 18, 22, 25-octabutoxypalladium phthalocyanine[PdPC(OBu)₈] and doxorubicin hydrochloride (DOX) (LPD) were prepared by (NH4)₂SO₄-gradient method. LPD was characterized with transmission electron microscopy (TEM), dynamic light scattering particle size, zeta potentials, photo-sensitive ROS-responsive DOX release behaviors and the serum stability in vitro. LPD cytotoxicity, DOX uptake and singlet oxygen production in MCF-7 cells were evaluated. The results showed that the particle size of LPD was (169.3 ±1.2) nm, PDI of LPD was 0.198 ±0.003 and zeta potentials of LPD was (-39.8 ±0.8) mV. The accumulated release of DOX reached 95.5% in 5 min under 730 nm laser irradiation (300 mW·cm^-2). The DOX uptake of liposome was increased and ¹O₂ was generated. The half inhibition concentration (IC₅₀) of DOX in LPD with irradiation group was decreased by 85.7% and no irradiation group was decreased by 67.9% compared with free DOX group in MCF-7 cells. Therefore, photo-sensitive ROS-responsive liposomes would be a promising drug delivery system for tumor therapy.

Calcium signaling plays a critical role in response to various abiotic and biotic stresses in plants. Preliminarily evidence showed that calcium signaling perceived and transduced the harmful signaling generated from continuous cropping stress in R. glutinosa. To investigate the roles of calcium signaling in continuous cropping injury formation, the key genes involved in calcium signaling transduction were identified in R. glutinosa transcriptome through bioinformatic methods. Furthermore, the calcium ion concentration in both normal and continuous cropping R. glutinosa root cells were measured by potassium pyroantimonate precipitation and calcium fluorescence method. As a result, a set of 84 calcium signaling-related genes, including 5 CaMs, 12 CBLs, 21 CDPKs, 21 CIPKs, 16 CMLs, and 9 CRKs were captured in R. glutinosa transcriptome. The analysis of expression profile in continuous cropping compared to normal growth R. glutinosa indicated that continuous cropping stress significantly increased the expression of calcium signaling-related genes in continuous cropping R. glutinosa. At the same time, the abundance levels of 12 calcium signaling-related genes quantified by qPCR further validated the high expression of calcium signaling-related genes presented in continous cropping R. glutinosa. In addition, the continuous cropping condition significantly promoted the accumulation of intracellular calcium ions in R. glutinosa based on two methods of potassium pyroantimonate precipitation and calcium fluorescence. This study verified the possible roles of calcium signaling in the formation of continuous cropping injury on molecular and cellular level, which lays a solid foundation for illuminating formation mechanism of continuous cropping injury on molecular level.

Jasmonic acid (JA) is an important signal molecule involved in plant resistance, and allene oxide synthase (AOS) is a key enzyme in the biosynthesis of jasmonates. In this study, a full-length cDNA of AsAOS1 gene was cloned from Aquilaria sinensis. Meanwhile, the sequence analysis, prokaryotic expression, purification, tissue-specific expression analysis and expression analysis under different abiotic stresses and hormone treatments were performed. The open reading frame (ORF) of AsAOS1 gene was 1 575 bp, encoding a protein of 524 amino acid residues, with a predicted molecular mass of 58.70 kDa. AsAOS1 protein possessed the conserved sequences of cytochrome P450 (CYP450). The phylogenetic analysis indicated that AsAOS1 protein had the highest level of homology with AOS protein of Citrus sinensis. The recombinant AsAOS1 protein was successfully expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsAOS1 and the recombinant AsAOS1 was purified by Ni²⁺ affinity chromatography. Expression analysis results in different tissues showed that AsAOS1 was primarily observed in stems, and then roots, followed by leaves. AsAOS1 transcript level was significantly induced after 12 h treatment of NaCl, cold temperature and CdCl₂. Furthermore, AsAOS1 expression level was enhanced upon methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. However, mannitol and gibberellin (GA₃) treatments had little influence on the expression level of AsAOS1. These results provides valuable insights into the role of JA in the mechanism of agarwood formation and plant resistance.