The interaction of drug and target protein is a critical part of new drug discovery. It is the premise for drugs to exert therapeutic effects by targeting specific binding sites of target proteins and thereby affecting its pharmacological activity. Currently, a variety of techniques are exploited to detect the interaction between drug ligands and target proteins. For example, cellular thermal shift assay (CETSA) and differential scanning fluorimetry (DSF) based on thermodynamics, mass spectrometry and nuclear magnetic resonance technology, etc. In addition, high-throughput ligand screening technology provides technical convenience for the search of specific ligand, and is a powerful tool to efficiently identify the interaction between drug ligand and target protein. Here, we summarize the detection techniques of interaction between small molecules and target proteins, and discuss the application of high-throughput ligand screening technology in drug research.

Target identification and verification of natural products is an important and challenging work in the field of chemical biology. It is also an important job for researchers to apply chemical proteomics technology to biomedicine in order to identify target proteins of natural products. Target identification is critical to understanding its mechanisms and developing natural products as molecular probes and potential therapeutic drugs. Traditional approaches of small molecule target identification based on affinity have been shown to be successful, such as click-chemical probes, radioisotope labeling or photosensitized small-molecule probes. Nevertheless, these technologies require purified candidate target proteins, and modified small molecules with probes or linkers, such as adding agarose beads, biotin labels, fluorescent labeling or photo-affinity labeling. Many structure-activity relationship studies should be performed to ensure that the addition of small molecule labels undisturbed the original biological activity of the small molecules. Unfortunately, all these modifications are likely to alter their biological activity or binding specificity. To overcome the bottleneck of "target recognition", researchers have developed a series of new techniques for unmodified drug target identification. In this article, we reviewed the target identification techniques of natural product without structural modification in order to provide reference for the development of natural products.

Adenofibroma ; metabolism ; pathology ; Antigens, CD34 ; metabolism ; Child, Preschool ; Diagnosis, Differential ; Female ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; surgery ; Neoplasms, Germ Cell and Embryonal ; metabolism ; pathology ; surgery ; Nephroma, Mesoblastic ; metabolism ; pathology ; Sarcoma, Clear Cell ; metabolism ; pathology ; Stromal Cells ; metabolism ; pathology ; Vimentin ; metabolism

Objective Benign childhood epilepsy with centrotemporal spikes (BECT) is the most common partial epilepsy syndrome in children, and responds well to carbamazepine (CBZ), oxcarbazepine (OXC), and valproic acid (VPA). The aim of this study is to investigate the therapeutic effect of OXC on BECT. Methods We retrospectively discussed a case of partial epilepsy in a 6-year-old boy with no abnormality on neuroradiologic examination. Results The patient′s seizures were easily controlled by administration of OXC, but electroencephalography (EEG) identified deterioration of the EEG features following the introduction of OXC monotherapy. Then OXC was gradually decreased in dose and substituted with VPA. When VPA was increased to the dose of 0.5g/d, the boy had no more seizures and exhibited normal EEG in the conscious state. Conclusion OXC may induce new types of seizure and aggravate EEG features although it is considered to be the first-line anti-epileptic drug (AED) and much better tolerated than either phenytoin or CBZ.

Objective To systematically review the efficacy and safety of repetitive transcranial magnetic stimulation (rTMS)for post-stroke dysphagia. Methods The clinical trials about rTMS for post-stroke dysphagia were searched on Pubmed,EMbase,MEDLINE,Cochrane library,China National Knowledge Infrastructure (CNKI),Chinese BioMedical Literature Database on disc (CBMdisc),and Wanfang database with computers. Two independent reviewers selected the literatures by the inclusion and exclusion criteria,data extraction and cross-checking,and then conducted the analysis. Results A total of seven trails (228 patients,136 of them in a rTMS group and 92 in a control group)were included. Four randomized controlled trials (RCTs)were used for Meta-analysis (54 patients in the rTMS group,42 in the control group). The other three non-RCTs were only reviewed systematically. (1)Meta-analysis showed that the improvement of swallowing function score after rTMS was better than that of the control group (SMD=1. 73,95%CI 0. 45 to 3. 01;P=0. 008). (2)After high-frequency rTMS (>1 Hz),there was significant difference in the improvement of swallowing function score between the two groups (SMD=1. 60,95%CI 0. 10 to 3. 11;P=0. 04). Compared to the control group,there were also significant differences in the improvement of some swallowing function scores in low-frequency rTMS (1 Hz),suggesting the potential advantages of low-frequency therapy. (3)After rTMS,there was no significant difference in the improvement of Barthel index in patients with stroke compared with the control group (MD= -21. 60,95%CI-36. 21 to-7. 00;P=0. 004). (4)In terms of adverse reactions,seven trials did not report any adverse events, such as headache,tinnitus or epilepsy,etc. Conclusion Using rTMS may significantly promote the recovery of swallowing function for patients with post-stroke dysphagia,and it is both safe and effective.

Objective To investigate the value and safety of biopsy guided by colposcopy in diagnosis of cervical diseases in pregnant women.Methods From Aug.2007 to Feb.2009.17 828 pregnant women who receive antenatal examination underwent cervical cytological screening thinprep cytology test(TCT)in Beijing Obstetrics and Gynecology Hospital.If abnormal cytological results were found,those preguant women were administered by eolposcopic examination and biopsy after they signed informed consent.Results (1)TCT:the abnormal TCT results of 1502 preguant women(8.425%,1502/17 828) were found in 17 828 cases.(2)Colposeopie examination:two hundred and four pregnant women underwent colposcopic examination.The rate of satisfied colposcopic imaging wag 92.6%(189/204),colposcopic examination identified 125 cages with cervical inflammation or cervical intraepithelial neoplasia (CIN)Ⅰ,25 cases with CIN Ⅱ and 54 cases with CIN Ⅲ or microinvasive squamous carcinoma (MIVC) of squamous cervical carcinoma(SCC).(3)The results of biopsy guided by colposcopy:among 204 cases,it was found 33 cases with cervical inflammation or wart,95 cases with CIN Ⅰ,28 CIN Ⅱ,36 cases with CIN Ⅲ and 12 cases with MIVC. (4) The rate of concordance: compared with biopsy pathologic examination, colposcopy examination found 113 cases with cervical inflammation and CIN Ⅰ , the rate of concordance was 90. 4%(113/125). And 54 cases with CIN Ⅲ or SCC diagnosed by colposcopy examination, however biopsy pathologic examination confirm 23 cases with CIN % and 10 cases with SCC at stage Ⅰ a, the concordance rate was 61% (33/54). (5) Complication: eight (3.9%, 8/204) pregnant women underwent cervical wound suturing due to continuous bleeding after colposcopy exam or biopsy. No other complications were recorded. Conclusions It is necessary that TCT should be performed in pregnant women without cytological screening within one year. Colposcopic examination and biopsy were indicated if pregnant woman with abnormal cytological result were found. Pregnant women with cervicitis or CIN Ⅰ diagnosed by colposcopy should be followed up. If pregnant woman was suspected with CIN Ⅱ or advanced disease, biopsy guided by colposcopy should be performed.

Objective To evaluate the maternal and fetal outcomes of planned delay in treatment for cervical microinvasive squamous cancer during pregnancy.Methods A prospective study of pregnant women was done from August 1,2007 to May 31,2010.Pregnant women who had not been carried out cervical cytological screening within one year were got thin-prep cytology test (TCT) screening at their initial prenatal visit.Patients with abnormal cytological results were performed colposcopic examination and directed biopsy.Women with cervical microinvasive cancer were followed up every 8 to 12 weeks.If lesion progression were suspected,compared with previous image,repeated biopsy directed by colposcopy should be performed.Once worsening invasive cancer was confirmed,the pregnancy should be terminated timely.All patients should be reevaluated 6 to 12 weeks postpartum with repeated colposcopic examination and biopsy.All mothers were performed cold knife conization (CKC) at 6 to 12 weeks postpartum.Results We totally diagnosed 17 cases cervical microinvasive squamous carcinoma during pregnancy.The positive rate is 6.2/10 000 (17/27 230).After informed consent,15 pregnant women decided to delay treatment until fetal maturation.The mean gestational age of initial diagnosis was (19.3 ± 5.9) weeks.The women were followed up 2 to 4 times during pregnancy.Only 1 patient was verified lesion progression by directed biopsy at 34 weeks and delivered by cesarean section.The progression rate during pregnancy was 1/15.The mean delivered time was (37.1 ± 1.8) weeks (ranged from 34 to 40 weeks).The mean diagnosis-to-delivery interval was (18.4 ± 5.2) weeks.All patients were delivered by cesarean section and all newborns had good outcomes.Finally we confirmed 1 case with cervical cancer stage Ⅰ a2,11 cases with stage Ⅰ al,3 cases with cervical intraepithelial neoplasia (CIN) Ⅲ by pathological diagnosis after CKC during 6 to 12 weeks postpartum.All cases were disease free after follow-up ranged from 22 to 48 months.Conclusions It is necessary to perform TCT screening for pregnant women who have not been carried out cervical cytology screening within 1 year.If cervical microinvasive squamous cancer were suspected during pregnancy,in order to achieve fetal maturity it is acceptable for the women who desired pregnancy to delay treatment under closely monitoring until postpartum.

OBJECTIVETo investigate the correlation between matrix metalloproteinase 9 (MMP-9) expression and tumor invasion and metastasis as well, and to explore the potential application of controlled expression of target gene in tumor gene therapy.

METHODSOne self-contained tetracycline-regulated retroviral vector containing anti-sense cDNA of MMP-9 was constructed and transfected into a metastatic human melanoma cell line WM451 which expressed a high level of MMP-9. Techniques such as growth rate measurment, MTT assay, (3)H-thymidine incorporation, colony forming ability in soft agar, invasion assay in Boyden chamber, as well as zymography and Western blot were applied to analyze the expression of MMPs and behaviors of tumor cells in vitro before and after gene transfection. Tumorigenecity and spontaneous metastasis were tested in nude mice.

RESULTSIn the presence of exogenous tetracycline, the transfected antisense MMP-9 did not affect the endogenous level of MMP-9 in WM451 cells, and showed no significant changes in cell behaviors in comparison with that of the vector-transfected control cells. Nevertheless, withdrawal of tetracycline from the medium caused a significant down-regulation of expression and activity of MMP-9. The capacity of cell growth in vitro, colony forming ability in soft agar, invasion through Matrigel all were inhibited remarkably when compared with the controls. Spontaneous metastasis in nude mice was significantly inhibited.

CONCLUSIONSTransfection of anti-sense MMP-9 can down-regulate the invasion and metastasis of melanoma cells both in vitro and in vivo, further clarifying the important role of MMP-9 in tumor progression.

Animals ; Cell Division ; Cell Line, Tumor ; DNA, Antisense ; DNA, Complementary ; genetics ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; Genetic Vectors ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Melanoma ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Transplantation ; Retroviridae ; genetics ; Tetracycline ; pharmacology ; Transfection

OBJECTIVETo identify the effect of NEP on Abeta -induced apoptosis in PC12 cells.

METHODSPC12 cells that stably express NEP is generated and the effect of NEP on the process of apoptosis induced by Abeta is analyzed, including the viability of the cells, the production of LDH, ROS and ATP, the activity of Caspase-3.

RESULTSNEP could improve the viability of cells and the production of ATP, inhibit the release of LDH and ROS. In the same time, the activity of caspase-3 descended (P < 0.05). But iNEP had not significant effect on cells apoptosis (P > 0.05).

CONCLUSIONNEP has the protective effect on Abeta-induced apoptosis in PC12 cells.

Amyloid beta-Peptides ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Endopeptidases ; genetics ; metabolism ; PC12 Cells ; Rats ; Transfection

Asian Continental Ancestry Group ; China ; Diabetes Mellitus, Type 2 ; ethnology ; genetics ; Genetic Variation ; Genotype ; Humans ; Insulin Receptor Substrate Proteins ; Intracellular Signaling Peptides and Proteins ; Phosphoproteins ; genetics