Rheumatoid Arthritis: Lin Z

Sun J, Lin Z, Feng J, Li Y, Shen B. · Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 30072, PR China. · Eur J Pharmacol. · Pubmed #18793632 No free full text.

Abstract: Since B cell activating factor belonging to tumor necrosis factor (TNF) family (BAFF) has been identified as a critical factor for B cell maturation and survival, convincing evidence indicates that deregulation of BAFF is involved in pathogenesis of B cell related autoimmune diseases. Blockade of BAFF activity significantly improves the symptoms of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis both in animal models and clinical trials. Therefore, BAFF-targeting therapy is a promising approach to treat B cell related autoimmune diseases.

Chang H, Qin W, Li Y, Zhang J, Lin Z, Lv M, Sun Y, Feng J, Shen B. · Institute of Basic Medical Sciences, Beijing, PR China. · Mol Immunol. · Pubmed #17485112 No free full text.

Abstract: Anti-TNF antibody has been an effective therapeutic strategy for the diseases related to aberrant production of TNF-alpha, such as rheumatoid arthritis (RA) and Crohn's disease. The limitations of large molecule inhibitors in the therapy of these diseases prompted the search for other potent novel TNF-alpha antagonists. Antagonistic peptides, derived directly or designed rationally from complementarity-determining regions (CDRs) of neutralizing antibodies against TNF-alpha, have been demonstrated for their ability of inhibiting TNF-alpha. However, their activity is very low. In this study, to increase the affinity and bioactivity, human antibody variable region was used as scaffold to display antagonistic peptides, which were designed on the interaction between TNF-alpha and its neutralizing monoclonal antibody (mAb Z12). Based on the previously designed domain antibody (framework V(H)5), framework V(kappa)1 was used as light chain scaffold. On the basis of computer-guided molecular design method, a novel human scFv fragment (named as TSA1) was designed. Theoretical analysis showed that TSA1 could bind to TNF-alpha with more hydrogen bonds and lower binding free energy than the designed domain antibody. The biological experiments demonstrated that TSA1 could directly bind with TNF-alpha, competitively inhibit the binding of mAb Z12 to TNF-alpha and block the binding of TNF-alpha to TNFR I and TNFR II. TSA1 could also inhibit TNF-induced cytotoxicity on L929 cells and TNF-mediated NF-kappaB activation on HEK-293T cells. The bioactivity of TSA1 was significantly increased over the domain antibody. This study indicated that the framework of antibody variable region could serve as an ideal scaffold for displaying the peptides and provides a novel strategy to design TNF-alpha inhibitors with the ability to block the deleterious biological effects of TNF-alpha.

Qin W, Feng J, Li Y, Lin Z, Shen B. · Research Institute of Nephrology, Jinling Hospital, Nanjing, PR China. · Mol Immunol. · Pubmed #17125837 No free full text.

Abstract: Neutralizing of TNF-alpha has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-alpha and it's neutralizing monoclonal antibody. On the basis of the structural character and physical-chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-alpha (named as ATD5) was designed as TNF-alpha antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-alpha was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-alpha and inhibit the binding of TNF-alpha to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-alpha-mediated cytotoxicity and inhibit TNF-alpha-mediated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-alpha inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-alpha using in vitro analysis systems in combination with the computer-aided modeling method.

Li H, Luo W, Zeng Q, Lin Z, Luo H, Zhang Y. · Shantou University Medical College, Central Laboratory, Shantou, Guangdong 515041, China. · J Chromatogr B Analyt Technol Biomed Life Sci. · Pubmed #16890029 No free full text.

Abstract: Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX+MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze-thaw cycle to break blood cells) was well mixed with 0.8 ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze-thaw cycle) was mixed with 80 microl ascorbic acid (114 mM) and incubated at 37 degrees C for 2h to enzymatically convert the MTXPG to MTX. Then 20 microl NaOH solution (0.5M) and 0.8 ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2 ml hydrochloric acid (10mM). Methylene chloride (0.2 ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6 ng/ml, respectively.

Qin W, Feng J, Li Y, Lin Z, Shen B. · Institute of Basic Medical Sciences, P.O. Box 130 (3), Taiping Road, Beijing 100850, PR China. · J Biotechnol. · Pubmed #16522339 No free full text.

Abstract: Tumor necrosis factor-alpha (TNF-alpha) antagonists have become therapeutic drugs for immunological diseases including rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, etc. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. Based on the 3-D complex structure of TNF-alpha with its neutralizing monoclonal antibody (Mab) Z12, an antagonistic peptide (AP) was rationally de novo designed. The designed AP possessed similar structural character and potential bioactivity with Mab Z12. AP could competitively inhibit the binding of Mab Z12 to TNF-alpha, TNF-alpha-meditated caspase activation and TNF-alpha-induced cytotoxicity on murine L929 cells with a dose-dependent fashion. This study highlights the potential of computation-aided method for the design of novel peptides with the ability to block the deleterious biological effects of TNF-alpha.