Screening And Optimization Of Peptide Inhibitors Targeting H₃₇Ra

Tuberculosis is a chronic communicable disease caused by Mycobacterium tuberculosis. Previous studies showed Mycobacterium tuberculosis were phagocytized by macrophage, then became persistent and sustained in cells which taken energy from glyoxylate bypass. Gloxylate bypass hasn’t been found in human and other mammals, except in mice till now. The effectiveness of existing drugs will be descended if patients’immunity decreased or drug was stopped. So there is an urgent demand to find new anti-tubercular drugs. Isocitrate lyase (ICL) is a key rate-limiting enzyme in the glyoxylate bypass when human are infected and ICL is a decisive factor while Mycobacterium tuberculosis which are persistent. So ICL was selected as the target for studying anti-tubercular drugs.Fmoc Solid-phase synthesis was used to synthese peptide inhibitors in this study. The synthesized peptides purfied by HPLC and detected by mass spectrometry. Through inhibition experiments, the inhibition effect of peptides was determined in different culture medium, and theirs minimal inhibitory concentration (MIC) was measured. Antibacterial effects of peptides were detected in cultures with different concentrations of Vc to optimize futher.In this study, four selected peptides all had antibacterial effect which correlated with dose. The results showed that polypeptide inhibited the growth of bacteria while concentration were between 800 and 1500μg/mL. When concentration was 500μg/mL, only one peptide could inhibit the growth of bacteria in the normal culture medium, but in other culture mediums which carbon source was limited could not inhibit the growth of bacteria. The inhibition effect of No.2 peptide was the best, the MIC were 200μg/mL in normal medium, and 500μg/mL in carbon limitations.According to the result of MIC, the experiments of antibacterial were showed that the inhibitory effects of peptide, RIF and INH were related to the dose.MTT was used to detect cell toxicity of No.2 peptide to Phagocytic cell THP-1. The MTT results showed that cytotoxicity was lower when the concentration of polypeptide was less than 2.0 mg/mL, this concentration was used to detect bioactivity of cells.The effect of peptide to survival rate of H37Ra in THP-1 cells showed that H37Ra had been effectively engulfed in THP-1 cells. Colony counts showed that H37Ra could proliferate in THP-1 cells, and the number of H37Ra increased correlates with the infecting time. The number of H37Ra decreased after 4 day s and 7 days when the concentration of peptide was 2.0mg/mL, the growth of H37Ra was significantly inhibited. Above all, the study showed that the peptide of No.2 could inhibit the growth of H37Ra intracellular at a lower concentration.Based on bioinformatics, peptide inhibitors were screened and optimizated which targeting to H37Ra according MIC, Cell toxicity test and Intracellular. The result provid expermiment foundation to develop new anti-TB inhibitiors.