| Staphylococcus epidermidis (S. epidermidis) is a normal inhabitant of human skin and mucous membranes that rarely causes pyogenic infections in healthy individuals. However, during the past two decades S. epidermidis has emerged as one of the major pathogens in nosocomial infections. The primary pathogenicity trait of S. epidermidis is associated with its ability to form biofilms on surfaces of medical devices, limiting severely the efficacy of many conventional antibiotics, and biofilms may also protect the bacteria against attacks from the host defence system. In parallel, the appearance of multi-drug resistant S. epidermidis strains has increased quickly due to the increasing use of antibiotics in hospitals, which urgently require design novel antibiotics against staphylococcus infections, especially in relation to biofilm development.The aim of our group is to look for the drug targets against S. epidermidis, which is consisted of two major parts:one is the discovery of lead compounds against S. epidermidis; the other is the discovery of molecular basis of biofilms formation in clinical isolates and the evaluation of feasibility of the potential anti-biofilm drug target.In this study, based on the discovery of Histidine Kinase YycG Inhibitors, we had modificated the structure of the Histidine Kinase YycG Inhibitors, and obtained several derivatives that had displayed significant activity on killing bacteria. Then, we care the derivatives’effection on biofilm. Last we preliminary study the mechanism of these derivations inacting with bcateria, and we think our findings will contribute to the development of anti-staphylococci drugs.Part.1 Structure-modification on inhibitors of the YycG histidine kinase:Screening derivatives with good activities to combat Staphylococcus epidermidis infectionsMany two-component signal transduction systems (TCSTSs) are ubiquitous in bacteria and are integral components of the adaptive regulatory processes utilized by pathogenic bacteria to sense the environment and coordinate the expression of the genes encoding virulence factors, making them attractive targets for antimicrobial therapy. Based on bioinformatics analysis,16 pairs of TCSTSs are found in the whole genome of S. epidermidis, most of which regulate multiple functions such as exoprotein production, adhesion and autolysis. Among these TCSTSs, two pairs (YycG/YycF and YhcS/YhcR) are required for bacterial growth. Subsequently, a series of novel inhibitors of the histidine kinase YycG protein of S. epidermidis had been discovered by structure-based virtual screening (SBVS) from a small molecular leading-compound library, followed by experimental validation, five compounds were screened out and displayed bactericidal effects on S. epidermidis cells.Based on those work, we colaberate with professer Han (Nanjing University of Technology), and modificate the structure of compound 2 and compound 5, then detect the biologic activity of these derivations. We obtained several derivatives that have displayed significant activity in inhibiting S. epidermidis growth (MIC values are 0.2～50μM). Furthermore, they displayed bactericidal effects on S. epidermidis cells (MBC values are 6.25～100μM).Part.2 The derivatives’effection on biofilm:blocking biofilm formation and killing bacteria embedded in old biofilmIt was reported that the biofilm could increase the bacteria’s resistance to antibiotic, so we detect these derivatives’effection on bacteria embedded in biofilm.First we study the effections on young biofilm (6 h)--blocking biofilm formation, and found that all these derivatives could inhibit the proliferation of young biofilm. Then we detect derivatives’effection on old biofilm (24 h), and found that no changes in morphology were detected, but when observed by confocal almost we found all bacteria embedded in old biofilm had been killed.In this study, we have found all derivatives have bacteriostatic activites. Our findings will contribute to the development of anti-staphylococci drugs.Part.3 The preliminary mechanism of derivatives’interaction with S. epidermidis and cytotoxicity & hemolysis assaysWe study the preliminary mechanism of derivatives’interaction with & epidermidis. First we care these derivatives’taget. So we detected the derivatives’ protease inhibition to YycG’s auto-phosphorylation, indicating that they are potential inhibitors of the YycG/YycF TCSTS, and found that those derivatives’IC50 valuses between 22.15～88.35μM. Then we study the specific amino acid of YycG that act with these derivatives by the method of screen the point mutation site of bacteria’s resistance to these derivatives.Because we detected all derivatives have good activities on inhibiting bacterial growth and killing bacteria. What is more, these derivatives could block biofilm formation and killing the bacteria embedded in old biofilm. So we now should care about the cytotoxicity on mammalian cells and hemolysis on the healthy human erythrocytes.We detected these derivatives’cytotoxicity on Vero cells by MTT (Cell Proliferation Kit I, Roche), and found CC50 Values of all derivatives are greater than 200μM, indicating that all derivatives have no toxicity on mammalian cells (CC50 represents the concentration that produce a 50% cytotoxicity effect on Vero cell, as the highest concentration tested corresponding to 200μM). When to the hemolysis on the healthy human erythrocytes, we found that all derivatives almost have no hemolysis at the concentration of MIC.
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