Discovery And Identification Of The Lead Inhibitors Of Streptococcus Pneumoniae Based On The Virtual Screening With The Homology-Modeled Structure Of Histidine Kinase (VicK)

Two-component signaling systems (TCSs) are main signaling transductional system in sensing, responding to various environmental stimuli, and thus regulating virulence expression and viability. In different bacteria, TCSs typically consist of two basic protein units: a sensor kinase (HK) and a response regulator (RR). Histidine kinase, which plays a major role in signal transduction in prokaryotes, is a multifunctional enzyme having autokinase, phosphotransferase and phosphatase activities, and most of them are transmembrane sensor proteins. As many signaling pathways, protein phosphorylation is a way to transmit information. In prokaryotes, different from the higher eukaryotes, amino acid residues tyrosine, serine or threonine serve as the substrate for phosphorylation, once being activated, histidine kinase autophosphorylated, and the phosphoryl group of the histidine residue is subsequently transferred to a conserved aspartate residue in the response regulator. This discrepancy between eukaryotes and prokaryotes makes them attractive targets for screening of novel antibiotic compounds.Histidine kinase VicK belongs to the family of Pho, which was first discovered in B.subtilis. Among 13 TCSs identified in S.pn, VicR/K is the only one essential for growth and has been designated as MicA/B, YycF/G or 492hk/rr. The VicR/K TCS is specific to low G + C Gram-positive bacteria, and it is highly conserved among this group of bacteria. The VicR/K TCS system is involved in and is essential for the proper regulation of bacterial cell wall, membrane composition in the recent years; it has attracted great attention in developing innovative antimicrobial agents. Now, it have been reported that some lead-compound against B.subtilis and Staphylococcus epidermidis (S.epidermidis) were screened out based on homology modeling and virtual screening, while it was not reported in S.pn. Therefore, based on homology modeling, this study was designed to screen out lead-compounds against the target VicK in S.pn. It includes the following parts:1. Histidine kinase VicK of S.pn: Homology modeling and analysis.3D model of VicK protein was first constructed by homology modeling, and then the reliability of the model was assessed using Procheck and Profile₃D softwares. Besides, the active-site cavity of VicK and the residues key for substrate interaction were analyzed by Autodock4.0. The results showed that VicK HATPase_c domain in S.pn (GenBank accession number: AAK75332.1) was similar with the domain of Thermotoga maritime (PDB entry: 2c2a), with 33% sequence identity and 57% conservative replacements. The constructed 3D model of VicK adopted a similar folding pattern to the template and the two matched well. The conservative amino acids in the substrate-binding pocked, such as Asn145, Asn149 and Lys152, as well as the hydrophobic residues at the bottom of the pocket played important role in binding and hydrolyzing substrate ADP. We have successfully constructed a reliable model of VicK protein. The model is fundamental for further designing antibacterial drugs.2. Virtual screening the lead compounds of histidine kinase VicK of S.pn.A primary screening was conducted by using the program DOCK4.0. Residues within a radius of 4? around the ATP-binding pocket of the VicK HATPase_c domain were used for constructing the grids for the docking screening. Secondly, the 10,000 compounds with the highest score as obtained by DOCK4.0 search were selected for a second round docking by using Cscore programs. Thirdly, the 2,425 compounds from last round were followed by our own filter of drug-ability to eliminate the non-drug-able molecules and were obtained 308 compounds in this round screening. Finally, we manually selected 105 molecules according to their molecular diversity, shape complementarities, and potential to form hydrogen bonds in the binding pocket of the VicK HATPase_c domain.3. Identification of the kinase activity of VicK' protein in S.pn. Domain analysis (http://smart.embl.de/smart/show_motifs) indicated that the VicK protein of S.pn contained one transmembrane segment and several domains: PAS, PAC, HisKA and HATPase_c. The VicK' gene fragment containing the cytoplasmic signal domains (the HATPase_c and HisKA domain) of VicK' (coding 200-449aa) was amplified by PCR. Subsequently, the fragment was ligated into the corresponding sites of pET28a to obtain a recombinant plasmid pET28a/VicK'. After being transformed into E.coli strain BL21 (DE3), this recombinant plasmid was induced to express the protein of VicK' by isopropyl-1-thio-β-D- galactopyranoside (IPTG). Cells were harvested and sonicated, and then the debris was removed by centrifugation. The fraction containing the cytoplasmic domain was isolated from the supernatant solution through a His-tagged column, as assessed by gel electrophoresis and Coomassie Blue staining. The product was identified by SDS-PAGE and Western blot assays, purified by a His-tagged Ni affinity column, and determined for kinase activity. The results showed that the product was about 35 kDa, reaching a purity of 95%, and has dose-dependent kinase activity of hydrolyzing ATP in vitro.4. In vitro and in vivo assayUsing the purified VicK' protein possessed the ATPase activity for obtained 105 molecules based on virtual screening, we initially screened 23 compounds of 105 candidate inhibitors decreased the ATPase activity of VicK' protein by more than 50%, and then investigated the bactericidal activity of these 23 compounds against S.pn. Finally, six of them could obviously inhibit the growth of S.pn, so they were further studied using a standard MIC, MBC and cytotoxicity assay.Survival of S.pn against 6 compounds of three concentrations was detected by spectrophotometer per hour, and hence drawing the time- and concentration-dependent curve. Based on the curve, the time and dose for animal was further determined by in vivo assay. Mouse sepsis model by S.pn (D39) was successfully established by intraperitoneal infection of 100μl S.pn (5×103 CFU/ml). Generally, these mice began to die within 24 hours and couldn't survive more than 48 hours unless they got appropriate therapeutic treatments. These mice were randomly assigned to 8 groups (10-12 per group, half in each sex): 6 compound-treated groups, one negative control and one positive control. The results showed that the bacteriostases of the 6 compounds were significantly increased as concentration increasing. Survival of the mouse infection models was monitored after treated differently for a period of an 8-day. Survival curves were analyzed by using the Kaplan-Meier Method. Significant treatment effects were found among the groups (P < 0.01) by an overall comparison. Pairwise comparisons revealed that compounds 1-6 prolonged could significantly prolong the survival time in mouse infection models as compared to negative control (p < 0.01). Compound 1, 2, 3 and 6 were less effective than positive control PNC (P < 0.05 or P < 0.1), while compound 4 and 5 were effective against S.pn infection.In this study, we got 6 small molecular compounds by screening, and all of them have in vivo and in vitro inhibition activity against S.pn by specifically inhibiting the target protein VicK. Moreover, this study established a basic method for further investigation of the antibacterial chemicals.