Structure-based discovery of inhibitors of the YycG histidine kinase: New chemical leads to combat Staphylococcus epidermidis infections
- Zhiqiang Qin†1,
- Jian Zhang†2,
- Bin Xu1,
- Lili Chen2,
- Yang Wu1,
- Xiaomei Yang1,
- Xu Shen2,
- Soeren Molin3,
- Antoine Danchin4,
- Hualiang Jiang2Email author and
- Di Qu1Email author
© Qin et al; licensee BioMed Central Ltd. 2006
Received: 01 July 2006
Accepted: 10 November 2006
Published: 10 November 2006
Coagulase-negative Staphylococcus epidermidis has become a major frequent cause of infections in relation to the use of implanted medical devices. The pathogenicity of S. epidermidis has been attributed to its capacity to form biofilms on surfaces of medical devices, which greatly increases its resistance to many conventional antibiotics and often results in chronic infection. It has an urgent need to design novel antibiotics against staphylococci infections, especially those can kill cells embedded in biofilm.
In this report, a series of novel inhibitors of the histidine kinase (HK) YycG protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular lead-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds displayed significant activity in inhibiting S. epidermidis growth. Furthermore, five of them displayed bactericidal effects on both planktonic and biofilm cells of S. epidermidis. Except for one, the compounds were found to bind to the YycG protein and to inhibit its auto-phosphorylation in vitro, indicating that they are potential inhibitors of the YycG/YycF two-component system (TCS), which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study.
These novel inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious than traditional screening technology.
In recent years, coagulase-negative strains of Staphylococcus epidermidis have become frequent causes of infections in connection with surgically implanted medical devices [1, 2]. In parallel, the appearance of multi-resistant and vancomycin-resistant S. epidermidis strains has increased quickly due to the increasing use of antibiotics in hospitals . The primary pathogenicity trait of S. epidermidis has been 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 [4, 5]. It has also been observed that aminoglycoside antibiotics may trigger biofilm formation in some bacteria . There is therefore an urgent need to design novel antibiotics against staphylococcus infections, especially in relation to biofilm development. Recently, the complete genome sequences of two S. epidermidis strains, viz. the non-biofilm-forming strain ATCC12228 and the biofilm-forming strain RP62A, have been published [7, 8], bringing about new opportunities to discover potential antimicrobial targets using in silico genome analyses.
Two-component system (TCS) control proteins, harboring histidine kinase (HK) and response transcription regulator activities, have been uncovered in most bacteria. Recently, the TCSs have attracted attention due to their potential as novel antibacterial targets, especially those required for regulation of bacterial growth and virulence in pathogenic microorganisms [9, 10]. One TCS, YycG/YycF, highly conserved and specific to low G+C Gram-positive bacteria has been shown to be essential for Bacillus subtilis and Staphylococcus aureus survival [11, 12]. Inhibitors of the YycG HK, such as synthetic imidazole and zerumbone derivatives, or aranorosinol B, obtained by screening acetone extracts from 4000 microbes, have been documented to be effective antibacterial agents against B. subtilis [13, 14]. Identification of this limited number of YycG inhibitors required laborious biological and chemical experiments, and the side-effects of these compounds on mammalian cells remain unclear. Moreover, B. subtilis may not be an optimal model organism to investigate biofilm formation, a process of major importance for the virulence of staphylococci. This prompted us to demonstrate that S. epidermidis possesses a homologous YycG/YycF TCS, and to investigate whether it would be an appropriate target for the design of novel antibacterial agents. As a prerequisite we set up a rapid and convenient procedure for screening novel inhibitors of the YycG/YycF TCS, testing the possible effects of these inhibitors on both planktonic and sessile bacteria, while using the extreme sensitivity of mammalian cells as a control to put aside compounds that would display a non-specific effect on membranes.
Upon binding, many small molecules may affect the functions of proteins. Functional analysis has been the basis of a variety of experiments, in which synthetic or purified small molecules have been used to probe the molecular mechanisms underlying the biological processes in which target proteins are involved. This chemistry-based approach has been coined "chemical biology" . Combinatorial chemistry and in vivo or in vitro High Throughput Screening (HTS) constitute preferred approaches for discovering active compounds against particular protein targets . A complementary approach is to use computational methods to identify active compounds (binders or hits) targeting the three-dimensional (3D) structure of the substrate binding pocket of a protein. This in silico approach is called Structure-Based Virtual Screening (SBVS) [16–19].
In the present study, we first identified the homologous YycG/YycF TCS in the genomes of the S. epidermidis ATCC12228 and RP62A strains. Next, a 3D structural model of the conserved HATPase_c domain of S. epidermidis YycG HK was constructed by using the homologous modeling approach. Subsequently, the SBVS method was used to search for potential YycG inhibitors from the SPECS chemical lead-compound database. Of the 76 candidates selected from the database by SBVS, seven compounds were active in inhibiting growth of S. epidermidis on plates or in liquid media. Five of these compounds displayed bactericidal effects on both planktonic and biofilm cells of S. epidermidis. Except for one, the compounds bound to the YycG protein and inhibited its auto-phosphorylation in vitro. These compounds displayed low cytotoxicity on mammalian cells and were not hemolytic, indicating that they may be good leads to develop new antibiotics against staphylococci infection.
In silico identification of the YycG/YycF TCS in S.epidermidis
A 3D model of the YycG HATPase_c domain of S. epidermidis
Discovery of potential inhibitors of the S. epidermidis YycG HK by Virtual Screening
The ATP-binding pocket formed by residues within a radius of 5 Å around the ATP site of the YycG HATPase_c model of S. epidermidis was used as the target site for high throughput virtual screening (HTVS). In a first step, 85,000 potential drug-like molecules, constituting an in-house database (named SPECS_1), were selected from the SPECS database using the drug-selection filter developed by Zheng et al. . The SPECS_1 database was searched for potential binding molecule structures using the program DOCK4.0 [28, 29] in a primary screening. The most optimal 10,000 structures were subsequently re-scored using the FlexX program  and CSCORE , a consensus scoring method that integrates five popular scoring functions. Two hundred molecules passed this highly selective filter. Finally, 100 molecules were manually selected from the latter sample as inhibitor candidates, according to their molecular diversity, their shape complementarity, and their potential for forming hydrogen bonds in the binding pocket of the YycG HATPase_c domain. Of those 100 candidates, 76 compound samples could be purchased from the SPECS Company for further experimental assays.
Antimicrobial activities of potential YycG inhibitors in vitro
Since the YycG/YycF TCS is essential for growth and survival in B. subtilis and S. aureus [11, 12], its conservation in sequence and in genome organization in S. epidermidis strongly suggests that it is essential in this organism as well. To test this possibility, we explored whether the potential YycG inhibitor candidates obtained by virtual screening could inhibit bacterial growth. At a concentration of 200 μM in liquid culture in a first screening procedure, seven out of the 76 candidates completely inhibited growth of S. epidermidis. The seven inhibitors belong to four different classes of chemical structures: three thiazolidinone analogs (compounds 2, 5, and 7), two benzamide analogs (compounds 1 and 3), one furan derivative (compound 4) and one derivative of pyrimidinone (compound 6), as shown in Figure 4.
Minimal inhibitory concentrations and minimal bactericidal concentrations of seven potential inhibitors of the YycG histidine kinase
Chemical inhibitor a
Minimal inhibitory concentration (MIC, μM)
Minimal bactericidal concentration (MBC, μM)
Killing biofilm cells of S. epidermidis by potential YycG inhibitors
Binding affinity of potential YycG inhibitors to the YycG' protein
Biological effects of seven potential inhibitors of the YycG histidine kinase
MICs (μM) a
K D value (μM) b
IC50 (μM) for YycG' c
CC50 (μM) on Vero cell d
Hemolysis (%) e
< 0.1 (< 0.1)
< 0.1 (0.5)
< 0.1 (< 0.1)
Inhibition of the YycG' protein ATPase activity in vitro
The common characteristic of HKs is ATP-dependent auto-phosphorylation, associated with the conserved HATPase_c domain. We measured the effects of our potential YycG inhibitors on the protein ATPase activity by using the Kinase-Glo™ Luminescent Kinase assay. Firstly, the putative kinase activity of YycG' protein was measured by quantifying the amount of ATP remained in solution after reaction. A direct relationship existed between the luminescence measured with the Kinase-Glo™ Reagent and the amount of ATP (see Additional File 2A), indicating the sensitivity of this assay is good. After adding purified YycG' protein into the reaction system (4 μg protein in each reaction system), the luminescence was decreased in all the groups containing different ATP concentrations, compared with parallel groups without YycG' treatment (see Additional File 2B), since the kinase can hydrolyze ATP for its auto-phosphorylation. Keeping the ATP concentration constant (50 μM), the luminescence was continuously decreased following the amounts of purified YycG' protein increased (see Additional File 2C). The results indicated that the purified YycG' protein possesses the ATPase activity in vitro. At a concentration of 50 μM, compounds 1–5 and 7 decreased the ATPase activity of 4 μg YycG' protein in the presence of 3 μM ATP by 52% to 86% (52%, 61%, 70%, 76%, 73% and 86%, respectively), indicating that the binding affinities of these compounds to YycG' correlate well with their inhibitory activities of auto-phosphorylation of YycG'. In contrast, compound 6, which binds poorly to the YycG' protein, showed almost no activity against YycG' ATPase activity (approximately 7% inhibition at 50 μM). The concentrations needed to decrease YycG' ATPase activity to 50% (IC50 values) by the 6 active compounds (1–5 and 7) were calculated by gradual dilution of these compounds with an invariable concentration of protein (4 μg) and ATP (3 μM), as shown in Table 2. The IC50 value of compound 6 was above 200 μM under the same reaction conditions. As a comparison, the fragment of another HK protein SrrB' containing homologous domains with YycG' in S. epidermidis  was also expressed and purified to observed the inhibitory effect on protein phosphotylation by these 6 potential YycG inhibitors (1–5 and 7) (Supple. Table 1). At the concentration of 50 μM, only compound 5 displays much lower inhibitory effect (20%) on SrrB' than it does (73%) on YycG', while the other 5 compounds have almost no inhibitory effects.
Cytotoxicity and hemolysis of the antimicrobial compounds in vitro
Interaction models of potential YycG inhibitors to the target protein
As potential YycG inhibitors, six compounds (1–5 and 7) selected by in silico screening bound with high affinity to the YycG' protein and inhibited its ATPase activity in vitro. To further investigate the sites of interaction between the compounds and the YycG protein, and to develop a strategy for designing novel inhibitors, models of the interaction of the compounds with the YycG protein were analyzed based on docking simulations.
The YycG/YycF TCS is conserved and specific for low G+C Gram-positive bacteria such as B. subtilis, S. aureus, Streptococcus pneumoniae and Listeria monocytogenes [11, 12, 37, 38]. This TCS has been shown to be essential in B. subtilis  and could not be inactivated by direct mutation in S. aureus , and which also occurs in S. epidermidis (this work). Moreover, several genes involved in cell-wall biosynthesis and metabolism, such as teichoic acid biosynthesis protein F , and cell wall synthesis protein YpfP  were predicted to be regulated by this TCS in S. epidermidis based on the presence of a potential YycF consensus DNA binding recognition sequence similar to the one described in B. subtilis and S. aureus [42, 43], although this predicted result needs to be further verified (Qin et al, unpublished data). We have therefore explored the possibility of using the YycG HK as a potential target in a screening for new antibiotics.
Compared with biological and chemical screening for new antibiotics, the advantage of SBVS technology is the rapid, economical and efficient throughput. Furthermore, lead-compound databases provide ample sources for screening, whereas chemical synthesis is time consuming and expensive. As a case in point, the use of lead-compound databases has already led to the discovery of the diarylquinoline lead-compound against Mycobacterium tuberculosis .
Screening 80,000 possible compounds in silico we finally retained 7, among which 6 were promising candidates as potential YycG HK inhibitors. These 6 compounds bound to the YycG protein in vitro and inhibited its ATPase activity, while they were also active antimicrobials against S. epidermidis. This does not prove, however, that YycG is the only target when compounds interact with bacteria. In fact, we have found a total of 16 putative TCSs in the genome of S. epidermidis, including YycG/YycF. Because they have many core features in common, we cannot exclude that some of the compounds may also bind to other HKs. This would in fact enhance the antibiotic effect of the compounds. To investigate this possibility, we expressed and purified the fragment of another HK of S. epidermidis – SrrB (designated as SrrB' containing similar domains with YycG' described in Methods), which is involved in the TCS SrrB/SrrA . We analyzed the effects of the 6 active compounds (1–5 and 7) on the auto-phosphorylation of this purified protein. We observed that only compound 5 (at 50 μM) inhibited the auto-phosphorylation of SrrB'. The inhibition was approximately 20% lower than that demonstrated here on YycG' (73%) under the same reaction conditions (see Additional File 3). This suggests that the compounds display a certain degree of specificity dependent on the characteristics of the HATPase_c domain structure in the different HKs.
One of the interesting and challenging goals of new searches for anti-microbial compounds is to identify potential drugs which are equally active against planktonic and sessile bacteria (biofilms). The YycG inhibitor compounds described here were found to have somewhat reduced bactericidal effect on mature biofilms of S. epidermidis. These compounds were, however, much more efficient against sessile bacteria than the commonly used staphylococcus antibiotic, vancomycin. In fact, vancomycin was almost without effect against biofilm cells (even at 128 μg/ml), as also reported by others . This was accounted for in S. aureus by reduced penetration of vancomycin and delay of the exposure of the bacteria in the biofilm deeper layers . However, it remains unknown whether the situation is the same with our compounds in S. epidermidis biofilms. Interestingly, seventeen compounds among the 76 candidates we retained for further studies were active in inhibiting biofilm formation without interfering with bacterial growth. Most of them did not bind to the YycG' protein nor inhibit its auto-phosphorylation in vitro, indicating that these compounds are not potential inhibitors of the YycG HK (data not shown). Their targets in S. epidermidis and the mechanisms of inhibiting biofilm formation are still under investigation.
In previous studies, some potential inhibitors of histidine kinase appeared to display trivial side effects, such as membrane disruption, excessive protein binding or limited bioavailability. This prevented their further development . In this study, the potential YycG inhibitors displayed low cytotoxicity and low hemolysis to mammalian cells at the effective concentrations we used in vitro. We are now in the process of finding out the more effective derivatives, using those as leads. In future work, an appropriate animal model also needs to be established to investigate the effect of these compounds in vivo.
In our study, these novel inhibitors of YycG histidine kinase are considered as promising lead-compounds for developing new reagents against staphylococci infections. Furthermore, the structure-based virtual screening (SBVS) technology can be widely used for discovery of potential bacterial TCSs inhibitors in both Gram-positive and Gram-negative species, even on other "drug target" proteins. And it is more rapid and efficacious than traditional screening technology.
Bacterial strains, media and reagents
Staphylococcus epidermidis ATCC12228 and RP62A strains were purchased from the American Type Culture Collection (ATCC, Manassas, USA). Staphylococcus aureus ATCC29213, Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were kindly provided by Dr. Bijie Hu at Zhongshan Hospital (Shanghai, China). S. epidermidis strains se527, se886, se847, seG203, Streptococcus pyogenes strain 144, and Streptococcus mutans strain 128 were all clinical isolates from Zhongshan Hospital and Huashan Hospital (Shanghai, China). If not stated otherwise, S. epidermidis and S. aureus strains were grown at 37°C in tryptic soy broth (TSB, Oxoid) containing 0.25% glucose, E. coli and P. aeruginosa strains were grown at 37°C in Luria-Bertani (LB, Oxoid).
All compounds used as inhibitor candidates were purchased from the SPECS Company in the Netherlands. Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO). All other chemicals were of reagent grade or ultra-pure quality and purchased from Sigma.
Domain analysis was performed based on the SMART database . The complete genome sequences of the two S. epidermidis strains, ATCC12228 (NC_004461) and RP62A (NC_002976) were accessed from the National Center for Biotechnology Information (NCBI) genome database . The homologous sequences with the YycG HATPase_c domain of S. epidermidis were searched from the Protein Data Bank (PDB) , using the BLASTp program . ClustalX was used to align the protein sequences .
3D structure modeling of the YycG HATPase_c domain
The sequence of S. epidermidis HK YycG was retrieved from GenBank (accession number AY864800). The Align123 module encoded in InsightII  was used in the pair-wise sequence alignment. Using the secondary structure information of EnvZ (PDB entry 1BXD) , the sequence alignment was adjusted manually to obtain a fine alignment for 3D structure construction. The 3D model of the YycG HATPase_c domain was generated by using the MODELLER program  encoded in InsightII. Finally, the whole structural models were optimized using the Discover_3 module of InsightII with CVFF force field. Several structural analysis softwares such as Prostat and Profile-3D  were used to check the structure quality. The Prostat module of InsightII was used to analyze the properties of bonds, angles, and torsions. The Profile-3D program was used to check the structure and sequence compatibility.
Structure-based virtual screening
Before docking the small molecules of interest on the model structure, we delineated the general features that the binding pocket should have. Major residues possibly composing the ATP-binding site of YycG HATPase_c domain were identified by the sequence alignment with the osmolarity sensor protein EnvZ of E. coli, and the ATP-binding pocket was probed on the optimized 3D model of YycG HATPase_c domain using the SiteID program encoded in Sybyl6.8 . The surface (electrostatic, hydrophobic and hydrogen bonding) properties of the binding pocket of the YycG HATPase_c domain were calculated using the MOLCAD program encoded in Sybyl6.8. The ATP-binding pocket of the YycG HATPase_c domain was used as a target for screening the SPECS database using the docking approach . The SPECS database contains the structural information of 280,000 small molecules. The SPECS Company supplies all the compound samples collected from different sources. As a first step, the SPECS database was submitted to our own filter of drug-ability , non-drug-able molecules were eliminated from the database, and finally 85,000 potential drug-able molecules were selected out for docking screening. The program DOCK 4.0 [28, 29] was used for primary screening. Residues within a radius of 5 Å around the ATP-binding pocket of the YycG HATPase_c domain were used for constructing the grids for the docking screening. During the docking calculations, Kollman-all-atom charges  were assigned to the protein, and Gasterger-Hückel charges [57, 58] were assigned to the small molecules. Conformational flexibility of the compounds from the database was implemented in the docking search. During DOCK simulation, the ligand-receptor binding energy was approximated by the sum of the van der Waals and electrostatic interaction energies. After an initial evaluation of orientation and scoring, a grid-based minimization was carried out for the ligand to locate the nearest local energy minimum within the receptor binding site. Position and conformation of each docked molecule were optimized using the single anchor search and torsion minimization method of DOCK 4.0. The 10,000 compounds with the highest score as obtained by DOCK search were selected for a second round docking using the FlexX program , and CSCORE  was used to re-score the compounds. The virtual screening was performed on a 392-processor Sunway-1 supercomputer at the Shanghai Supercomputer Center.
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays
MIC assays for the antibacterial activities of the compounds were performed according to the broth micro-dilution (in tubes) method of the Clinical and Laboratory Standards Institute (CLSI) of America . The Minimal Bactericidal Concentration (MBC) was obtained by subculturing 100 μl from each negative (no visible bacterial growth) tube from the MIC assay, onto substance-free Mueller-Hinton agar plates. The plates were incubated at 37°C for 24 hours, and the MBC was defined as the lowest concentration of substance which produced subcultures growing no more than five colonies on each plate.
Killing biofilm cells of S. epidermidis by potential YycG inhibitors
An overnight culture of S. epidermidis strain RP62A was diluted 1:100 in TSB containing 0.25% glucose, then 1 ml bacterial suspension was inoculated into the wells of sterile 12-well polystyrene microtiter plates (Falcon) incubated at 37°C for 24 h. The plates with mature biofilm were washed gently four times with sterile PBS before adding fresh TSB containing the various compounds at their MBC values, and incubated at 37°C for 24 h. The plates were washed again four times with sterile PBS, then the biofilm cells were scraped from the plate and resuspended in 1 ml PBS, and violently vortexed to disintegrate clumps of cells. Next, the suspension was diluted gradually with sterile PBS and subcultured onto substance-free Mueller-Hinton agar plates, incubated at 37°C for 24 h, and the colonies were counted. The fresh medium containing vancomycin at the MBC value and substance-free fresh medium served as the controls. The experiment was repeated three times.
Cloning, expression and purification of the YycG' protein
The YycG' fragment containing the cytoplasmic signal domains (the HATPase_c and HisKA domain, see Figure 1A) of YycG (370aa to 610aa) was amplified by PCR (with the chromosomal DNA of S. epidermidis ATCC12228 as the template, and (5' GCGGATCCACAACAACAAGTCGAACGTGAAC 3') and (5' GCCTCGAGTTATTCATCCCAATCACCGTCT 3') as the primers. Subsequently, the fragment was digested with BamHI and XholI (TAKARA, Japan) and ligated into the corresponding sites of pET28a (Promega, Madison, USA) to obtain pETYycG'. The expressed YycG' protein was purified with the ProBond™ Purification System (Invitrogen, California, USA), according to the manufacturer's protocol.
Compound-YycG' protein binding assay
The in vitro binding affinities of the inhibitor compounds to the YycG' protein was determined using surface plasmon resonance (SPR) biosensor technology on the dual flow cell Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) with a similar method as described in our previous study . Immobilization of the YycG' protein to the hydrophilic carboxymethylated dextran matrix of the sensor chip CM5 (Biacore) was carried out by the standard primary amine coupling reaction. The protein to be covalently bound to the matrix was diluted in 10 mM sodium acetate buffer (pH 4.2) to a final concentration of 0.3 mg/ml. Equilibration of the baseline was completed by a continuous flow of HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA and 0.005% (v/v) surfactant P20, pH 7.4) through the chip for 1–2 hours. Biacore data were collected at 25°C with HBS-EP as running buffer at a constant flow of 20 ml/min. Sensorgrams were processed by using automatic corrections for non-specific bulk refractive index effects. The equilibrium constants (KD) evaluating the protein-ligand binding affinity were determined by the steady state or the kinetic state affinity fitting model encoded in the Biacore analysis software.
Inhibition assay for the ATPase activity
The inhibitory activities of the compounds for the ATPase activity of the YycG' protein was measured using the Kinase-Glo™ Luminescent Kinase Assay (Promega, Madison, USA). Briefly, 4 μg YycG' protein was pre-incubated with a series of dilution of compounds in the reaction buffer (40 mM Tris [pH 7.5], 20 mM MgCl2, and 0.1 mg/ml BSA), at 25°C for 30 min. Then 3 μM ATP was added for another incubation of 30 min at 25°C, and Kinase-Glo™ Reagent was added to detect the rest amount of ATP, as recorded from luminescence measurements (RLU). In parallel, theYycG' protein with no addition of compounds was used as the control. The rate of inhibiting protein phosphorylation (Rp) by the compounds was calculated from equation 1:
IC50 (the concentration of inhibition of 50% YycG' protein autophosphorylation) was obtained by using the Origin v7.0 software (OriginLab, Northampton, USA).
Cytotoxicity and erythrocyte hemolysis assays
Cytotoxicity of the antibacterial compounds on cultured Vero cell was measured by using the Cell Proliferation Kit I (MTT) (Roche, Indianapolis, USA) according to the manufacturer's protocol. Addition of DMSO (1%) in the medium produced a slight cytotoxicity on Vero cell, which could easily be corrected for. Each assay was performed in quadruplicate and repeated three times. The results were converted to percentage of the control (cells only treated with 1% DMSO) and CC50 (concentrations that produce a 50% cytotoxicity effect on Vero cell) was calculated by the Origin v7.0 software (OriginLab, Northampton, USA). Hemolytic activities of the compounds were determined by using healthy human erythrocytes . The erythrocytes were washed three times in sterile saline and resuspended to 5% prior to the assay. Then a volume of 200 μl cell suspension containing MIC or 4×MIC concentrations of the compounds was added in quadruplicate to the wells of 96-well microtiter plates (Falcon). Cells without compound treatment and cells with 1% Triton-100 treatment were used as 0% and 100% hemolysis controls, respectively. The cell suspensions were incubated for 1 hour at 37°C and centrifuged at 1000×g for 10 min. Volumes of 100 μl supernatants were transferred to another sterile plate and hemoglobin release from the cells was determined at 570 nm. Addition of DMSO (1%) in the medium did not affect the integrity of erythrocyte membrane. The hemolysis assays were repeated twice.
Pharmacophore model building
The best conformations of the six inhibitor outputs from Dock4.0 were superimposed using the DISCO program  encoded in Sybyl6.8. Multi-conformations were generated for the Flex Searches using Multisearch and REJECT features. The REJECT feature removes duplicates to leave a set of unique low energy conformers. DISCO produces a number of possible pharmacophores. The hypotheses were grouped on the basis of the assignment of atoms to features. Four features containing two hydrogen donor and two hydrophobic centers were detected by DISCO.
The abbreviations used are
structure-based virtual screening
high throughput screening
minimal inhibitory concentration
surface plasmon resonance.
This work was supported by the State Key Program of Basic Research of China (973) (2002CB512802, 2002CB512803 and 2002CB428), the Hi-Tech Program of China (863) (2002AA229041, 2004AA223080), the Scientific Technology Development Foundation of Shanghai (02DJ14002, 055407069), and the National Natural Science Foundation of China (30400017). AD wishes to acknowledge support from the European Network of Excellence "Europathogenomics" LSHB-CT-2005-512061.
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