BACKGROUND
s. pneumoniae is a major risk factor with high morbidity and mortality world-widely, especially in the elderly and children.
it is believed to be one of the four major infectious disease killers  <cit> .
meanwhile, an increasing number of bacterial strains with resistance are encountered in the clinic nowadays, among which antibiotic-resistant s. pneumoniae has caused many deaths due to antibiotics abusage in hospitals.
therefore, it is urgent to develop new types of antibiotics.
in prokaryotes, the two-component signaling systems , each pair of which are typically composed of histidine kinase  and response regulator , play important roles in drug-resistance, pathogenesis and bacterial growth  <cit> .
the regulation of tcs on histidine phosphorylation in signal transduction distinct from that on serine/threonine and tyrosine phosphorylation in higher eukaryotes  <cit> .
for some tcss, both the hk and rr are essential for bacterial viability in several gram-positive pathogens, including bacillus subtilis , enterococcus faecalis and staphylococcus aureus   <cit> , and thus received attention as potential targets for antimicrobials  <cit> .
in s. pneumoniae, although at least  <dig> tcss were identified, only tcs <dig>  is essential for bacteria viability, which can be a potential target for antimicrobial intervention.
to be detailed, in tcs <dig>  only functional vicr appears to be essential for s. pneumoniae  <cit> , without which s. pneumoniae can't grow or act as a pathogen  <cit> .
however, the crystal structure of vicr is unsuitable for structure-based virtual screening because the active site is too shallow to dock a small molecule  <cit> .
the reason that vick does not seem to be essential for s. pneumoniae viability, was supposed to be that some currently unknown hks also participate in the activation of vicr by phosphorylation  <cit> .
however, among these hks, vick it is best-known one with definite action on vicr.
moreover, recent researches showed a high-degree homology in the catalytic domain of these hks  <cit> .
thus theoretically, selective inhibitors to vick, a representative of hks, can interrupt the phosphorylation of vicr and ultimately reduce the viability of s. pneumoniae.
the structure-based virtual screening , an approach used widely in drug design and discovery, possesses many advantages, such as rapidness, economization, efficiency and high-throughput.
in the recent years, sbvs has attracted great attention in developing innovative antimicrobial agents.
a case in point is the discovery of a lead-compound named diarylquinoline against mycobacterium tuberculosis  <cit> .
our study here was designed to search the compound database for potential inhibitors targeting the vick protein of s. pneumoniae by using in silico and experimentalmethods, which may provide much valuable information to develop new antibiotics against pneumococcal infection.
RESULTS
sequence analysis of the vick tcs in s. pneumoniae
domain analysis http://smart.embl.de/smart/show_motifs.pl?id=q9s1j <dig> indicated that the vick protein of s. pneumoniae contained one transmembrane segment and several domains: pas, pac, hiska and hatpase_c.
multi-alignment of the hatpase_c domain sequences showed that in most bacteria the sequences around the atp binding site of vick hks are similar and have four conserved motifs: the n box, g <dig> box, f box and g <dig> box  <cit> .
this high homology of atp binding domain of hks in bacteria makes it reasonable to screen antibacterial agents by using this domain as a potential target  <cit> .
compared with vick hatpase_c domain in s. pneumoniae , the most homologous sequence in the structural protein data bank  was the similar domain of thermotoga maritime   <cit> , a tcs molecule, with 33% sequence identity and 57% conservative replacements .
this domain is the entire cytoplasmic portion of a sensor hk protein.
the x-ray crystal structure of the domain of thermotoga maritima was therefore used as a template for modeling the 3d structure of the vick hatpase_c domain of s. pneumoniae.
a 3d model of the vick hatpase_c domain of s. pneumoniae
based on the x-ray diffraction crystal structure of the homologous domain of the thermotoga maritima, a 3d model for the vick hatpase_c domain of s. pneumoniae was constructed.
figure 2a shows the final structure of this model that were checked and validated using structure analysis programs prosa and profile-3d  <cit> .
this model of 3d structure contains five stranded β-sheets and four α-helices, which form a two-layered α/β sandwich structure.
figure 2b indicates that the model superposed well with the homologous domain of thermotoga maritima, with a root-mean-square deviation  of the cα atoms being about  <dig>  Ǻ. the surface shape and general electrostatic feature of the hatpase_c domain of vick were shown in figure 2c.
the atp binding site consists of a relatively hydrophobic inner cavity and a larger hydrophilic outer cavity.
both cavities are connected by a gorge-like channel, and are consisted of highly conserved residues which can bind and fix the substrate.
the inner part lack of polar amino acid residues can accommodate the adenosine, while the outer one rich in charged residues can bind the triphosphate.
discovery of potential inhibitors of the s. pneumoniae vick hk by virtual screening
the target site for high throughput virtual screening  was the atp-binding pocket of the vick hatpase_c model of s. pneumoniae, which consisted of residues within a radius of  <dig> Ǻ around the atp site.
in the primary screening, the database specs containing about  <dig>  molecules was searched for potential binders using the program dock <dig>   <cit> .
subsequently, structures ranked in the first  <dig>  were re-scored by using the autodock  <dig>  program  <cit> .
as a result, about  <dig> molecules were filtered out by these highly selective methods.
finally, we manually selected  <dig> molecules according to their molecular diversity, shape complementarities, and the potential to form hydrogen bonds and hydrophobic interactions in the binding pocket of the vick hatpase_c domain.
inhibition of the vick' protein atpase activity in vitro
in order to confirm the interaction of the potential vick inhibitors with their putative target protein, we expressed and purified his-tagged vick' protein by using the pet28a plasmid in bl <dig> as shown in figure 3a.
the kinase activity of vick' protein was measured by quantifying the amount atp remained in solution after the enzymatic reaction .
these results indicated that the purified vick' protein possessed the atpase activity, which can hydrolyze atp in vitro.
using the purified active vick', we obtained  <dig> compounds from the  <dig> candidate inhibitors which could decrease the atpase activity of vick' protein by more than 50%, indicating these compounds may also be potential vick inhibitors in s. pneumoniae.
antimicrobial activities of potential vick' inhibitor and cytotoxicity of the antimicrobial compounds in vitro
we investigated the bactericidal activity of these  <dig> compounds against s. pneumoniae using a standard minimal bactericidal concentration assay  .
six compounds , each inhibiting the vick' activity by more than 50%  , could obviously inhibit the growth of s. pneumoniae, with mic values below  <dig> μm.
moreover, their mic values were positively correlated with the corresponding ic <dig>  values , which indicates that the bactericidal effects of these chemicals were realized by disrupting the vick/r tcs system in s. pneumoniae.
chemical structures of these  <dig> compounds are shown in figure  <dig>  which belong to three different classes of chemicals: one imidazole analogue, four furan derivatives and one derivative of thiophene .
a 3-- <dig>  5-diphenyl tetrazolium bromide  assay was carried out on vero cell line to determine the cc <dig> values of these compounds.
as shown in table  <dig>  the cc <dig> values of all these six compounds were larger than  <dig> μm and than their respective mic values, indicating low cytotoxicity effects on vero cell.
collectively, these compounds inhibited bacterial growth with low toxic effects.
time- and concentration-dependent growth curve
while several compounds identified in our study could be used as excellent drug leads in vitro, the best and most valuable ways would be in vivo validation.
the following results of the time- and concentration-dependent effects of the lead inhibitors on the growth of s. pneumoniae further illustrated their antibacterial characteristics, and would be an important guide for in vivo administration.
as shown in figure  <dig>  the similar curves of compounds  <dig>   <dig>   <dig> and  <dig> indicated that these compounds have significant activity against s. pneumoniae at concentration of about  <dig> μm, and this activity could last at least  <dig> hours.
the most efficient inhibitor identified was compound  <dig>  which had bactericidal effect against s. pneumoniae even at concentration of as low as  <dig>  μm.
however, even at concentration of  <dig> μm, compound  <dig> was not likely to have bactericidal effect, but it seemed to have delayed the multiplication of s. pneumoniae.
therapeutic effects of the lead compounds in mouse s. pneumoniae infections
mouse sepsis models by s. pneumoniae  were successfully established by intraperitoneal injection of  <dig> μl s. pneumoniae .
generally, these mice began to die within  <dig> hours and couldn't survive more than  <dig> hours unless they got appropriate therapeutic treatments.
for facilitation of comparisons between the effects of these compounds and positive control , the concentration of penicillin used in this study almost equaled to that of the lead compounds.
to rule out the direct antibacterial effects that may compromise with the efficiency of this model, the lead compounds and penicillin were administrated through caudal vein.
as shown in figure  <dig>  these compounds were able to decrease, though slightly, the mortality of the infected mice in the first  <dig> hours as compared to negative control  .
significant treatment effects were found among the groups  by an overall comparison.
pairwise comparisons revealed that compounds 1– <dig> prolonged survival time in mouse sepsis models as compared to negative control .
however, compound  <dig>   <dig>   <dig> and  <dig> were less effective than positive control pnc .
although these compounds could not reverse the fatal pneumococcal infection with concentration used in this study, in vivo antibacterial activity of these six compounds suggested that it would be promising to develop lead-compound-based drugs against pneumococcal infection.
molecular modeling of vick' protein and its potential inhibitors
in order to get insight into the mechanism of inhibition, further studies were carried out to verify the interaction modes between six compounds and the modeled structure of vick' protein.
autodock  <dig>  software was used for the docking simulation.
the binding conformations of these inhibitors in the atp-binding pocket of the vick hatpase_c domain were shown in figure  <dig>  although these structures are diverse, the binding models of six potential inhibitors are similar, especially in the inner part of the conserved domain.
the surface of the binding pocket  is divided into two parts, one is hydrophobic inner part composed of residues ile <dig>  ile <dig>  leu <dig>  ile <dig>  phe <dig>  and the other is the outer hydrophilic part consisted of residues asn <dig>  lys <dig>  tyr <dig>  arg <dig>  arg <dig>  all six compounds bind in the pocket with rigid aromatic ring parts inserting into the inner part.
in the large and flexible outer part, these compounds adopt different interactions.
all of them have hydrogen bond acceptors in the binding outer part.
they could form hydrogen networks with the polar residues to stabilize the substrate interactions.
their binding models resemble natural substrate atp much.
discussion
in bacteria, hks have fundamental roles in tcs signal transduction pathways.
thus they are major targets for antibacterial drug development.
high structural and sequence homology of this kinase gene family makes the hks ideal targets for homology modeling and structure based virtual screening.
sbvs is an approach based on the three-dimensional structures of macromolecular to identify chemical entities binding to the targets and to elicit potential biological mechanisms with the advantages of speed, efficiency and high-throughput.
the availability of the small molecular lead-compound library and the modeled 3d target structure makes it possible to use sbvs to screen out a limited number of promising candidates that can interrupt the tcs signal transduction by interacting with the hks substrate of s. pneumoniae.
hks, as novel antibacterial targets, have attracted many attentions due to their essentiality in the viability of microbes and their deficiency in animals.
hks are involved in the regulation of bacterial growth and virulence in many bacterial species.
previously, a hk named vick has been used to screen lead compound inhibitors in b. subtilis and s. epidermidis.
we here for the first time obtained  <dig> candidate chemical compounds directly aiming at s. pneumoniae vick by screening  <dig>  possible compounds in silico.
compounds that can bind to the purified target protein vick' and compete with its substrate atp were further verified by in vitro and in vivo antibacterial assays.
eventually, we obtained  <dig> compounds with antibacterial activity that may be used as novel drug leads.
commonly, the response regulator yycf and the histidine kinase yycg are the only essential tcs for viability in b. subtilis and s. aureus  <cit> .
in s. pneumoniae, the vicr/k tcs regulates the expression of several critical genes, such as those encoding surface proteins and virulence factors  <cit> .
however, only the response regulator vicr was found to be essential  <cit> .
the signal transduction of vick was possibly bypassed by other tcs hks  <cit> .
vick has conserved atp-dependent hatpase_c domains accounting for autophosphorylation.
even non-cognate hks from other bacteria can phosphorylate the purified vicr from s. pneumoniae  <cit> .
in a previous study  <cit> , the mic values of the lead compounds screened out by sbvs targeting the yycg of s. epidermidis were almost equal to the corresponding ic <dig>  values, with a correlation coefficient of  <dig> , which suggested that inhibition of 50% the yycg protein activity would interfere with the growth of s. epidermidis.
if this case is true in s. pneumoniae, the result that the mic values of the lead-compounds were far less than the corresponding ic <dig> values may be explained as bypass effects of these compounds on other hks.
in a word, these lead compounds are most likely having a "cross-inhibition" on other hks in s. pneumoniae, which can enhance their antibacterial effects, although they were not verified in this study.
although the vick protein in s. pneumoniae can be homologous to yycg in other gram-positive strains, such as s. epidermidis, enterococcus faecalis and s. aureus, different strains generally have different characteristics of the hatpase_c domain structure of hks.
these characteristics will determine the binding specificity of the lead compounds screened out by sbvs.
moreover, a different template for homologous modeling and different parameters for sbvs were used, which can guarantee the specificity of the lead compounds binding to the vick' discovered.
what's more,  <dig> compounds can inhibit the purified vick' protein activity by more than 50%,  <dig> of which displayed different degrees of antibacterial effects in vitro and in vivo.
regretfully, the in vivo activities of these compounds were not quite consistent with their corresponding in vitro activity, and some compounds displayed obvious cytotoxicity, which would challenge our future investigation.
moreover, it seems to be a paradox that compound  <dig> have less bactericidal effects in the time- and concentration-dependent antibacterial assays, but demonstrated significant therapeutic effects in mice infected by s. pneumoniae.
however, due to the vick' is not essential in s. pneumoniae, this chemical may have a possibility to interrupt the invasion and virulence rather than cause numerous death of the bacterium, which decreases the selection pressure and contributes to the maintenance of species diversity, thus reduces the emergence of drug-resistant strains.
anyway, the subtle mechanisms need our future work.
CONCLUSIONS
to summarize, we have successfully found out several promising lead compounds for further drug development in this study, which also can be used as inhibitors to explore the mechanism of autophosphorylation by vick as well as other hks.
important work in future would be validation of their antibacterial effects in different strains and structural modification for more effective derivatives with less in vivo toxicity, and investigation into whether they can bind to other atp-dependent kinase is also necessary.

 s. pneumoniae is a major risk factor with high morbidity and mortality world-widely, especially in the elderly and children. it is believed to be one of the four major infectious disease killers. meanwhile, an increasing number of bacterial strains with resistance are encountered in the clinic nowadays, among which antibiotic-resistant s. pneumoniae has caused many deaths due to antibiotics abusage in hospitals. therefore, it is urgent to develop new types of antibiotics. in prokaryotes, the two-component signaling systems, each pair of which are typically composed of histidine kinase and response regulator, play important roles in drug-resistance, pathogenesis and bacterial growth. the regulation of tcs on histidine phosphorylation in signal transduction distinct from that on serine/threonine and tyrosine phosphorylation in higher eukaryotes. for some tcss, both the hk and rr are essential for bacterial viability in several gram-positive pathogens, including bacillus subtilis, enterococcus faecalis and staphylococcus aureus, and thus received attention as potential targets for antimicrobials. in s. pneumoniae, although at least tcss were identified, only tcs is essential for bacteria viability, which can be a potential target for antimicrobial intervention. to be detailed, in tcs only functional vicr appears to be essential for s. pneumoniae, without which s. pneumoniae can't grow or act as a pathogen. however, the crystal structure of vicr is unsuitable for structure-based virtual screening because the active site is too shallow to dock a small molecule. the reason that vick does not seem to be essential for s. pneumoniae viability, was supposed to be that some currently unknown hks also participate in the activation of vicr by phosphorylation. however, among these hks, vick it is best-known one with definite action on vicr. moreover, recent researches showed a high-degree homology in the catalytic domain of these hks. thus theoretically, selective inhibitors to vick, a representative of hks, can interrupt the phosphorylation of vicr and ultimately reduce the viability of s. pneumoniae. the structure-based virtual screening, an approach used widely in drug design and discovery, possesses many advantages, such as rapidness, economization, efficiency and high-throughput. in the recent years, sbvs has attracted great attention in developing innovative antimicrobial agents. a case in point is the discovery of a lead-compound named diarylquinoline against mycobacterium tuberculosis. our study here was designed to search the compound database for potential inhibitors targeting the vick protein of s. pneumoniae by using in silico and experimentalmethods, which may provide much valuable information to develop new antibiotics against pneumococcal infection.  sequence analysis of the vick tcs in s. pneumoniae domain analysis http://smart.embl.de/smart/show_motifs.pl?id=q9s1j indicated that the vick protein of s. pneumoniae contained one transmembrane segment and several domains: pas, pac, hiska and hatpase_c. multi-alignment of the hatpase_c domain sequences showed that in most bacteria the sequences around the atp binding site of vick hks are similar and have four conserved motifs: the n box, g box, f box and g box. this high homology of atp binding domain of hks in bacteria makes it reasonable to screen antibacterial agents by using this domain as a potential target. compared with vick hatpase_c domain in s. pneumoniae, the most homologous sequence in the structural protein data bank was the similar domain of thermotoga maritime, a tcs molecule, with 33% sequence identity and 57% conservative replacements. this domain is the entire cytoplasmic portion of a sensor hk protein. the x-ray crystal structure of the domain of thermotoga maritima was therefore used as a template for modeling the 3d structure of the vick hatpase_c domain of s. pneumoniae. a 3d model of the vick hatpase_c domain of s. pneumoniae based on the x-ray diffraction crystal structure of the homologous domain of the thermotoga maritima, a 3d model for the vick hatpase_c domain of s. pneumoniae was constructed. figure 2a shows the final structure of this model that were checked and validated using structure analysis programs prosa and profile-3d. this model of 3d structure contains five stranded β-sheets and four α-helices, which form a two-layered α/β sandwich structure. figure 2b indicates that the model superposed well with the homologous domain of thermotoga maritima, with a root-mean-square deviation of the cα atoms being about Ǻ. the surface shape and general electrostatic feature of the hatpase_c domain of vick were shown in figure 2c. the atp binding site consists of a relatively hydrophobic inner cavity and a larger hydrophilic outer cavity. both cavities are connected by a gorge-like channel, and are consisted of highly conserved residues which can bind and fix the substrate. the inner part lack of polar amino acid residues can accommodate the adenosine, while the outer one rich in charged residues can bind the triphosphate. discovery of potential inhibitors of the s. pneumoniae vick hk by virtual screening the target site for high throughput virtual screening was the atp-binding pocket of the vick hatpase_c model of s. pneumoniae, which consisted of residues within a radius of Ǻ around the atp site. in the primary screening, the database specs containing about molecules was searched for potential binders using the program dock. subsequently, structures ranked in the first were re-scored by using the autodock program. as a result, about molecules were filtered out by these highly selective methods. finally, we manually selected molecules according to their molecular diversity, shape complementarities, and the potential to form hydrogen bonds and hydrophobic interactions in the binding pocket of the vick hatpase_c domain. inhibition of the vick' protein atpase activity in vitro in order to confirm the interaction of the potential vick inhibitors with their putative target protein, we expressed and purified his-tagged vick' protein by using the pet28a plasmid in bl as shown in figure 3a. the kinase activity of vick' protein was measured by quantifying the amount atp remained in solution after the enzymatic reaction. these results indicated that the purified vick' protein possessed the atpase activity, which can hydrolyze atp in vitro. using the purified active vick', we obtained compounds from the candidate inhibitors which could decrease the atpase activity of vick' protein by more than 50%, indicating these compounds may also be potential vick inhibitors in s. pneumoniae. antimicrobial activities of potential vick' inhibitor and cytotoxicity of the antimicrobial compounds in vitro we investigated the bactericidal activity of these compounds against s. pneumoniae using a standard minimal bactericidal concentration assay. six compounds, each inhibiting the vick' activity by more than 50%, could obviously inhibit the growth of s. pneumoniae, with mic values below μm. moreover, their mic values were positively correlated with the corresponding ic values, which indicates that the bactericidal effects of these chemicals were realized by disrupting the vick/r tcs system in s. pneumoniae. chemical structures of these compounds are shown in figure which belong to three different classes of chemicals: one imidazole analogue, four furan derivatives and one derivative of thiophene. a 3-- 5-diphenyl tetrazolium bromide assay was carried out on vero cell line to determine the cc values of these compounds. as shown in table the cc values of all these six compounds were larger than μm and than their respective mic values, indicating low cytotoxicity effects on vero cell. collectively, these compounds inhibited bacterial growth with low toxic effects. time- and concentration-dependent growth curve while several compounds identified in our study could be used as excellent drug leads in vitro, the best and most valuable ways would be in vivo validation. the following results of the time- and concentration-dependent effects of the lead inhibitors on the growth of s. pneumoniae further illustrated their antibacterial characteristics, and would be an important guide for in vivo administration. as shown in figure the similar curves of compounds and indicated that these compounds have significant activity against s. pneumoniae at concentration of about μm, and this activity could last at least hours. the most efficient inhibitor identified was compound which had bactericidal effect against s. pneumoniae even at concentration of as low as μm. however, even at concentration of μm, compound was not likely to have bactericidal effect, but it seemed to have delayed the multiplication of s. pneumoniae. therapeutic effects of the lead compounds in mouse s. pneumoniae infections mouse sepsis models by s. pneumoniae were successfully established by intraperitoneal injection of μl s. pneumoniae. generally, these mice began to die within hours and couldn't survive more than hours unless they got appropriate therapeutic treatments. for facilitation of comparisons between the effects of these compounds and positive control, the concentration of penicillin used in this study almost equaled to that of the lead compounds. to rule out the direct antibacterial effects that may compromise with the efficiency of this model, the lead compounds and penicillin were administrated through caudal vein. as shown in figure these compounds were able to decrease, though slightly, the mortality of the infected mice in the first hours as compared to negative control. significant treatment effects were found among the groups by an overall comparison. pairwise comparisons revealed that compounds 1– prolonged survival time in mouse sepsis models as compared to negative control. however, compound and were less effective than positive control pnc. although these compounds could not reverse the fatal pneumococcal infection with concentration used in this study, in vivo antibacterial activity of these six compounds suggested that it would be promising to develop lead-compound-based drugs against pneumococcal infection. molecular modeling of vick' protein and its potential inhibitors in order to get insight into the mechanism of inhibition, further studies were carried out to verify the interaction modes between six compounds and the modeled structure of vick' protein. autodock software was used for the docking simulation. the binding conformations of these inhibitors in the atp-binding pocket of the vick hatpase_c domain were shown in figure although these structures are diverse, the binding models of six potential inhibitors are similar, especially in the inner part of the conserved domain. the surface of the binding pocket is divided into two parts, one is hydrophobic inner part composed of residues ile ile leu ile phe and the other is the outer hydrophilic part consisted of residues asn lys tyr arg arg all six compounds bind in the pocket with rigid aromatic ring parts inserting into the inner part. in the large and flexible outer part, these compounds adopt different interactions. all of them have hydrogen bond acceptors in the binding outer part. they could form hydrogen networks with the polar residues to stabilize the substrate interactions. their binding models resemble natural substrate atp much. discussion in bacteria, hks have fundamental roles in tcs signal transduction pathways. thus they are major targets for antibacterial drug development. high structural and sequence homology of this kinase gene family makes the hks ideal targets for homology modeling and structure based virtual screening. sbvs is an approach based on the three-dimensional structures of macromolecular to identify chemical entities binding to the targets and to elicit potential biological mechanisms with the advantages of speed, efficiency and high-throughput. the availability of the small molecular lead-compound library and the modeled 3d target structure makes it possible to use sbvs to screen out a limited number of promising candidates that can interrupt the tcs signal transduction by interacting with the hks substrate of s. pneumoniae. hks, as novel antibacterial targets, have attracted many attentions due to their essentiality in the viability of microbes and their deficiency in animals. hks are involved in the regulation of bacterial growth and virulence in many bacterial species. previously, a hk named vick has been used to screen lead compound inhibitors in b. subtilis and s. epidermidis. we here for the first time obtained candidate chemical compounds directly aiming at s. pneumoniae vick by screening possible compounds in silico. compounds that can bind to the purified target protein vick' and compete with its substrate atp were further verified by in vitro and in vivo antibacterial assays. eventually, we obtained compounds with antibacterial activity that may be used as novel drug leads. commonly, the response regulator yycf and the histidine kinase yycg are the only essential tcs for viability in b. subtilis and s. aureus. in s. pneumoniae, the vicr/k tcs regulates the expression of several critical genes, such as those encoding surface proteins and virulence factors. however, only the response regulator vicr was found to be essential. the signal transduction of vick was possibly bypassed by other tcs hks. vick has conserved atp-dependent hatpase_c domains accounting for autophosphorylation. even non-cognate hks from other bacteria can phosphorylate the purified vicr from s. pneumoniae. in a previous study, the mic values of the lead compounds screened out by sbvs targeting the yycg of s. epidermidis were almost equal to the corresponding ic values, with a correlation coefficient of, which suggested that inhibition of 50% the yycg protein activity would interfere with the growth of s. epidermidis. if this case is true in s. pneumoniae, the result that the mic values of the lead-compounds were far less than the corresponding ic values may be explained as bypass effects of these compounds on other hks. in a word, these lead compounds are most likely having a "cross-inhibition" on other hks in s. pneumoniae, which can enhance their antibacterial effects, although they were not verified in this study. although the vick protein in s. pneumoniae can be homologous to yycg in other gram-positive strains, such as s. epidermidis, enterococcus faecalis and s. aureus, different strains generally have different characteristics of the hatpase_c domain structure of hks. these characteristics will determine the binding specificity of the lead compounds screened out by sbvs. moreover, a different template for homologous modeling and different parameters for sbvs were used, which can guarantee the specificity of the lead compounds binding to the vick' discovered. what's more, compounds can inhibit the purified vick' protein activity by more than 50%, of which displayed different degrees of antibacterial effects in vitro and in vivo. regretfully, the in vivo activities of these compounds were not quite consistent with their corresponding in vitro activity, and some compounds displayed obvious cytotoxicity, which would challenge our future investigation. moreover, it seems to be a paradox that compound have less bactericidal effects in the time- and concentration-dependent antibacterial assays, but demonstrated significant therapeutic effects in mice infected by s. pneumoniae. however, due to the vick' is not essential in s. pneumoniae, this chemical may have a possibility to interrupt the invasion and virulence rather than cause numerous death of the bacterium, which decreases the selection pressure and contributes to the maintenance of species diversity, thus reduces the emergence of drug-resistant strains. anyway, the subtle mechanisms need our future work.  to summarize, we have successfully found out several promising lead compounds for further drug development in this study, which also can be used as inhibitors to explore the mechanism of autophosphorylation by vick as well as other hks. important work in future would be validation of their antibacterial effects in different strains and structural modification for more effective derivatives with less in vivo toxicity, and investigation into whether they can bind to other atp-dependent kinase is also necessary. 
to our knowledge, these compounds are the first reported inhibitors of hk with antibacterial activity in vitro and in vivo, and are novel lead structures for developing new drugs to combat pneumococcal infection. using a structure-based virtual screening method, compounds were computationally identified as potential inhibitors of the histidine kinase vick protein from the compound library specs. two-component systems are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. six of them were then validated in vitro to be active in inhibiting the growth of s. pneumoniae without obvious cytotoxicity to vero cell. in mouse sepsis models, these compounds are still able to decrease the mortality of the mice infected by s. pneumoniae and one compound even has significant therapeutic effect. due to the widespread abusage of antibiotics, antibiotic-resistance in streptococcus pneumoniae has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. among the pairs of tcss proteins presenting in s. pneumoniae, vicr/k is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against s. pneumoniae infection.
 due to the widespread abusage of antibiotics, antibiotic-resistance in streptococcus pneumoniae has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. two-component systems are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. among the pairs of tcss proteins presenting in s. pneumoniae, vicr/k is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against s. pneumoniae infection.  using a structure-based virtual screening method, compounds were computationally identified as potential inhibitors of the histidine kinase vick protein from the compound library specs. six of them were then validated in vitro to be active in inhibiting the growth of s. pneumoniae without obvious cytotoxicity to vero cell. in mouse sepsis models, these compounds are still able to decrease the mortality of the mice infected by s. pneumoniae and one compound even has significant therapeutic effect.  to our knowledge, these compounds are the first reported inhibitors of hk with antibacterial activity in vitro and in vivo, and are novel lead structures for developing new drugs to combat pneumococcal infection. 