the need for new antibiotics 
 has arisen due to the widespread resistance 
 to current drugs . despite this need , the 
 antibiotic pipeline in the past few decades has been relatively dry 
 in terms of new antibacterial classes when compared with progress 
 against other diseases . one strategy to 
 fight bacterial resistance is to inhibit enzymes that are not the 
 targets of current antibiotics but , instead , act in the same pathways 
 as existing drugs since this might enable the restoration of drug 
 sensitivity via combination therapy . the undecaprenyl diphosphate product ( upp ) 
 is essential for bacterial cell growth because of its role in the 
 formation of bacterial cell wall peptidoglycan , scheme 1 , and it is not produced by humans . smithkline beecham screened their compound collection 
 against upps 
 but reported no chemically tractable low micromolar hits . novartis pursued tetramic and tetronic acids 
 and dihydropyridin-2-ones , but noted issues associated with human 
 serum albumin binding and a lack of in vivo activity . previously , we reported several potent upps inhibitors together 
 with x - ray crystallographic ( or modeled ) binding modes for a variety 
 of chemical classes including lipophilic bisphosphonates , phthalic acids , diketo 
 acids , anthranilic acids , benzoic acids , aryl phosphonates , bis - amines , and bis - amidines . the most promising of these compounds , a bis - amidine , was 
 shown to have potent activity in biochemical assays , in cellular assays , 
 and in a murine model of mrsa infection . since upps must bind multiple substrates ( ipp , fpp , or more 
 elongated 
 prenyl - pp intermediates ) and many inhibitors are to some degree substrate 
 mimics , it is common to observe numerous inhibitors simultaneously 
 bound to upps , with up to 4 binding sites being occupied . however , it is unclear whether inhibitory activity 
 is due to binding to one specific site or to multiple sites . it has 
 been shown that some inhibitors occupy only site 4 , an allosteric 
 site distant from the catalytic center , while others bind to site 
 1 , the substrate binding site , complicating 
 docking studies and , regardless of the inhibitor - binding mode , the 
 flexibility of upps creates challenges for virtual screening . here , 
 to help reduce these problems we employed the 12 crystallographic 
 structures described in previous work to select 
 those that provided maximal enrichment in retrospective virtual screening studies . we then made prospective predictions using these structures , leading to novel upps inhibitors , 
 some with promising antibacterial activity . following 
 the methods described 
 in previous work , we docked 112 known 
 upps inhibitors having ic50 values < 100 m , together 
 with 1000 decoys from the schrdinger decoy collection ( having 
 an average molecular weight of 400 da ) , to escherichia coli upps ( hereafter , ecupps ) . docking was performed using the glide program , and compounds were ranked by their glide xp score . the 
 proteins were prepared by stripping water and ligand molecules , capping , 
 and neutralizing any unsolved loops , followed by preparation with 
 the schrdinger protein preparation wizard using standard parameters . after docking , compounds were ranked by their 
 docking score , and then area under the curve ( auc ) analyses were performed . retrospective enrichment was quite good for 2/12 structures ( pdb codes 2e98 and 4h3a ) , so we docked into 
 these structures for the prospective studies ( figure 1 ) . 2e98 is an ecupps x - ray structure containing four lipophilic bisphosphonates 
 ( bph-629 ; ic50 300 nm ) , which bind to sites 14 , 
 one inhibitor to each site.4h3a is an ecupps structure 
 containing a diketo acid inhibitor ( bph-1330 ) which has a 2 m 
 ic50 , and the inhibitor binds ( in the solid state ) only 
 to site 4 . these structures thus have significant 
 differences : only site 4 is occupied in 4h3a , while in 2e98 , all four sites are occupied and the 
 protein is in a  wide - open  conformation ( figure 2 ) . stereo presentation of 
 the x - ray structures chosen for further 
 virtual screening from docking and roc analysis . ( b ) 4h3a showing one inhibitor bound to site 4 . to find new inhibitors , we began with a library 
 of 450,000 
 commercially available compounds , the chembridge experimental library . the library was filtered to exclude compounds that had undesired , 
 toxic or reactive functional groups ; known promiscuous binders ; mw 
 > 460 da or mw < 250 da ; more than 4 chiral centers ; polar surface 
 area ( psa ) > 150  or psa < 50  ; number of rotatable bonds > 10 ; or clogp > 5 or clogp 2 . next , the selected compounds ( 100,000 ) 
 were loaded into schrdinger s virtual screening workflow , 
 where they were prepared with ligprep and then docked using the filtering 
 procedure for efficiency and only retaining the top 20% of compounds 
 in the two rapid , initial docking modules htvs ( top 20% retained ) 
 and sp ( top 20% retained ) . finally , glide xp was used to assign a 
 final docking score to each molecule . auc analyses on active and decoy 
 data sets were previously performed using the glide xp module , however , 
 this was impractical for the large filtered chembridge library . therefore , 
 we relied on htvs and sp modules to provide early filtering before 
 employing the more time intensive xp protocol . we then extracted 
 the 400 top scoring compounds ( docking 
 score less than 7 kcal / mol ) . binary molprint2d fingerprints 
 were generated using canvas , and 40 clusters were generated using k - means clustering . of these 40 clusters , the top 
 scoring compounds from each cluster were visually inspected and a 
 representative was chosen from each cluster , resulting in a final 
 list of 100 compounds . these were purchased from chembridge ( chembridge 
 corporation , san diego , ca ) and then assayed for upps inhibition activity . similarity searches 
 based on these active compounds were then performed using pubchem 
 and scifinder , and additional compounds were obtained and tested . molecular weights and purities were verified by mass spectrometry 
 and sds  page , respectively . briefly , the condensation of fpp with ipp catalyzed by upps was 
 monitored by using a continuous spectrophotometric assay in 96-well plates with 200 l reaction 
 mixtures containing 400 m 2-amino-6-mercapto-7-methylpurine 
 ribonucleoside ( mesg ) , 350 m ipp , 35 m fpp , 20 mm trishcl 
 buffer ( ph 7.5 ) , 0.01% v / v the ic50 values were obtained by fitting the inhibition 
 data to a rectangular hyperbolic dose  response function using 
 graphpad prism 4.0 software ( graphpad software , san diego , ca ) . the 
 ic50 values for the most active hits were verified using 
 a radiometric assay with 2.5 m 
 fpp , 25 m [ h]ipp , and 0.01% v / v triton x-100 . subtilis ( atcc 6051 ) , escherichia coli ( atcc 
 29425 ) , and saccharomyces cerevisiae ( atcc 208352 ) 
 were purchased from the american type culture collection . bacillus subtilis strain 168 , bacillus anthracis strain sterne , listeria monocytogenes strain 4b 
 f2365 , staphylococcus aureus usa300 ( methicillin - resistant ) , e. coli mc400 , pseudomonas putida , and enterococcus faecalis u503 ( vancomycin - resistant ) were from 
 our laboratory strain collection . ic50 values for e. coli growth inhibition 
 were determined by using a microbroth dilution method . the culture 
 was then diluted 500-fold into fresh lb medium , and 100 l was 
 inoculated into a 96-well flat bottom culture plate ( corning inc . , the starting concentration of each compound was 0.3 
 mm , and this was 2-fold serially diluted . plates were incubated for 
 3 h at 37 c to midexponential phase . a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ( mtt ) cell proliferation assay ( atcc ) ten microliters 
 of mtt reagent was added into each well , followed by incubation for 
 24 h until a purple precipitate was visible . then , 100 l 
 of detergent reagent was added and plates were further incubated in 
 the dark at 23 c for 2 h. the absorbance was recorded at 570 
 nm . for each inhibitor , two independent experiments were performed 
 and the ic50 values found were averaged . a 16 
 h culture of b. subtilis was diluted 50-fold 
 into fresh luria the culture was then diluted 500-fold into 
 fresh lb medium , and 100 l was inoculated into a 96-well flat 
 bottom culture plate ( corning inc . , the starting concentration 
 of each compound was 0.5 mm and was then serially diluted . a nonlinear regression analysis was carried out 
 on the data obtained using origin 6.1 . for each inhibitor , two independent 
 experiments were performed and the ic50 values found were 
 averaged . the protocol was the same as with the b. subtilis assay protocol except that ypd medium was used and the 96-well plate 
 was incubated for 36 h instead of 1216 h. b. subtilis strain 168 , b. anthracis strain sterne , e. coli mc4100 , 
 and p. putida were grown to stationary phase in 10 
 ml of lb broth at 37 c . s. aureus usa300 ( methicillin - resistant ) , e. faecalis u503 ( vancomycin - resistant ) , and l. 
 monocytogenes strain 4b f2365 were grown to stationary phase 
 in 10 ml of brain  heart infusion ( bhi ) medium at 37 c . the cultures were adjusted to an od600 of 0.016 in the 
 designated medium before being added to 96-well microplates . successive 
 2-fold dilutions of compounds 1 and 4 were 
 added to the cultures ( 0.2564 g ml ) . as a control , kanamycin ( 132 g ml ) was added to samples of e. coli , b. subtilis , b. anthracis , p. putida , and l. monocytogenes . gentamycin was used as a control for s. aureus and e. faecalis with dilutions 
 from 1 to 64 g ml . as a negative control , 
 an equal volume of dmso the minimum inhibitory 
 concentration ( mic ) that suppressed at least 99% of bacterial growth 
 was established based on culture turbidity in the microbroth dilution 
 assay . the assay was repeated in three replicates , and values were 
 averaged , or a range was reported . the purities of the key compounds investigated , obtained from chembridge 
 ( 1 and 4 ) , were determined by high - performance 
 liquid chromatography and structures verified by nmr spectroscopy 
 and high resolution mass spectrometry ( supporting 
 information figures s2s9 ) and were consistent with 
 the structures provided by the vendor . in previous work , we obtained moderate correlations 
 between enzyme 
 inhibition activity and docking scores within a congeneric series 
 of upps inhibitors ( lipophilic bisphosphonates ) using 
 docking methods , so we first examined whether we could obtain similar 
 correlations between docking scores and experimentally determined 
 ic50 values for the 112 known actives . there was no significant 
 correlation between docking scores and pic50 values ( pic50 = logic50 ) , figure s1 in the supporting information . the wide variety of potential 
 binding modes ( sites 1 , 2 , 3 , and 4 ) and protein 
 conformations would be expected to make it difficult to achieve a 
 good correlation between a scoring function and the experimentally 
 determined pic50 values , in addition to the assumptions 
 made in scoring functions that cause inaccuracy when compared to experimental 
 affinities . nevertheless , docking studies can provide enrichment of 
 active compounds from large libraries , even though docking scores 
 rarely correlate well with activity when structurally diverse compounds 
 are involved . we thus next employed an area - under - the - curve ( auc ) 
 analysis , also known as the receiver - operating - characteristic ( roc ) , 
 a method that has been shown to be useful in validating structure - based 
 virtual screening protocols and is a 
 standard method for evaluating such protocols . we therefore tested 12 ecupps x - ray structures for 
 their ability 
 to separate actives ( ic50 < 100 m ) from decoys 
 ( presumed inactive compounds in the decoy library ) . several ecupps 
 x - ray structures showed a good separation of active from decoy compounds , 
 with auc values of 0.8 . these structures also demonstrated 
 early enrichment , as evidenced by the steep initial slope of the curve . this means that the best scores were given primarily to active compounds 
 and suggests that , in screening a large compound library , the best 
 scoring compounds would be enriched in upps inhibitors . we thus picked 
 the two x - ray structures ( pdb codes 2e98 and 4h3a ) that provided significant early enrichment 
 and a high auc in the validation studies , for predictive studies . 
 using these two x - ray structures , we screened the chembridge express - pick 
 compound library ( after filtering ) and determined 400 hits 
 with glidexp scores less than 7 kcal / mol ( lower energy is 
 better ) . since many highly ranked compounds were chemically very similar , 
 we clustered the top scoring compounds and selected representatives 
 from each cluster to ensure chemical diversity among the compounds 
 to be tested . the screening 
 of the chembridge express - pick compound library using the validated 
 docking protocol resulted in the discovery of three new upps inhibitor 
 classes : the 4-oxo-2-thioxo-1,3-thiazolidines , also known as rhodanines 
 ( e.g. , compound 1 ) , dihydroxyphenyls ( the resorcinol , 
 compound 2 ) , and pyrimidinetrione ( a barbiturate analogue , 
 compound 3 ) . all three compounds are predicted to bind in 
 either site 1 or 3 of the 2e98 crystal structure ( figure 3 ) , although x - ray crystallographic studies will be required to confirm 
 this binding mode ( and our attempts to obtain crystal structures of 
 these systems have not been successful ) . in any case , the three new 
 inhibitors discovered represent upps inhibitors with  drug - like physicochemical properties , passing the common drug - like filters as 
 described in the methods and materials section . the most potent of the 3 compounds was the 4-oxo-2-thioxo-1,3-thiazolidine 1 ( ic50 2.6 m against s. 
 aureus upps ) , which in an initial screen for bioactivity 
 was also found to be active against b. subtilis , 
 mic ( minimal inhibitory concentration ) 3 g / ml ( table 1 ) . for this reason , we chose to next investigate 
 analogues based on the 4-oxo-2-thioxo-1,3-thiazolidine core . three new classes 
 of upps inhibitors discovered via virtual screening : 
 ( a ) chemical structure and computed docking mode of compound 1 , a rhodanine derivative ; ( b ) chemical structure and docking 
 mode of compound 2 , a resorcinol derivative ; ( c ) same 
 for compound 3 , a barbiturate . we next obtained 16 additional compounds 
 from chembridge , from sigma - aldrich , and from the drug synthesis and 
 chemistry branch , developmental therapeutics program , division of 
 cancer treatment and diagnosis , national cancer institute ( 419 , table 1 ) , containing 
 the 4-oxo-2-thioxo-1,3-thiazolidine core and tested them for activity 
 against saupps and ecupps , as well as a preliminary activity screen 
 against b. subtilis , e. coli , and s. cerevisiae ( the latter as a general cytotoxicity control , 
 since it lacks upps ) . the alkyl carboxylic acid containing compounds 
 with the 4-oxo-2-thioxo-1,3-thiazolidine core were active in assays 
 against b. subtilis , and the most potent compound 
 was 4 ( an analogue of 1 ) . 4 was roughly equipotent against saupps and ecupps with an ic50 of 2 m . additionally , 4 was 
 active against b. subtilis with an mic 0.43 
 g / ml and was very weakly active ( 200 g / ml ) against s. cerevisiae , indicating that 4 was not generally 
 cytotoxic . since 1 and 4 showed significant 
 activity in enzymatic and bacterial growth assays , we subsequently 
 tested them against several pathogens . both 1 and 4 gave mic values in the high ng / ml to low g / ml range 
 against b. anthracis sterne , mrsa , vre , and l. monocytogenes , table 2 . this promising 
 antibacterial activity suggested the potential utility of these upps 
 inhibitors in synergizing with other cell wall agents but where significant 
 resistance has emerged , such as with methicillin ( mrsa ) and vancomycin 
 ( vre ) . the compounds were tested against 
 a panel of both gram - positive ( top five ) and gram - negative ( lower 
 two ) bacteria . to investigate the possibility 
 of synergistic interactions with known cell wall biosynthesis inhibitors , 
 we determined the fractional inhibitory concentration index ( fici ) 
 values for three systems : mrsa , using 1 + methicillin ; 
 vre , using 1 + vancomycin ; and b. anthracis , using 1 + ampicillin . the fici is defined aswhere fic(a ) and fic(b ) 
 are the fractional 
 inhibitory concentrations of drugs a and b , mic(a ) and mic(b ) are 
 the mic values of drugs a and b acting alone , and mic(ab ) and mic(ba ) 
 are the mic values of the most effective combination of drug a or 
 b in the presence of drug b or a. using this method , fici values of 
 < 0.5 represent synergism , > 0.5 and < 1.0 represent additivity , 
 > 1 and < 2 represent an indifferent effect , and > 2 represents 
 drug 
 antagonism . , the fici for 1 + methicillin in mrsa is 0.11 , which indicates strong synergism 
 during late stage growth . however , with both vre ( 1 + 
 vancomycin ) and b. anthracis ( 1 + ampicillin ) 
 the fici values are in the 12 range , which indicates an indifferent 
 effect . in vitro synergy assays . isobolograms for growth inhibition of 
 vre , mrsa , and b. anthracis strain sterne . ( a ) 1 and vancomycin inhibition of e. faecalis u503 ( vancomycin - resistant , vre ) . fici = 1.78 . ( b ) 1 and methicillin inhibition of s. aureus ( usa300 ) what is particularly interesting about the most active species 
 investigated here ( 1 ) is that it has a structure that 
 is very similar to that found in the drug epalrestat , an aldolase reductase inhibitor that 
 is used to treat diabetic neuropathy , and is approved for clinical 
 use in japan , china , and india . this is encouraging because rhodanines 
 as a class are known to often have activity in widely different assays , 
 and indeed computer programs such as pains categorize , e.g. , 14 ( as well 
 as epalrestat ) as possible this can mean that the compounds cause false positives in assays , 
 or that they may be multitarget inhibitors . in some cases multitargeting may be undesirable ; however , in the context of anti - infective development , 
 multitargeting is expected to increase efficacy as well as decrease 
 the possibility of resistance development , both very desirable features . first , we carried out an in silico screen of 100 known upps 
 inhibitors and 1000 decoys using 12 reported upps x - ray structures . the two x - ray structures providing the best enrichment in an auc - roc 
 analysis were then used to screen a subset of 100,000 compounds 
 selected for drug - like activity from an initial chembridge library 
 of 450,000 compounds . we then tested the 100 in silico 
 hits in vitro against saupps and ecupps , leading to several m 
 upps inhibitors ( as deduced from both ppi release and radioactive 
 assays ) . the most potent lead was 1 , which is structurally 
 quite similar to epalrestat , in clinical use to treat diabetic neuropathy . 1 ( and its analogue 4 ) inhibited the growth of 
 gram positives ; they did not inhibit the growth of gram negatives 
 ( important with e. coli in the context of maintaining 
 commensal microflora ) , and they had no activity against s. 
 cerevisiae . activity against b. anthracis , a vancomycin - resistant enterococcus spp . , and listeria monocytogenes was good , in the 0.1254 g / ml 
 range , and there was very strong synergy ( fici = 0.11 ) with methicillin 
 and 1 in a mrsa strain of s. aureus , 
 suggesting that 1 could be a promising lead ( in combination 
 therapies ) for treating staph infections .
there 
 is a significant need for new antibiotics due to the rise 
 in drug resistance . 
 drugs such as methicillin and vancomycin target 
 bacterial cell wall biosynthesis , but methicillin - resistant staphylococcus aureus ( mrsa ) and vancomycin - resistant enterococci ( vre ) have now arisen and are of major concern . 
 inhibitors acting on new targets in cell wall biosynthesis are thus 
 of particular interest since they might also restore sensitivity to 
 existing drugs , and the cis - prenyl transferase undecaprenyl 
 diphosphate synthase ( upps ) , essential for lipid i , lipid ii , and 
 thus , peptidoglycan biosynthesis , is one such target . 
 we used 12 upps 
 crystal structures to validate virtual screening models and then assayed 
 100 virtual hits ( from 450,000 compounds ) against upps from s. aureus and escherichia coli . 
 the most 
 promising inhibitors ( ic50 2 m , ki 300 nm ) had activity against mrsa , listeria monocytogenes , bacillus anthracis , and a vancomycin - resistant enterococcus sp . with 
 mic or ic50 values in the 0.254 g / ml range . 
 moreover , one compound ( 1 ) , a rhodanine with close structural 
 similarity to the commercial diabetes drug epalrestat , exhibited good 
 activity as well as a fractional inhibitory concentration index ( fici ) 
 of 0.1 with methicillin against the community - acquired mrsa usa300 
 strain , indicating strong synergism .