Patent Application: US-52646105-A

Abstract:
a method for identifying antibacterial agents comprises depleting bacteria of a strain comprising a luxab construct of ca 2 + , incubating the ca 2 + depleted bacteria with an agent the antibacterial effect of which shall be determined , recording the light emitted by the bacteria upon addition of an aldehyde , the incubation being carried out at a temperature which is at least 10 ° c . higher than the temperature at which the light is emitted by the bacteria . also disclosed are corresponding probes and antibacterial agents identified by the method .

Description:
general . a compound collection consisting of 9 , 400 unique substances ( chembridge , diverset f ) was screened for inhibition of the luxab reporter signal using the non - virulent strain pib29el . the compounds were screened in duplicate at a final concentration of 20 μg / ml . assay interfering compounds , e . g . potential luciferase inhibitors , were identified by addition of hits from the primary screen to assay wells 10 - 20 min prior to addition of n - decanal . compounds showing activity in one of the duplicate wells were re - screened to confirm activity or the lack thereof . the complete screening campaign gave about 60 compounds that caused at least 40 % inhibition ( duplicate average ) of the luciferase light signal . for most of these compounds a dose / response relationship was established , and inhibition of bacterial growth was examined . a number of the compounds were found to have an ic 50 for inhibition of the luciferase light signal of 50 μm or less . compounds 3 - 5 are representative examples of compounds that show a clear selectivity for inhibition of the luciferase light signal over growth inhibition indicating that these substances possibly act on targets directly or indirectly regulating expression of the yops , but are not required for growth ( fig4 a - c ). several of the compounds from the primary screening belong to a class of acylated hydrazones of salicylic aldehydes of which compound 3 is the most potent . the bacterial targets for compounds 3 - 5 are unknown but compounds closely related to 4 have been found to inhibit bacterial two - component systems 29 . detailed investigations were also carried out for the majority of the active compounds although most emphasis was put on compounds 3 - 5 . all experiments based on luciferase - based readout were carried out in triplicate or quadruplicate in the same manner as in primary screening . as a first step the compounds were assayed with the wild - type control strain , pib102al , with luxab downstream of the promoter for yera , the chaperone for yope . as seen in fig5 a and 5b compounds 3 and 5 show a stronger inhibitory effect on the wild - type strain pib102el with luxab under control of the yope promoter than on the control strain . this selectivity suggests that compounds 3 and 5 target secretion or the ca 2 + dependent regulation involving lcrq rather than the temperature dependent lcrf cascade ( cf . fig1 a ). compound 4 on the other hand show no selectivity for any of these two strains as illustrated in fig5 c . in order to zoom in on potential targets and to further support a selective mode of action for compounds 3 - 5 two additional reporter gene strains were employed . based on the regulatory model in fig1 a compounds that inhibit the signal from the yera reporter gene should also inhibit a reporter gene signal under control of the lcrf promoter . the yope and yera promoters are both positively regulated by lcrf but the yera promoter is not regulated by the negative ca 2 + control loop . in pib102fδhlhl the protein lcrf lacks a negative self - regulating helix - loop - helix sequence thus resulting in enhanced levels of lcrf . consistent with the regulatory model in fig1 a compound 4 inhibits the reporter gene signal all four strains pib102el , pib102al , pib102fl , and pib102fδhlhl strains to more or less the same extent ( fig5 c ). these results suggest that compound 4 targets lcrf directly or act on regulatory elements upstream lcrf . compound 3 on the other hand was found to enhance the reporter gene signal from both pib102fl , and pib1102fδhlhl . the effect was most dramatic for pib102fδhlhl for which the signal was more than doubled at concentrations that almost completely inhibit the signal from pib102el ( fig5 a ). compound 5 displays yet a different pattern showing minor inhibition of the signal from pib102fl and modest enhancement of the signal for pib1102fδhlhl ( fig5 b ). the investigation was continued by western analysis of inhibition of actual yop secretion in presence of compounds 3 - 5 . the wild - type strain pib102el was grown at ambient temperature ( ca . 21 ° c .) in presence or absence of different compound concentrations for one hour and then the temperature was raised to 37 ° c . for induction and secretion of the yops . after three hours at 37 ° c . the bacteria were removed by centrifugation , and the protein content in the supernatant , i . e . the surrounding media , was examined by western analysis using a polyclonal serum active against all secreted proteins . as seen in fig6 a compound 3 efficiently inhibits yop secretion in a dose dependent manner similar to the light inhibition data obtained using the strain with luxab under the yope promoter . this dose dependent inhibition of secretion was also observed for compound 4 and 5 , i . e . ic 50 for inhibition of the luciferase readout is similar to the ic 50 for inhibition secretion ( data not shown ). for compound 3 the total yop content , i . e . yops present in both bacteria and the surrounding media , was examined at various compound concentrations . as seen in fig6 b yops can be observed at concentrations that completely inhibit secretion suggesting that compound 3 targets events in the actual secretion rather than yop transcription or translation , i . e . the bands in the 50 and 100 μm lanes originate from intracellular proteins . bacterial strains and growth condition . all strains used are y . psedotuberculosis serotype iii ( ypiii ) and in the following text strains are only labelled with the name of the virulence plasmid . strains with the luxab contruct were prepared from either the non - virulent yoph and yada mutant y . pseudotuberculosis pib29 28 or the wild type yada mutant pib102 27 by constructing yope - luxab , yer - luxab , lcrf - luxab and lcrfδhlh - luxab operon fusions essentially as described previous 26 . the resulting strains pib29el ( yope - luxab ), pib29al ( yera - luxab ), pib102el ( yope - luxab , wild - type ), pib102al ( yer - luxab , wild - type ), pib102fl ( lcrf - luxab , wild - type ), and pib1102fδhlhl ( lcrfδhlh - luxab , wild - type ) strains were struck and grown at room temperature on lb - plates containing chloramphenicol ( sigma ) at a final concentration of 50 μg / ml . from plates not older than one week , bacteria for experiments were grown in liquid brain / heart infusion ( bhi ) broth ( oxoid ; unipath ltd ., basington , uk ) containing 2 . 5 mm cacl 2 or 20 mm mgcl 2 and 5 mm egta for ca 2 + depletion . compounds , antibiotics and antibodies . the chemical library that consists of 9 , 400 unique compounds in 96 - well plate format was purchased from chembridge ( diverset f ). the compounds were dissolved in dmso to give a stock solution of 2 mg / ml . for compounds further characterized in the described experiments additional 5 or 10 mg samples were purchased from chembridge . streptomycin ( sigma ), carbenicillin ( sigma ), polymyxin b ( mixture of polymyxin b1 and b2 , fluka ), and nalidixic acid ( sigma ) were dissolved in water . antibodies against the different yersinia proteins were available from other projects at the department of molecular biology , umeå university . the synthesis of compound 5 has been reported previously ( 2 -[ benzo - 2 , 1 , 3 - thiadiazole - 4 - sulfonyl ) amino ] benzoic acids as synthetic intermediates for antihelmintics . mikhailitsyn f . s ., drusvyatskaya s . k ., uvarova , n . a , patent no . su1685935 ; search for new antiparasitic agents 3 . synthesis of haloid - containing sulfamidobenzamides with benzo - 2 , 1 , 3 ,- dithiazole residue in sulfonamide group and study of their acute toxicity . mikhailitsyn f . s ., lebedeva m . n ., kozyreva n . p ., lychko n . d . drusvyatskaya s . k ., uvarova , med . parazitol . parazit . bolezni . 1991 ( 2 ) 36 - 38 ). acylated hydrazones of salicylaldehyde e . g . compound 3 and analogs are prepared from the corresponding acylhydrazides and aldehydes as described previously ( cytotoxicity of salicylaldehyde benzoylhydrazone analogs and their transition metal complexes : quantitative structure - activity relationships . ainscough e . w , brodie a . m ., denny w . a ., finlay g . j ., gothe s . a ., ranford j . d ., j . inorg . biochem . 1999 ( 77 ) 125 - 133 ). compound 4 and analogs are prepared according to a known method ( method of producing 2 - acetoxy - 4 ′- chloro - 3 , 5 - diiodobenzanilide . mikhailitsyn f . s ., petyrov y . f ., sapozhnikova l . a ., ussr patent no . su 1226807 ). general screening and assay conditions . an overnight culture grown at room temperature in bhi medium containing 20 mm mgcl 2 and 5 mm egta for ca 2 + depletion was diluted to od 600 = 0 . 15 - 0 . 25 . in parallel the compounds to be tested were dispensed into the wells of a 96 - well plate ( polysorp fluoronunc ™ modules , nunc ) containing 50 μl of medium per well . to each well 50 μl of the bacterial solution was added . for compounds dissolved in dmso the final dmso concentration was kept below 2 %. the plate was incubated on an orbital shaker at room temperature ( ca . 21 ° c .) for 1 h . the temperature was then shifted to 37 ° c . and incubation with orbital shaking was continued for 2 h . subsequently the temperature was shifted back to room temperature and the plate was incubated for 2 h without shaking . finally 100 μl freshly made n - decanal emulsion ( sigma , 10 μl / 100 ml water , emulsified by vigorous shaking ) was added to each well and the light emission was measured within 1 - 3 minutes after addition . light emission was recorded with a light - sensitive charge - coupled device ( ccd ) camera , diana chemoluminescense detection module ( raytest , isotopenmessgeräte , gmbh ). the intensity of the light signals was quantified using the computer program tina ( version 2 . 0 ). primary screening of the compound library was carried out in duplicate with a final concentration of 20 μg / ml . other experiments were carried out in triplicate and quadruplicate with modifications as indicated in the text and figure legends . for antibody experiments 5 μl monoclonal mouse ascites solution was used . results were typically reproduced in at least three independent experiments . growth inhibition experiments . growth inhibition by compounds identified in the screening process was measured by growing bacteria at 37 ° c . in the presence of different compound concentrations in 96 - well plates containing 100 μl bacterial culture in bhi medium with 2 . 5 mm cacl 2 per well . the experiments were carried out in a molecular device spectramax 340 plate reader with continuous shaking at 37 ° c . and periodical determination of absorption at 600 nm . experiments were carried out in triplicate or quadruplicate , and the results were typically reproduced in at least three independent experiments . values for percent growth inhibition were calculated from differences in growth rate in presence or absence of compound over a time period with approximately linear growth . western analysis of protein secretion . an overnight culture grown at room temperature in bhi medium containing 20 mm mgcl 2 and 5 mm egta for ca 2 + depletion , was diluted to od 600 = 0 . 15 - 0 . 25 . in parallel the compounds to be tested were dispensed into the wells of a 96 - well plate containing 50 μl of medium per well . to each well 50 μl of the bacterial solution was added . the plate was incubated on an orbital shaker at room temperature for 1 h . the temperature was then shifted to 37 ° c . and incubation with orbital shaking was continued for 3 h . subsequently the cultures were transferred to micro - centrifuge tubes . after brief centrifugation the supernatants were investigated by standard western analysis ( 12 % sds - page ) using a rabbit polyclonal total ant - yop serum . motility was measured as the movement of bacterial cells through semisolid motility agar ( luria broth containing 0 . 25 % bactoagar ). around 10 7 y . pseudotuberculosis ypiii pib102el bacteria in a 2 . 5 μl drop were placed at the centre of each well in a 6 - well plate ( falcon multiwell ) containing 5 ml motility agar supplemented with 5 mm egta , 20 mm mgcl 2 and different concentrations of test substance in 0 . 1 ml dmso per well . the plates were incubated at 22 ° c . for 18 h to allow for the bacteria to grow and to move out from the centre of the plate . to induce yop and luxab expression , plates were shifted to 37 ° c . 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