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BioAssay: AID 624061

K5 Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis

Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. more ..
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 624061
Data Source: Southern Research Specialized Biocontainment Screening Center (ECPhage_K5_01)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-04-11
Hold-until Date: 2013-04-10
Modify Date: 2013-04-10

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
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AIDNameTypeComment
488966Primary and Confirmatory Screening for Inhibitors of Bacterial Capsule Biogenesisconfirmatoryprimary and confirmatory screen
602408Confirmatory Screen for Inhibitors of Bacterial Capsule Biogenesis (2)confirmatoryConfirmatory screen
493020Cytotoxicity screening for potential Inhibitors of Bacterial Capsule BiogenesisconfirmatoryCytotoxicity counter screen
504769Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (2)confirmatoryCytotoxicity counter screen
504831Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (3)confirmatoryCytotoxicity counter screen
588399Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)confirmatoryCytotoxicity counter screen
602414Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (5)confirmatoryCytotoxicity counter screen
504358Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 controlconfirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
504543Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (2)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
504675Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (3)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
504768Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (4)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
588321Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (5).confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189
588386Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (6)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189
588395Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (7)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189
504349Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis InhibitionconfirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504538Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (2)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504676Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (3)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504767Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (4)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
588322Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (5)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504733Orcinol Secondary Screening for Inhibitors of Bacterial Capsule BiogenesisotherOrcinol Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis
488970Screening for Inhibitors of Bacterial Caspule Biogenesis - Summarysummary
652021Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)confirmatory
Description:
Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise, driving efforts to elucidate vulnerable targets in the molecular pathogenesis of infection. New insights into the roles of K capsules in UPEC virulence during UTI make capsules an attractive target.

During UTI, UPEC lives in intracellular and extracellular locales. UPEC adheres to the apical bladder epithelium and then invades this layer of cells. Within the bladder epithelium, UPEC typically replicates in a biofilm-like state called intracellular bacterial communities (IBC). After maturation of IBCs, UPEC disperses away from the IBC and exits the infected cells. Extracellular UPEC must then re-adhere, initiating the invasion and intracellular replication phases again. Past studies have revealed bacteria encased in the IBC within a complex matrix of fibrous protein assemblies and polysaccharides. Prior studies have also shown that disruption of the IBC pathway aborts experimental UTI, highlighting the importance of this intracellular lifecycle. A detailed study of urine samples from women with acute UTI demonstrated IBC in shed bladder epithelial cell, showing that the pathway is conserved in humans. Investigations have shown that K capsule contributes to multiple aspects of pathogenesis, including IBC formation.

Of the different K types, the Group 2 and Group 3 capsules are most prevalent among UPEC isolates, with K1 and K5 being leading types. Although the capsules have different compositions, they are synthesized, assembled, and exported by functionally homologous factors, leading us to hypothesize that we can develop small molecular inhibitors of K-type encapsulation that target the most medically important K types. This secondary assay will identify the phenotypic specificity of hits from which we may determine the optimal targets for capsule biogenesis inhibition and develop analogues with pharmacologically optimized properties.
Protocol
Secondary Assay Protocol: This assay was performed only on the probe candidate, and determined if compounds considered active in the T7 and orcinol secondary assays were able to also inhibit K5 capsule biogenesis. This assay was performed in a method identical to the T7 assay test, but a different bacterial test strain and bacteriophage are used. In this validation test, we used E. coli strain DS17, a pyelonephritis clinical isolate expressing a K5 capsule. DS17 is highly susceptible to K5 phage-mediated lysis. Thus, compounds that were active in the K1F phage assay but did not inhibit phage in the T7 phage assay were analyzed using this assay. This secondary assay was conducted in the 96 well plate format. Bacterial cultures of E. coli strain DS17 were grown and prepared immediately prior to use. Overnight starting cultures of DS17 were grown at 37 degrees C and diluted 1:100 in LB. Compounds were added to plates in quadruplicate at 50 and 100 uM, followed by addition of 100 microliters of bacterial culture. 1% DMSO (final well concentration) was included. The plates were tape sealed and shaken vigorously for 1.5 hr. An initial OD600 reading was measured to identify compounds that cause growth retardation or bacterial killing in the absence of phage. Next, K5 bacteriophage (5 microliters) was added to all of the test wells. The plates were resealed and shaken vigorously at 37 degrees C, and measurements of OD600 for phage-mediated lysis were taken after 3 hr. True inhibitors of capsule yielded bacteria that were not susceptible to K5 bacteriophage and did not show lysis within 2 hr of the addition of phage. The positive control drug C7 (100 micromolar final well concentration) was used in this screen.

Endpoint Read: The plates were read at ambient temperature from the bottom for absorbance at A600 in a BioTek Quantplate reader and the degree of inhibition of phage-mediated lysis was determined based on the absorbance.
Comment
Outcome: Inhibition of capsule formation was calculated relative to the mean of the bacterial (no drug) control. Inhibition at 50 and 100 micromolar were tested. Compounds that showed <50% capsule formation were considered Active.

Score: In this secondary assau using purified compounds, active compounds were scored on a scale of 81-100 based on thier ability to inhibit capsule formation. Compounds that were not confirmed as active were given the score 0.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: selectivity

BAO: bioassay specification: assay biosafety level: BSL2

BAO: assay format: cell-based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: format detail: reagent: inducer: none

BAO: meta target: molecular target: protein target:enzyme

BAO: meta target: biological process target: biofilm formation

BAO: meta target detail: binding reporter specification: interaction: protein-drug

BAO: assay design: viability reporter: cell number

BAO: detection technology: spectrophotometry: absorbance

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Percent Bacterial Growth @ 100 uMFloat%
2Percent Bacterial Growth @ 50 uMFloat%
Additional Information
Grant Number: 1 R03 MH090791-01

Data Table (Concise)
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