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BioAssay: AID 504769

Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (2)

Cytoxicity Assay Rationale and Summary: 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 more ..
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 Tested Compounds
 Tested Compounds
All(9)
 
 
Active(9)
 
 
 Tested Substances
 Tested Substances
All(9)
 
 
Active(9)
 
 
 Related BioAssays
 Related BioAssays
AID: 504769
Data Source: Southern Research Specialized Biocontainment Screening Center (Phage_Tox2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-05-20
Hold-until Date: 2012-05-18
Modify Date: 2012-05-22

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 9
Related Experiments
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AIDNameTypeComment
488970Screening for Inhibitors of Bacterial Caspule Biogenesis - SummarySummarydepositor-specified cross reference
504831Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (3)Confirmatorydepositor-specified cross reference
588321Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (5).Confirmatorydepositor-specified cross reference
588322Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (5)Confirmatorydepositor-specified cross reference
588386Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (6)Confirmatorydepositor-specified cross reference
588395Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (7)Confirmatorydepositor-specified cross reference
588399Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)Confirmatorydepositor-specified cross reference
602408Confirmatory Screen for Inhibitors of Bacterial Capsule Biogenesis (2)Confirmatorydepositor-specified cross reference
602414Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (5)Confirmatorydepositor-specified cross reference
624055Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (6)Confirmatorydepositor-specified cross reference
624056Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (7)Confirmatorydepositor-specified cross reference
624060Orcinol Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis (2)Otherdepositor-specified cross reference
624061K5 Secondary Screening for Inhibitors of Bacterial Capsule BiogenesisOtherdepositor-specified cross reference
652019Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen) (6)Confirmatorydepositor-specified cross reference
652021Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)Confirmatorydepositor-specified cross reference
488966Primary and Confirmatory Screening for Inhibitors of Bacterial Capsule BiogenesisConfirmatorysame project related to Summary assay
493020Cytotoxicity screening for potential Inhibitors of Bacterial Capsule BiogenesisConfirmatorysame project related to Summary assay
504349Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis InhibitionConfirmatorysame project related to Summary assay
504358Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 controlConfirmatorysame project related to Summary assay
504538Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (2)Confirmatorysame project related to Summary assay
504543Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (2)Confirmatorysame project related to Summary assay
504675Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (3)Confirmatorysame project related to Summary assay
504676Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (3)Confirmatorysame project related to Summary assay
504733Orcinol Secondary Screening for Inhibitors of Bacterial Capsule BiogenesisOthersame project related to Summary assay
504767Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (4)Confirmatorysame project related to Summary assay
504768Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (4)Confirmatorysame project related to Summary assay
Description:
Cytoxicity Assay Rationale and Summary: 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. Uropathogenic Escherichia coli (UPEC) produces 80% of community-acquired urinary tract infections (UTI). UPEC is also a leading cause of nosocomial UTI, the most prevalent hospital acquired infection. Dissemination of UPEC from the lower urinary tract is associated with morbidity and mortality through infection of the kidneys, bloodstream, and central nervous system. In recent years, the treatment of outpatient and inpatient UTI has been severely compromised by the rising incidence of antibiotic-resistant UPEC.

Investigators have found that encapsulation is an important UPEC virulence factor. The K1 capsule type is closely associated with pathogenic isolates; not only is it the leading type in UTI, but it also accounts for much of the extra-urinary tract complications. Animals studies of E. coli K1 sepsis demonstrated that injection of K1 capsule degrading enzyme abrogates infection. However, the enzyme treatment is immunogenic; accordingly, chemical inhibition may prove to be a superior approach.

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. Furthermore, the medically important infectious agents Campylobacter jejuni, Hemophilus influenzae, Neisseria meningitides, and Salmonella typhimurium among others, use these homologues in the biogenesis of their capsules. By exploiting the properties of a K-type specific phage, we performed a small scale high-throughput screen of >2,100 molecules from the NCI that uncovered several promising inhibitors of K1 and K5 encapsulation. This assay will identify a larger number of inhibitors with different mechanisms of action from which we may determine the optimal targets for capsule biogenesis inhibition and develop analogues with pharmacologically optimized properties.

Chemical modulators of K1 encapsulation might represent a new avenue to combat the catastrophic effects K1 diseases. To this end, this team has successfully developed a 1536-well high-throughput primary screen suitable for the discovery of novel capsule biogenesis inhibitors. This 384-well format counter-screen for hit toxicity in Bladder 5637 cells, along with the secondary assays for target validation will allow a prioritization of "hits" from the primary screen.
Protocol
Bladder 5637 Cell Cytotoxicity Dose Response Screen. The dose response testing established the compound cytotoxicity and CC50 data necessary to determine the compounds advanced to secondary screens and target localization studies. Compounds were plated in 96-well microplates in duplicate. Bladder carcinoma 5637 cells were added to the compounds, and 72 hrs later cell viability was measured using CellTiter Glo. Hyamine was used as a positive cytotoxic control. Compound cytotoxicity and the 50% toxic concentration (CC50) was determined and compared to IC50 to calculate the therapeutic index.
Cell viability was calculated relative to the mean of the cell control. Viability at each of eight tested concentrations ranging from 300 uM to 2.3 uM. Compounds that showed cell viability < 80% were considered Active (or Toxic). CC50 values were calculated using XLFit.
Comment
Compounds that showed cell viability < 80% were considered Active (or Toxic).
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In confirmatory (dose response) screens active compounds are scored on a scale of 41-80 based on the CC50 result compounds that did not show activity were given the score 0. Active purified or synthesized compounds are scored on a scale of 81-100 based on the CC50 result with inactive compounds receiving the score of 0.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Type: Toxicity
Assay Cell Type: 5637
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50*Cytotoxic concentrationFloatμM
3% Cell Viability @ 300 uM (300μM**)Float%
4% Cell Viability @ 150 uM (150μM**)Float%
5% Cell Viability @ 75 uM (75μM**)Float%
6% Cell Viability @ 37.5 uM (37.5μM**)Float%
7% Cell Viability @ 18.75 uM (18.75μM**)Float%
8% Cell Viability @ 9.38 uM (9.38μM**)Float%
9% Cell Viability @ 4.69 uM (4.69μM**)Float%
10% Cell Viability @ 2.34 uM (2.34μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH090791-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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