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BioAssay: AID 1677

Broad Institute MLPCN Streptokinase Expression Inhibition Project

The goal of the project is to identify and develop novel antibiotic small molecules through inhibiting streptokinase (SK) expression in group A streptococcus (GAS). Based on the critical role of SK in GAS pathogenicity, we are attempting to identify chemical compounds that specifically reduce the expression of bacterial SK, without interfering with bacterial viability. Specifically, a probe is a more ..
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Probe(3)
 
 
Active(3)
 
 
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Probe(3)
 
 
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AID: 1677
Data Source: Broad Institute (2014)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-04-13
Modify Date: 2011-03-04

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: streptokinase A precursor [Streptococcus pyogenes M1 GAS]
Description ..   
Protein Family: Staphylokinase/Streptokinase family

Gene:SKA     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: Chemical Probe: 3    Active: 3
Related Experiments
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AIDNameTypeComment
1662MLPCN Streptokinase Expression InhibitionScreeningdepositor-specified cross reference: 2014-01 - MLPCN Streptokinase Expression Inhibition
1900Luminescence Microorganism-Based Dose Confirmation HTS to Identify Compounds Cytotoxic to SK(-)GAS Group A StreptococcusConfirmatorydepositor-specified cross reference: Secondary Screen of 3225 compounds
1902Luminescence Microorganism-Based Dose Confirmation HTS to Identify Inhibitors of Streptokinase Promotor ActivityConfirmatorydepositor-specified cross reference: Dose Retest of 3227 compounds
1914Absorbance Microorganism-Based Dose Response HTS to Identify Inhibitors of Streptokinase ExpressionConfirmatorydepositor-specified cross reference: Secondary Screen of 3264 compounds
1915Luminescence Microorganism-Based Dose Response HTS to Identify Compounds Cytotoxic to StreptococcusConfirmatorydepositor-specified cross reference: Secondary Screen of 3264 compounds
2137Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase ExpressionConfirmatorydepositor-specified cross reference: Secondary Screen of 63 compounds
2138Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to StreptococcusConfirmatorydepositor-specified cross reference: Secondary Screen of 63 compounds
2470Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase Expression.Confirmatorydepositor-specified cross reference: Secondary Screen of 14 compounds
2477Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcus.Confirmatorydepositor-specified cross reference: Secondary Screen of 14 compounds
504351Dose responses of compound inhibitions on streptokinase expression and effects on viability in Group A streptococci Measured in Microorganism System Using Plate Reader - 2014-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorydepositor-specified cross reference: 58 drypowder compounds at dose in SK Expression measuring activity set 3
504396Dose responses of compound inhibitions on streptokinase expression and effects on viability in Group A streptococci Measured in Microorganism System Using Plate Reader - 2014-03_Inhibitor_Dose_DryPowder_Viability_Set3Confirmatorydepositor-specified cross reference: 58 drypowder compounds at dose in SK Expression measuring viability set 3
Description:
Broad Institute of MIT and Harvard, Cambridge MA

NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number 1R03DA026214-01

Internal Project ID: 2014

Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence

Primary Collaborators:

Hongmin Sun, University of Missouri-Columbia, sunh@health.missouri.edu

Probes:
ML126, ML134, ML135

Project Overview:

The goal of the project is to identify and develop novel antibiotic small molecules through inhibiting streptokinase (SK) expression in group A streptococcus (GAS). Based on the critical role of SK in GAS pathogenicity, we are attempting to identify chemical compounds that specifically reduce the expression of bacterial SK, without interfering with bacterial viability. Specifically, a probe is a small molecule that shows no less than one hundred fold selection for the primary strain (SKKanGAS, see below) vs a control strain SK(-)GAS (see below) in growth-based assays and shows significant inhibition of SK secretion in abiochemical assay that measure SK activity. The more potent and specific probe will be used to further explore the role of SK in GAS pathogenicity.

Primary Assay Summary:

Internal Assay ID: 2014-01

Assay: The strain used for this project is a GAS strain carrying the kanamycin resistant gene under the control of the streptokinase promoter (SKKanGAS). Compounds that inhibit streptokinase promoter activity lead to a decrease of the kanamycin resistant gene product and cell death in the presence of kanamycin (Sigma K1637). Briefly, SKKanGAS at OD600 equal to 0.015 were plated onto 384-well plates (Corning 3570) and incubated with test compounds (7.5 uM, 0.2%DMSO) in the presence of 40 ug/ml of kanamycin in Todd-Hewitt Broth medium for 6 hours before cell survival is assessed by BacTiterGlo reagent (Promega G8233). Selectivity is determined by comparing growth inhibition between SKKanGAS and another GAS strain, SK(-)GAS where the kanamycin resistant gene is under the control of an SK-independent promoter.

Outcome: A decrease in the luminescent signal will identify compounds that either inhibit the SK promoter, or inhibit the cell growth independent of inhibiting the SK promoter. Normalization of the HTS data across runs and calibration to the positive control was performed as described below. The results are reported as % inhibition. Specificity for SK promoter inhibition will be determined in a secondary assay where a GAS strain carrying kanamycin resistant gene which is not controlled by the SK promoter will be used.

PubChem AID 1662

Number compounds evaluated: 303545

Active Compounds : Activity_Score >=50

Number of active compounds: 3959

Inconclusive Compounds: Activity_Score < 50 with at least one replicate activity score >=50.

Number of inconclusive compounds: 795


Dose Confirmation HTS - AID# 1900
Assay Biology/Type: Cell-Based (SK(-)GAS).
Readout Technology: Plate Reader/Luminescence.
Target Pathway: Streptokinase (SK) expression.
Test concentration(s): 6-point Dose Titration (0.06, 0.18, 0.56, 1.60, 5.00 and 15.00 microM).
Activity criterion: EC50 <= 1 log over the highest tested concentration.
Number of Substances evaluated: 3229
Number of Substances active: 2881


Dose Confirmation HTS - AID# 1902
Assay Biology/Type: Cell-Based (SKKanGAS).
Readout Technology: Plate Reader/Luminescence.
Target Pathway: Streptokinase (SK) expression.
Test concentration(s): 6-point Dose Titration (0.06, 0.18, 0.56, 1.60, 5.00 and 15.00 microM).
Activity criterion: EC50 <= 1 log over the highest tested concentration.
Number of Substances evaluated: 3227
Number of Substances active: 2946


Dose Confirmation HTS - AID# 1914
Assay Biology/Type: Cell-Based (GAS UMAA2166).
Readout Technology: Plate Reader/Luminescence.
Target Pathway: Streptokinase (SK) expression.
Test concentration(s): 6-point Dose Titration (0.06, 0.18, 0.56, 1.60, 5.00 and 15.00 microM).
Activity criterion: EC50 <= 1 log over the highest tested concentration.#
Number of Substances evaluated: 3266
Number of Substances active: 774


Dose Confirmation HTS - AID# 1915
Assay Biology/Type: Cell-Based (GAS UMAA2166).
Readout Technology: Plate Reader/Luminescence.
Target Pathway: Streptokinase (SK) expression.
Test concentration(s): 6-point Dose Titration (0.06, 0.18, 0.56, 1.60, 5.00 and 15.00 microM).
Activity criterion: EC50 <= 1 log over the highest tested concentration.
Number of Substances evaluated: 3266
Number of Substances active: 2220
Comment
The following compounds [SID] were purchased or synthesized for this project:
85281113 85281114 85281115 85281116 85281117 85281118 85281119 85281120 85281121 85281122 85281123 85281124 85281125 85281126 85281127 85281128 85281129 85281130 85281131 85281133 85281134 85281136 85281137 85281138 85281139 85281140 85281141 85281143 85281144 85281145 85281146 85281147 85281148 85281149 85281150 85281152 85281153 85281155 85281157 85281158 85281159 85281161 85281163 85281168 85281169 85281170 85281173 85281174 85281175 87334020 87334021 87334023 87334024 87334025 87334026 87334027 87334028 87334029 87334030 87334031 87334032 85281132 85281135 85281142 85281148 85281151 85281154 85281156 85281160 85281162 85281164 85281165 85281166 85281167 85281171 85281172
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 3
MLP Probe ML# for probe 1: ML126
PubChem Substance ID (SID) for probe 1: 85281160
PubChem Compound ID (CID) for probe 1: 576989
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 3600
Target for probe 1: Streptokinase expression (gi: 15675770)
Disease relevance for probe 1: Antiinfectant
Anti-target for probe 1: Bacterial cell viability
Fold selectivity for probe 1: 3.6 uM vs inactive
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK56228/ (ID: 2509080)
Grant number for probe 1: DA026214-01
MLP Probe ML# for probe 2: ML134
PubChem Substance ID (SID) for probe 2: 85281155
PubChem Compound ID (CID) for probe 2: 1612038
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 5200
Target for probe 2: SK expression (gi: 15675770)
Disease relevance for probe 2: Antiinfectant
Anti-target for probe 2: Bacterial cell viability
Fold selectivity for probe 2: inactive
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK56227/ (ID: 2508840)
Grant number for probe 2: DA026214-01
MLP Probe ML# for probe 3: ML135
PubChem Substance ID (SID) for probe 3: 85281146
PubChem Compound ID (CID) for probe 3: 2769229
Probe type for probe 3: Inhibitor
IC50/EC50 (nM) for probe 3: 2500
Target for probe 3: SK expression (gi: 15675770)
Disease relevance for probe 3: Antiinfectant
Anti-target for probe 3: Bacterial cell viability
Fold selectivity for probe 3: inactive
NCBI Book chapter link for probe 3: http://www.ncbi.nlm.nih.gov/books/NBK56240/ (ID: 2510382)
Grant number for probe 3: DA026214-01
NCBI Book chapter title for probe 1: Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1
NCBI Book chapter title for probe 2: Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 2
NCBI Book chapter title for probe 3: Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 3
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML_NUMML number assigned to compound.String
Additional Information
Grant Number: 1R03DA026214-01

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