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BioAssay: AID 2389

qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1: Summary

The caspases (Cysteine Aspartyl Protease) comprise a related family of 14 dimeric proteases that are critical mediators of apoptosis (programmed cell death) and inflammation. The caspases represent an important class of drug targets for stroke, ischemia, cancer and inflammatory diseases. Comparison of crystal structures of procaspases and caspases have revealed that the subunits of the homodimer are composed of subdomains which have highly conserved architecture and mechanism, and show large structural transitions between procaspase and active conformers. ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Probe(1)
 
 
Active(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Probe(1)
 
 
Active(6)
 
 
AID: 2389
Data Source: NCGC (CASP146)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-22
Modify Date: 2010-05-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 1    Active: 6
Related Experiments
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AIDNameTypeComment
888Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: CatalaseConfirmatorydepositor-specified cross reference: Caspase-1 inhibition counterscreen: testing for inhibition in the presence of catalase
896Confirmation Concentration-Response Assay for Allosteric/Competitive Inhibitors of Caspase-1Confirmatorydepositor-specified cross reference: Caspase-1 inhibition confirmation assay
900qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1Confirmatorydepositor-specified cross reference: Caspase-1 inhibition primary assay
923qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1: Spectroscopic Profiling in AFC Spectral RegionConfirmatorydepositor-specified cross reference: Caspase-1 inhibition counterscreen: fluorescence interference of the AFC fluorophore
929Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: CysteineConfirmatorydepositor-specified cross reference: Caspase-1 inhibition counterscreen: redox behavior
996Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: SummaryConfirmatorydepositor-specified cross reference: Caspase-1 inhibition counterscreen: testing for inhibition in the presence of catalase
2494Confirmation Assay for Allosteric/Competitive Inhibitors of Caspase-1Confirmatorydepositor-specified cross reference
504731Allosteric/Competitive Inhibitors of Caspase 1: Metabolic Stability ProfilingOtherdepositor-specified cross reference
504732Allosteric/Competitive Inhibitors of Caspase 1: Caco-2 Permeability ProfilingOtherdepositor-specified cross reference
Description:
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: 1 X01 MH078950-01
PI Name: Dr. James Wells,
Dr. Enrique Perez-Paya, Dr.Janice Williams, Dr. Dennis Wolan, Mr. Brandon Butler
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence

NCGC Assay Overview:

The caspases (Cysteine Aspartyl Protease) comprise a related family of 14 dimeric proteases that are critical mediators of apoptosis (programmed cell death) and inflammation. The caspases represent an important class of drug targets for stroke, ischemia, cancer and inflammatory diseases. Comparison of crystal structures of procaspases and caspases have revealed that the subunits of the homodimer are composed of subdomains which have highly conserved architecture and mechanism, and show large structural transitions between procaspase and active conformers.
The goal of this project was to identify compounds that act as selective inhibitors caspase 1. To identify both allosteric and competitive inhibitors of caspase 1, the enzyme was screened using a pro-fluorescent substrate in a qHTS format (AIDs: 896, 900). Compounds were checked for fluorescence interference of the AFC fluorophore used in the primary assay (AID: 923) as well for redox behavior by switching the reducing agent in the buffer from DTT to cysteine (AID: 929), or testing for inhibition in the presence of catalase (AIDs: 888, 996).
The compound (CID: 44620939) has been shown to be selective against other caspases using a panel of 9 other caspases (Reaction Biology; http://www.reactionbiology.com).

All IC50#s (given in nM) for the capases listed were determined using 10-doses of compound in a 3-fold serial dilution series starting at 10 uM (final assay concentration). Fluorescent substrates were at a 5 uM final assay concentration which is less then or equal to their KM values. Compound potency against Caspases 3, 6 and 7 were determined in 50 mM HEPES pH 7.4, 100mM NaCl, 0.01% CHAPS, 0.1 mM EDTA. Compound potency against Caspases 1, 4, 5, 8, 9, 10, and 14 were determined in 50 mM HEPES pH 7.4, 1 M Na Citrate, 100 mM NaCl, 0.01% CHAPS, 0.1 mM EDTA. The protocol involved delivering 2X enzyme, and then compounds were added in 100% DMSO by Acoustic technology (Echo550). The reaction was started by adding 2X substrate and the increase in fluorescence intensity was measured on a PE EnVision microtiter plate reader at 5 min intervals, 25 times (2 hours total). Analysis was performed by determining the slope (signal/min) for the initial linear part of the reaction. Data was normalized relative to controls appropriate for each caspase enzyme (see Reaction Biology web site for more details). Empty cells in the data table indicate no inhibition or compound activity that could not be fit to an IC50 curve.
Protocol
Please refer to AIDs (888, 896, 900, 923, 929, and 996) for detailed assay protocols.
Comment
This summary is written for the purposes of summarizing the probe activities from the project. MLPCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules are given a score of 50. Molecules pending validation are given a score of 10. Inactive compounds are given a score of 0. We have identified a probe in this project.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML132
PubChem Substance ID (SID) for probe 1: 87544173
PubChem Compound ID (CID) for probe 1: 44620939
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 0.023
Target for probe 1: Caspase 1 (gi: 15431328)
Disease relevance for probe 1: Ischemic disorders, Huntington's disease, amyotrophic lateral sclerosis
Anti-target for probe 1: 9 Caspase panel
Fold selectivity for probe 1: >100
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK56241/ (ID: 2510508)
Grant number for probe 1: MH078950-01
PubMed Publication ID (PMID) for probe 1: 20229566
NCBI Book chapter title for probe 1: A small molecule inhibitor of Caspase 1
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed based on the compound activity in caspase-1 confirmation assay.String
2Potency in Caspase-1The concentration of sample yielding half-maximal inhibition in caspase-1 confirmation assay.FloatnM
3Potency in Caspase-3The concentration of sample yielding half-maximal inhibition in caspase-3 confirmation assay.FloatnM
4Potency in Caspase-4The concentration of sample yielding half-maximal inhibition in caspase-4 confirmation assay.FloatnM
5Potency in Caspase-5The concentration of sample yielding half-maximal inhibition in caspase-5 confirmation assay.FloatnM
6Potency in Caspase-6The concentration of sample yielding half-maximal inhibition in caspase-6 confirmation assay.FloatnM
7Potency in Caspase-7The concentration of sample yielding half-maximal inhibition in caspase-7 confirmation assay.FloatnM
8Potency in Caspase-8The concentration of sample yielding half-maximal inhibition in caspase-8 confirmation assay.FloatnM
9Potency in Caspase-9The concentration of sample yielding half-maximal inhibition in caspase-9 confirmation assay.FloatnM
10Potency in Caspase-10The concentration of sample yielding half-maximal inhibition in caspase-10 confirmation assay.FloatnM
11Potency in Caspase-14The concentration of sample yielding half-maximal inhibition in caspase-14 confirmation assay.FloatnM
Additional Information
Grant Number: 1 X01 MH078950-01

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