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.

Please refer to AIDs (888, 896, 900, 923, 929, and 996) for detailed assay protocols.

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.

Grant Number: 1 X01 MH078950-01