AID	Name	Type	Probe	Comment
1018	Chemical Antagonists IAP-family anti-apoptotic proteins	confirmatory
1449	Chemical Antagonists of IAP-family anti-apoptotic proteins confirmation	confirmatory
1513	Counter Screen using XIAP-Bir3 for Chemical Antagonists of Bir1/2 domains of IAP-family anti-apoptotic proteins	confirmatory
1514	Counter Screen using XIAP-Bir3 of the Chemical Antagonists of IAP-family anti-apoptotic proteins confirmation assay	confirmatory
1638	Summary of Chemical Antagonists IAP-family anti-apoptotic proteins	summary	3

Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Proposal Number: MH081277-01
Assay Provider: John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA

This dose response assay is developed and performed as a counter screen to compounds in the Chemical Antagonists of IAP-family anti-apoptotic proteins confirmation (AID 1449) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally. This assay was performed in the assay providers' laboratory.

Apoptosis plays an essential role in many aspects of normal development and physiology, becoming dysregulated in myriad diseases characterized by insufficient or excessive cell death. Caspases are intracellular proteases that are suppressed by Inhibitor of Apoptosis Proteins (IAPs), a family of evolutionarily conserved anti-apoptotic proteins. Proteins released from mitochondria (SMAC and HtrA2) can competitively displace IAPs from the Caspases, thus helping to drive apoptosis. It has been shown that only a few residues at the N-terminus of activated SMAC protein (4'mer) are sufficient to affect the release of IAPs from Caspases. Thus, it is plausible to identify chemical compounds that mimic the effect of SMAC in antagonizing IAPs by causing them to release Caspases. Non-peptidyl chemical inhibitors would have advantages over SMAC peptides, in terms of cell permeability, stability, and in vivo pharmacology. Thus, the goal of this project is to generate small-molecule chemical probe compounds that mimic the effects of SMAC peptides, inhibiting the function of IAPs.

Basis of the assay is disruption of fluorescence polarization resulting from binding of a XIAP-BIR3 (bacoloviral IAP repeat, "Bir3") domain protein derived from two of the three conserved caspase binding "BIR" domains of XIAP to a rhodamine tagged 7-mer N-terminal SMAC peptide.

BIR3 assay materials:
1) Bir3 protein was provided by Prof. John C. Reed (Sanford-Burnham Medical Research Institute, San Diego, CA) and SMAC-rhodamine peptide (AVPIAQK-rhodamine) was provided by Richard Houghten (Torrey Pines Institute for Molecular Studies, San Diego, CA).
2) Assay buffer: 33.35 Hepes @ pH 7.5, 1.33 mM TCEP, 0.00667 % Tween 20, 0.265 uM BIR3 and 0.0267 uM SMAC-rhodamine.

BIR3 protocol:
1) 15 ul of assay buffer was added to a Molecular Devices black 96 well plate (number 42-000-0117) in columns 1 through 10.
2) Five ul of compounds were added to the plate in columns 1 through 10 with a 16 point curve generated by 2 fold dilutions of each compound. Final compound concentration in the assay ranged from 100 uM to 0.00612 uM via two fold dilutions starting with 400 uM maximum concentration.
3) Positive controls without protein were in column 12. Negative controls with protein were in column 11.
4) The AVPF peptide was present with the same concentration range as the compounds on each plate as an internal control.
5) Final concentrations of the components were 25 mM Hepes @ pH 7.5/ 1 mm TCEP/0.005% Tween 20/20 nM rhodamine-SMAC/0.2 uM BIR3. Compound was diluted in water starting at 400 uM and diluted serially by a factor of 2. Final concentrations of compounds were one fourth the concentration of the initial compound curve.
6) Plates were then centrifuged at 1000 rpm for 1 minute before reading on an LJL Analyst using an excitation filter of 530 nm with emission at 580 nm and a dichroic mirror at 565 nm.
7) Resultant data was fit to a nonlinear curve in Prism and IC50 values and Hill coefficients were determined. BIR3 assays often had a Hill coefficient greater than 1.

Compounds with an IC50 < 100 uM are considered "active."

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the Bir1/2 assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the Bir1/2 assay is as follows:

1) First tier (0-40 range) is reserved for primary screening data-and is not applicable in this assay.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound's activatory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the XIAP assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 84 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 90 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

A score of 84 is given to active compounds selected from plates:
a) That do not have a Hill coefficient associated with them and have a qualifier of < or >.
or
b) The value of + 3*(pIC50-3)*QC, is < 0.500

Active compounds will have a score >= 84.

Grant Number: MH081277-01