Chemical Antagonists IAP-family anti-apoptotic proteins
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) more ..
BioActive Compounds: 17
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
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 his-tagged-BIR1-BIR2 (bacoloviral IAP repeat, Bir1/2) 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.
Bir1/2 assay materials:
1) Bir1/2 protein and rhodamine-SMAC peptide (AVPIAQK-rhodamine) were provided by Prof. John Reed (Sanford-Burnham Medical Research Institute, San Diego, CA)
2) Assay buffer: 31.25 mM HEPES-NaOH, pH 7.5, 1.25 mM TCEP, 0.00625% Tween 20.
3) Bir1/2 working solution contained 2.5 uM Bir1/2 in the assay buffer.
4) Rhodamine-SMAC working solution contained 50 nM Rhodamine-SMAC in the assay buffer.
Bir1/2 HTS protocol:
1) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well black small-volume plates (784076). Columns 1 and 2 were added with 4 uL of 10% DMSO.
2) Positive control wells, that contained no Bir1/2, were assigned to column 1 and were added 8 uL of assay buffer using WellMate bulk dispenser (Matrix).
3) 8 uL of Bir1/2 working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Negative control wells that contained no compounds were assigned to column 2.
4) Plates were briefly spinned down and incubated for 1h at room temperature.
5) 8 uL of Rhodamine-SMAC working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
6) Final concentrations of the components in the assay were as follows:
a. 25 mM HEPES-NaOH, pH 7.5, 1 mM TCEP, 0.005% Tween 20.
b. 20 nM Rhodamine-SMAC (columns 1-24)
c. 1 uM Bir1/2 (columns 2-24)
d. 2 % DMSO (columns 1-24)
e. 20 uM compounds (columns 3-24)
7) Plates were incubated for 30 min at room temperature protected from direct light.
8) Fluorescence polarization was measured on an EnVision plate reader (Perkin Elemer) using rhodamine filters: excitation filter - 540 nm, emission filter 590 nm, dichroic mirror 560 nm. The signal for each well was acquired for 100 ms.
9) Data analysis was performed using CBIS software (ChemInnovations, Inc).
10) Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.
Compounds with greater than 50% displacement of Rhodamine-SMAC in the Bir1/2 assay at 20 uM concentration and F_ratio parameter less than 1.5 are defined as actives of the primary screening.
For dose-response compound testing the same steps basic protocol was utilized. Compounds were serially diluted in DMSO to have duplicate 10-point curves with 2-fold dilution between concentrations. Positive and negative control wells were in columns 1-2 and 23-24 respectively. Compounds with an IC50 < 100 uM were 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 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-the score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % displacement is less than 0%, then the assigned score is 0
b. If primary % displacement is greater than 100%, then the assigned score is 40
c. If primary % displacement is between 0% and 100%, then the calculated score is (% Displacement)*0.4
d. If the F_ratio is >=1.50 then the score is set to 5.
e. Compounds with no fluorescence interference and >50% displacement have the score >= 20.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
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 TNAP activation 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 = 44 + 6*(pEC50 - 3)*QC,
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 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 44 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 >.
b) The value of + 6*(pEC50/IC50-3)*QC, is < 0.500
Active compounds will have a score >= 44.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)