Counterscreen for procaspase-3 activators: absorbance-based primary biochemical high throughput screening assay to identify activators of procaspase-7
Name: Counterscreen for procaspase-3 activators: absorbance-based primary biochemical high throughput screening assay to identify activators of procaspase-7. ..more
BioActive Compounds: 302
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Paul Hergenrother, University of Illinois at Urbana-Champaign
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 CA120439-01
Grant Proposal PI: Paul Hergenrother, University of Illinois at Urbana-Champaign
External Assay ID: PROCASPASE7_ACT_EPIABS_1536_1X%ACT CSRUN
Name: Counterscreen for procaspase-3 activators: absorbance-based primary biochemical high throughput screening assay to identify activators of procaspase-7.
Cancer progression depends upon evasion of the programmed cell death (apoptosis) machinery that normally kills an unneeded or rogue cell (1). Although apoptosis induction using chemotherapeutics is a common anti-cancer treatment, cancer cells often survive because of defects in the pro-apoptotic proteins activated by these drugs (2). The main effector proteins involved in the apoptosis pathway are the cysteine-aspartic acid proteases (caspases). Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme (3,4). Caspase 3 is a major executioner enzyme, and is responsible for proteolysis of hundreds of cellular substrates, including caspases 6, 7 and 9. Procaspase 3 itself is processed by caspases 8, 9 and 10. Studies demonstrating that most mutations in which upstream proapoptotic signals are improperly transmitted to activate executioner caspases lead to cancers and that levels of procaspase 3 protein are elevated in certain cancers (5, 6), suggest that procaspase 3 may play a role in resistance to chemotherapy. As a result, the identification of procaspase 3 activators may identify molecular mechanisms of apoptosis-resistant cancers, and elucidate the role of procaspase 3 activation in tumorigenesis (7).
1. McConkey, DJ and Zhu, K, Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist Updat, 2008. 11(4-5): p. 164-79.
2. Cory, S and Adams, JM, Killing cancer cells by flipping the Bcl-2/Bax switch. Cancer Cell, 2005. 8(1): p. 5-6.
3. Roy, S, Bayly, CI, Gareau, Y, Houtzager, VM, Kargman, S, Keen, SL, Rowland, K, Seiden, IM, Thornberry, NA and Nicholson, DW, Maintenance of caspase-3 proenzyme dormancy by an intrinsic "safety catch" regulatory tripeptide. Proc Natl Acad Sci U S A, 2001. 98(11): p. 6132-7.
4. Yang, X, Chang, HY and Baltimore, D, Autoproteolytic activation of pro-caspases by oligomerization. Mol Cell, 1998. 1(2): p. 319-25.
5. Putt, KS, Chen, GW, Pearson, JM, Sandhorst, JS, Hoagland, MS, Kwon, JT, Hwang, SK, Jin, H, Churchwell, MI, Cho, MH, Doerge, DR, Helferich, WG and Hergenrother, PJ, Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol, 2006. 2(10): p. 543-50.
6. Svingen, PA, Loegering, D, Rodriquez, J, Meng, XW, Mesner, PW, Jr., Holbeck, S, Monks, A, Krajewski, S, Scudiero, DA, Sausville, EA, Reed, JC, Lazebnik, YA and Kaufmann, SH, Components of the cell death machine and drug sensitivity of the National Cancer Institute Cell Line Panel. Clin Cancer Res, 2004. 10(20): p. 6807-20.
7. Peterson, QP, Goode, DR, West, DC, Ramsey, KN, Lee, JJ and Hergenrother, PJ, PAC-1 activates procaspase-3 in vitro through relief of zinc-mediated inhibition. J Mol Biol, 2009. 388(1): p. 144-58.
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The purpose of this assay is to identify compounds that increase the activity of procaspase-7, an enzyme with a high degree of structural similarity to caspase-3. This assay also serves as a counterscreen for procaspase-3 activators. This assay employs a procaspase-7 triple-mutant enzyme (D23A/D198A/D206A) (PC-7 D3A) which is unable to autoproteolyze itself because its aspartic acid cleavage sites have been mutated to alanines. The mutant has a fully functional active site that can process the peptidic Ac-DEVD-pNA chromogenic substrate. Cleavage of this substrate by procaspase-7 hydrolyzes the bond between the aspartic acid and p-nitroaniline, leading to release of yellow p-nitroaniline and an increase in well absorbance at 405 nm. In this assay, the enzyme is pre-incubated with test compounds, followed by addition of substrate and measurement of well epi-absorbance. As designed, compounds that activate procaspase-7 activity will increase substrate hydrolysis, leading to an increase in well absorbance. Compounds are tested in singlicate at a nominal concentration of 8.5 uM.
Prior to the start of the assay, 2.5 uL of zinc-free Assay Buffer (50 mM HEPES, 300 mM NaCl, pH 7.4, 0.01% Triton-X 100) containing 2 uM of PC-7 D3A protein were dispensed into 1536 microtiter plates. Next, 43 nL of test compound in DMSO or DMSO alone (0.8% final concentration) were added to the appropriate wells and incubated for 1 hour at 25 C.
The assay was started by dispensing 2.5 uL of 400 uM Ac-DEVD-pNA in Assay Buffer to all wells. Plates were centrifuged and after 2 hours of incubation at 25 C, epi-absorbance was read on the EnVision plate reader using a photometric filter set (excitation = 405 nm, emission = 450 nm) and a dichroic mirror with the 425 nm cutoff. Fluorescence emission was read at 10 flashes per well at two time points (0 minutes and 120 minutes).
Prior to further calculations, the following formula was used to calculate absorbance:
Absorbance = ( -Log10 * ( [Raw2] / [Mean Reference2] ) ) - ( -Log10 * ( [Raw1] / [Mean Reference] ) )
Raw1 is defined as the read at T0 minutes.
Raw2 is defined as the read at T120 minutes.
Mean Reference is defined as a mean of values from wells containing buffer only at T0.
Mean Reference2 is defined as a mean of values from wells containing buffer only at T120.
The percent activation for each compound was calculated as follows:
% Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing DMSO and 5 uM PC-7.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-14, and for inactive compounds 14-0.
List of Reagents:
Recombinant Procaspase 7 enzyme (Assay Provider)
Assay Buffer (Assay Provider)
Chromogenic Substrate (Ac-DEVD-pNA) (Assay Provider)
1536 SWSN plates (Grainer, part 789175 )
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)