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BioAssay: AID 652107

Actin Polymerization inhibition by compounds from Cdc42 Probe Extension Project

Extended Characterization of Cdc42 Probe: Mechanism of Actions and Application to Cancer and Infectious Disease ..more
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 652107
Data Source: NMMLSC
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-03-14
Modify Date: 2013-03-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 2
Depositor Specified Assays
AIDNameTypeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasessummarySummary report for GTPase target project
Description:
University of New Mexico Assay Overview:
Assay Support: NIH 1R03 MH081231 01
Extended Characterization of Cdc42 Probe: Mechanism of Actions and Application to Cancer and Infectious Disease
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Implementation: Lin Hong, Ph.D.
Target Team Leader for the Center: Larry Sklar (lsklar@salud.unm.edu)
UNM Cheminformatics: Cristian Bologa, Ph.D., Oleg Ursu, Ph.D.

Chemistry: University of Kansas Specialized Chemistry Center
KU Specialized Chemistry Center PI: Jeff Aube, Ph.D.
KU SCC Project Manager: Jennifer E. Golden. Ph.D.
KU SCC Chemists on this project: Chad Schroeder, M.S., Denise Simpson, Ph.D., Julica Noeth, B.S.

Assay Background and Significance:

GTPases are known to be involved in actin polymerization. This assay will test a compound's effect on this cellular process.
Protocol
Granulocytes were separated from primary human blood cells by a sterile preparation of Mono-Poly(TM) resolving media. In one step the primary bloods cells are added to the sterile media, then spun down for isolation of mononuclear and polymorphonuclear leucocytes from whole blood in two distinct bands (reference: Kalmar, J.R., et al., J. Immunol. Meth., 110: 275-281 (1988)).

Cells are treated with 10 microM concentration of compounds for 10 min at 37 degree in Hanks'-BSA buffer. The cells in 1 milliL suspension are stirred and stimulated with fMLF. Cell aliquots are diluted 1:1 with a fixative solution containing 7.4% formaldehyde and buffer without calcium or magnesium. Samples are mixed and held at room temperature for 10-20 min before staining for 60 min with an equal volume of 3.7% formaldehyde with 0.2 mg/mL lysophosphatidylcholine and 20 microM FITC-phalloidin in buffer without calcium or magnesium. Actin data will be acquired in linear mode on the FL1 channel of a FACScan. The data will be analyzed using FCSQuest software developed by Dr. Bruce Edwards (UNM).

Calculations:

Measured readings of FL1 median are normalized to the original time point (t=0 sec);
NormFL1 = SampleFL1/FL1@t=0sec

Area under the curve of these time course values of normalized FL1 are calculated by the trapezoidal rule from t=0 to t~200sec. And the %Inhibition elicited by the sample compound are calculated by comparison to the area under the curve for negative control (1 microL DMSO) by the following equation:
%Inhibition = 100*(1-SampleArea/DMSOArea)
PUBCHEM_ACTIVITY_SCORE is based on the calculated % Inhibition:
SCORE = %Inhibition
Active compounds have SCORE > = 10
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PercentInhibition_10uM (10μM**)Percent Inhibition of actin polymerization in the presence of 10 microM test compoundFloat%

** Test Concentration.
Additional Information
Grant Number: NIH I RO3 MH081231-01

Data Table (Concise)
Classification
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