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
BioActive Compounds: 2
Depositor Specified Assays
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 (firstname.lastname@example.org)
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.
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).
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
** Test Concentration.
Data Table (Concise)