Bookmark and Share
BioAssay: AID 602148

Active GTPase Screen Compounds affects on binding of VLA-4 specific ligand

Activation of GTPases (in particular Rho family) are involved in the signal transduction cascades that commences with stimulation of formyl peptide receptors and resulting in the increase ligand-affinity of VLA-4 integrins. Assessment of this increased affinity can be made with , fluorescently labeled specific ligand for VLA-4, LDV-FITC. By utilizing U937 cells expressing non-desensetizing, more ..
_
   
 Tested Compounds
 Tested Compounds
All(20)
 
 
Active(10)
 
 
Inactive(10)
 
 
 Tested Substances
 Tested Substances
All(20)
 
 
Active(10)
 
 
Inactive(10)
 
 
AID: 602148
Data Source: NMMLSC (VLA4_LDVFITC_U937_Cells_GTPaseCompounds )
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-12
Hold-until Date: 2012-12-05
Modify Date: 2012-12-05

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 10
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 I RO3 MH081231-01
HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Implementation: Peter Simons, Ph.D., 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:
Activation of GTPases (in particular Rho family) are involved in the signal transduction cascades that commences with stimulation of formyl peptide receptors and resulting in the increase ligand-affinity of VLA-4 integrins. Assessment of this increased affinity can be made with , fluorescently labeled specific ligand for VLA-4, LDV-FITC. By utilizing U937 cells expressing non-desensetizing, deltaST, formyl peptide receptors, VLA-4 remains activate for a longer period of time post-stimulation. Hence a compound that interferes with the prolonged activation of GTPase would result in decreased ligand affinity.
Protocol
Cells (U937 cells expressing deltaST, non-desentizied formyl peptide receptors) are incubated at 37 degrees for 10-20 min. Flow cytometer data are acquired for 1024 s at 37 degrees. After establishing a baseline for unstained cells, LDV-FITC (VLA-4-specific ligand 4-((N'-2-methylphenyl)ureido)-phenylacetyl-l-leucyl-l-aspartyl-l-valyl-l-prolyl-l-alanyl-l-alanyl-l-lysine (LDV) FITC-conjugated) is added at sub-saturating concentration (~4 nanoM). Approximately 2 min later, fMLFF peptide (100 nanoM) is added to induce the cell stimulation. When the fluorescence increase stabilizes, different concentrations of compounds are added. The mean channel fluorescence is recorded. Nonspecific binding of LDV-FITC is determined by an excess of unlabeled LDV.

Response Assessment:
Compounds that induce a temporal decrease in LDV-FITC binding are considered to be active. Three distinct activity levels were assigned to each compounds; 0 no activity, 1 weak activity, and 2 strong activity.

PUBCHEM_ACTIVITY_SCORE is based on the different levels of activity:
Non-active compounds, SCORE = 0
Weak active compounds, SCORE = 33
Strong active compounds, SCORE = 66
Active compounds have SCORE > 0
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1VLA4_Affinity_Level (50μM**)Level of change in VLA-4 affinity due to test compoundInteger

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

Data Table (Concise)
Classification
PageFrom: