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BioAssay: AID 602146

EGFR degradation assay in SCC-12F cells

Stimulation by EGF (Epidermal Growth Factor) results in the internalization of EGFR (Epidermal Growth Factor Receptor) and trafficked in a Rab7-depended manner to the lysosome and subsequently degraded. Thus compounds that affect the activity of Rab family GTPases would decrease the rate of EGFR degradation. ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(5)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(5)
 
 
Inactive(1)
 
 
AID: 602146
Data Source: NMMLSC (EGFR_Degradation_SCC12Fcells_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 ):           View Active Data    View All Data
Target
BioActive Compounds: 5
Related Experiments
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AIDNameTypeProbeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasesSummary5 depositor-specified cross reference: Summary report for GTPase target project
757HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeScreening same project related to Summary assay
758HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeScreening same project related to Summary assay
759HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeScreening same project related to Summary assay
760HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeScreening same project related to Summary assay
761HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeScreening same project related to Summary assay
764HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantScreening same project related to Summary assay
1333Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
1334Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatory same project related to Summary assay
1335Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
1336Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
1337Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
1339Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantConfirmatory same project related to Summary assay
1340Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeConfirmatory same project related to Summary assay
1341Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
1758Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_wtScreening same project related to Summary assay
1759Activator binding kinetics on Ras and Ras-related GTPases, specifically Ras_wtScreening same project related to Summary assay
1760Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab7_wtScreening same project related to Summary assay
1761Activator binding kinetics on Ras and Ras-related GTPases, specifically Ras_act (activated mutant)Screening same project related to Summary assay
1762Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_act (activated mutant)Screening same project related to Summary assay
1763Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab2_wtScreening same project related to Summary assay
1769Profiling Assay to determine GST-GSH interactions in multiplex bead-based assaysConfirmatory same project related to Summary assay
2009Oxadiazole SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
2019Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype proteinConfirmatory same project related to Summary assay
2020Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
2021Additional SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
2022Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatory same project related to Summary assay
2027Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatory same project related to Summary assay
2031Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
2033Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
2036Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
2037Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatory same project related to Summary assay
2038Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
2039Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatory same project related to Summary assay
2040Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatory same project related to Summary assay
2041Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
2042Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
2043Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
2045Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
2046Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
2047Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
2048Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatory same project related to Summary assay
2050Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
2051Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatory same project related to Summary assay
2053Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
2055Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatory same project related to Summary assay
2372Compound effect on equilibrium binding with Cdc42 under varying GTP conditions: nucleotide depletion with Mg bufferOther same project related to Summary assay
2373Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with Mg bufferOther same project related to Summary assay
2374PAK-Effector Pull-down Assay for Quantification of Rac/Cdc42 Activation Using 3T3 CellsOther same project related to Summary assay
2375Panel results of Cell based ELISA Assessing Activation of Cdc42 and Rac1Other same project related to Summary assay
2376Dose Response of compounds with constant GTP under the condition of nascent nucleotide depletion and Mg bufferConfirmatory same project related to Summary assay
2378Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with EDTA bufferOther same project related to Summary assay
2393Dose Response of compounds for six proteins with constant GTP under the condition of Mg bufferOther same project related to Summary assay
2418Assessment of Cdc42 inhibitors on Bradykinin activation of 3T3 cellsOther same project related to Summary assay
588369Cellular toxicity assessed by Trypan Blue for compounds active in GTPase screenOther same project related to Summary assay
588373SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutant, round 1Confirmatory same project related to Summary assay
588377SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 1Confirmatory same project related to Summary assay
588381SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras activated mutant, round 1Confirmatory same project related to Summary assay
588383SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtype, round 1Confirmatory same project related to Summary assay
588384SAR compounds for Rab7 project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutant, round 1Confirmatory same project related to Summary assay
588385SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 1Confirmatory same project related to Summary assay
588387SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras activated mutant, round 1Confirmatory same project related to Summary assay
588388SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras wildtype, round 1Confirmatory same project related to Summary assay
588393SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 1Confirmatory same project related to Summary assay
588394SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 1Confirmatory same project related to Summary assay
588410Cellular toxicity assessed by Trypan Blue for compounds in active GTPase screen, set2Other same project related to Summary assay
588427Cellular toxicity assessed by Trypan Blue for compounds in active GTPase screen, Set1Other same project related to Summary assay
588477Dose Response of round 1 SAR compounds for Cdc42 probe extension project tested by multiplex of eight GTPase proteins under the condition of Mg Buffer, non-chelator bufferOther same project related to Summary assay
588479Dose Response of round 1 SAR compounds for GTPase inhibitor project tested by multiplex of eight GTPase proteins under the condition of Mg Buffer, non-chelator bufferOther same project related to Summary assay
588622Dose Response of round 2 SAR compounds for GTPase inhibitor project tested by multiplex of eight GTPase proteins under the condition of Mg Buffer, non-chelator bufferOther same project related to Summary assay
588624SAR compounds for Rab7 project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutant, round 2Confirmatory same project related to Summary assay
588626SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 2Confirmatory same project related to Summary assay
588628SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras activated mutant, round 2Confirmatory same project related to Summary assay
588630SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras wildtype, round 2Confirmatory same project related to Summary assay
588631SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 2Confirmatory same project related to Summary assay
602137Counter screen to determine effect of SAR compounds for Rab7 project on GST-GSH binding, round 1Other same project related to Summary assay
602145Counter screen to determine effect of SAR compounds for Rab7 project on GST-GSH binding, round 2Other same project related to Summary assay
602148Active GTPase Screen Compounds affects on binding of VLA-4 specific ligandOther same project related to Summary assay
651732Cellular toxicity assessed by Trypan Blue for compounds in active Cdc42 Probe Extension ProjectOther same project related to Summary assay
652107Actin Polymerization inhibition by compounds from Cdc42 Probe Extension ProjectOther same project related to Summary assay
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: 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:
Stimulation by EGF (Epidermal Growth Factor) results in the internalization of EGFR (Epidermal Growth Factor Receptor) and trafficked in a Rab7-depended manner to the lysosome and subsequently degraded. Thus compounds that affect the activity of Rab family GTPases would decrease the rate of EGFR degradation.
Protocol
For degradation assay, SCC-12F cells grown in a six-well plate are serum starved for 2 hours in DMEM containing 10-100 microM of compound and then stimulated with 20 nanoM EGF. At different time points (0-2hours), cells are lysed with SDS lysis buffer containing a cocktail of proteinase inhibitors. Cell debris is removed by centrifugation, and the total amount of proteins is quantified by BSA assay. The amounts of EGFR are analyzed by SDS-PAGE and Western blot.
Response Assessment:
Western blots bands were scanned and normalized to the initial time point. The time course of protein degradation in the presence of compound were compared with degradation in presence of DMSO. Based on the decrease rate of degradation compared with DMSO sample, the compounds were assesses as active. And based on the magnitude difference in this degradation rate, the PUBCHEM_ACTIVITY_SCORE were given. An active compound has a SCORE > 50.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: SCC-12F
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AVERAGE_DSMO_0MIN (0μM**)Average western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 0MINFloat
2STDEV_DSMO_0MIN (0μM**)Standard deviation western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 0MINFloat
3AVERAGE_DSMO_15MIN (0μM**)Average western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 15MINFloat
4STDEV_DSMO_15MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 15MINFloat
5AVERAGE_DSMO_30MIN (0μM**)Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 30MINFloat
6STDEV_DSMO_30MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 30MINFloat
7AVERAGE_DSMO_60MIN (0μM**)Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 60MINFloat
8STDEV_DSMO_60MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 60MINFloat
9AVERAGE_DSMO_120MIN (0μM**)Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 120MINFloat
10STDEV_DSMO_120MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 120MINFloat
11AVERAGE_10uM_0MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 0MINFloat
12STDEV_10uM_0MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 0MINFloat
13AVERAGE_10uM_15MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 15MINFloat
14STDEV_10uM_15MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 15MINFloat
15AVERAGE_10uM_30MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 30MINFloat
16STDEV_10uM_30MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 30MINFloat
17AVERAGE_10uM_60MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 60MINFloat
18STDEV_10uM_60MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 60MINFloat
19AVERAGE_10uM_120MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 120MINFloat
20STDEV_10uM_120MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 120MINFloat
21AVERAGE_30uM_0MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 0MINFloat
22STDEV_30uM_0MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 0MINFloat
23AVERAGE_30uM_15MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 15MINFloat
24STDEV_30uM_15MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 15MINFloat
25AVERAGE_30uM_30MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 30MINFloat
26STDEV_30uM_30MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 30MINFloat
27AVERAGE_30uM_60MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 60MINFloat
28STDEV_30uM_60MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 60MINFloat
29AVERAGE_30uM_120MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 120MINFloat
30STDEV_30uM_120MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 120MINFloat
31AVERAGE_100uM_0MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 0MINFloat
32STDEV_100uM_0MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 0MINFloat
33AVERAGE_100uM_15MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 15MINFloat
34STDEV_100uM_15MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 15MINFloat
35AVERAGE_100uM_30MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 30MINFloat
36STDEV_100uM_30MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 30MINFloat
37AVERAGE_100uM_60MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 60MINFloat
38STDEV_100uM_60MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 60MINFloat
39AVERAGE_100uM_120MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 120MINFloat
40STDEV_100uM_120MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 120MINFloat

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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