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
Target
| Sequence: epidermal growth factor receptor [Homo sapiens] Description ..   Protein Family: Catalytic domain of the Protein Tyrosine Kinase, Epidermal Growth Factor Receptor Gene:EGFR Related Protein 3D Structures |
BioActive Compounds: 5
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 1772 | Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases | summary | Summary 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: 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.
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.
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.
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | AVERAGE_DSMO_0MIN (0μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 0MIN | | Float | ||
| 2 | STDEV_DSMO_0MIN (0μM**) | Standard deviation western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 0MIN | | Float | ||
| 3 | AVERAGE_DSMO_15MIN (0μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 15MIN | | Float | ||
| 4 | STDEV_DSMO_15MIN (0μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 15MIN | | Float | ||
| 5 | AVERAGE_DSMO_30MIN (0μM**) | Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 30MIN | | Float | ||
| 6 | STDEV_DSMO_30MIN (0μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 30MIN | | Float | ||
| 7 | AVERAGE_DSMO_60MIN (0μM**) | Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 60MIN | | Float | ||
| 8 | STDEV_DSMO_60MIN (0μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 60MIN | | Float | ||
| 9 | AVERAGE_DSMO_120MIN (0μM**) | Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 120MIN | | Float | ||
| 10 | STDEV_DSMO_120MIN (0μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 120MIN | | Float | ||
| 11 | AVERAGE_10uM_0MIN (10μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 0MIN | | Float | ||
| 12 | STDEV_10uM_0MIN (10μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 0MIN | | Float | ||
| 13 | AVERAGE_10uM_15MIN (10μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 15MIN | | Float | ||
| 14 | STDEV_10uM_15MIN (10μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 15MIN | | Float | ||
| 15 | AVERAGE_10uM_30MIN (10μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 30MIN | | Float | ||
| 16 | STDEV_10uM_30MIN (10μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 30MIN | | Float | ||
| 17 | AVERAGE_10uM_60MIN (10μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 60MIN | | Float | ||
| 18 | STDEV_10uM_60MIN (10μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 60MIN | | Float | ||
| 19 | AVERAGE_10uM_120MIN (10μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 120MIN | | Float | ||
| 20 | STDEV_10uM_120MIN (10μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 120MIN | | Float | ||
| 21 | AVERAGE_30uM_0MIN (30μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 0MIN | | Float | ||
| 22 | STDEV_30uM_0MIN (30μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 0MIN | | Float | ||
| 23 | AVERAGE_30uM_15MIN (30μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 15MIN | | Float | ||
| 24 | STDEV_30uM_15MIN (30μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 15MIN | | Float | ||
| 25 | AVERAGE_30uM_30MIN (30μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 30MIN | | Float | ||
| 26 | STDEV_30uM_30MIN (30μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 30MIN | | Float | ||
| 27 | AVERAGE_30uM_60MIN (30μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 60MIN | | Float | ||
| 28 | STDEV_30uM_60MIN (30μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 60MIN | | Float | ||
| 29 | AVERAGE_30uM_120MIN (30μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 120MIN | | Float | ||
| 30 | STDEV_30uM_120MIN (30μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 120MIN | | Float | ||
| 31 | AVERAGE_100uM_0MIN (100μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 0MIN | | Float | ||
| 32 | STDEV_100uM_0MIN (100μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 0MIN | | Float | ||
| 33 | AVERAGE_100uM_15MIN (100μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 15MIN | | Float | ||
| 34 | STDEV_100uM_15MIN (100μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 15MIN | | Float | ||
| 35 | AVERAGE_100uM_30MIN (100μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 30MIN | | Float | ||
| 36 | STDEV_100uM_30MIN (100μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 30MIN | | Float | ||
| 37 | AVERAGE_100uM_60MIN (100μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 60MIN | | Float | ||
| 38 | STDEV_100uM_60MIN (100μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 60MIN | | Float | ||
| 39 | AVERAGE_100uM_120MIN (100μM**) | Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 120MIN | | Float | ||
| 40 | STDEV_100uM_120MIN (100μM**) | Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 120MIN | | Float |
** Test Concentration.
Additional Information
Grant Number: NIH I RO3 MH081231-01
Data Table (Concise)
Classification
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