Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET
Abstract Dysregulation of integral membrane proteins (IMPs) has been linked to a myriad of diseases, making these proteins an attractive target in drug research. Whilst PROTAC technology has had a significant impact in scientific research, its application to IMPs is still limited. Limitations of the...
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Nature Portfolio
2025-01-01
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| Online Access: | https://doi.org/10.1038/s41598-024-84217-2 |
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| author | Camilla Ruffilli Sascha Röth Noam Zelcer Kevin Moreau |
| author_facet | Camilla Ruffilli Sascha Röth Noam Zelcer Kevin Moreau |
| author_sort | Camilla Ruffilli |
| collection | DOAJ |
| description | Abstract Dysregulation of integral membrane proteins (IMPs) has been linked to a myriad of diseases, making these proteins an attractive target in drug research. Whilst PROTAC technology has had a significant impact in scientific research, its application to IMPs is still limited. Limitations of the traditional approach of immunoblotting in PROTAC research include the low throughput compared to other methods, as well as a lack of spatial information for the target. Here we compare orthogonal antibody based approaches, i.e. immunoblotting, flow cytometry and immunofluorescence, to measure PROTAC mediated degradation of two established, endogenous targets, epidermal growth factor receptor (EGFR) and hepatocyte growth-factor receptor (c-MET). We discuss advantages and limitations of each methodology for the assessment of PROTAC efficacy on IMPs. Overall, we recommend the use of immunofluorescence and flow cytometry, for an increased accuracy with both a qualitative and quantitative insight into degradation efficacy and a critical distinction between cell membrane-localized and intracellular IMP protein pools. |
| format | Article |
| id | doaj-art-c167773d1384460f8e6d3212803f3ccd |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-c167773d1384460f8e6d3212803f3ccd2025-01-05T12:14:55ZengNature PortfolioScientific Reports2045-23222025-01-0115111210.1038/s41598-024-84217-2Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-METCamilla Ruffilli0Sascha Röth1Noam Zelcer2Kevin Moreau3Safety Innovation and PROTAC Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZenecaSafety Innovation and PROTAC Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZenecaDepartment of Medical Biochemistry, Amsterdam UMC, University of AmsterdamSafety Innovation and PROTAC Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZenecaAbstract Dysregulation of integral membrane proteins (IMPs) has been linked to a myriad of diseases, making these proteins an attractive target in drug research. Whilst PROTAC technology has had a significant impact in scientific research, its application to IMPs is still limited. Limitations of the traditional approach of immunoblotting in PROTAC research include the low throughput compared to other methods, as well as a lack of spatial information for the target. Here we compare orthogonal antibody based approaches, i.e. immunoblotting, flow cytometry and immunofluorescence, to measure PROTAC mediated degradation of two established, endogenous targets, epidermal growth factor receptor (EGFR) and hepatocyte growth-factor receptor (c-MET). We discuss advantages and limitations of each methodology for the assessment of PROTAC efficacy on IMPs. Overall, we recommend the use of immunofluorescence and flow cytometry, for an increased accuracy with both a qualitative and quantitative insight into degradation efficacy and a critical distinction between cell membrane-localized and intracellular IMP protein pools.https://doi.org/10.1038/s41598-024-84217-2PROTACTargeted protein degradationIntegral membrane proteinEGFRc-MET |
| spellingShingle | Camilla Ruffilli Sascha Röth Noam Zelcer Kevin Moreau Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET Scientific Reports PROTAC Targeted protein degradation Integral membrane protein EGFR c-MET |
| title | Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET |
| title_full | Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET |
| title_fullStr | Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET |
| title_full_unstemmed | Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET |
| title_short | Orthogonal validation of PROTAC mediated degradation of the integral membrane proteins EGFR and c-MET |
| title_sort | orthogonal validation of protac mediated degradation of the integral membrane proteins egfr and c met |
| topic | PROTAC Targeted protein degradation Integral membrane protein EGFR c-MET |
| url | https://doi.org/10.1038/s41598-024-84217-2 |
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