Defining proteoform-specific interactions for drug targeting in a native cell signalling environment
Understanding the dynamics of membrane protein–ligand interactions within a native lipid bilayer is a major goal for drug discovery. Typically, cell-based assays are used, however, they are often blind to the effects of protein modifications. In this study, using the archetypal G protein-coupled rec...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Nature Research
2025
|
| _version_ | 1824459187794477056 |
|---|---|
| author | Lutomski, CA Bennett, JL El-Baba, TJ Wu, D Hinkle, JD Burnap, SA Liko, I Mullen, C Syka, JEP Struwe, WB Robinson, CV |
| author_facet | Lutomski, CA Bennett, JL El-Baba, TJ Wu, D Hinkle, JD Burnap, SA Liko, I Mullen, C Syka, JEP Struwe, WB Robinson, CV |
| author_sort | Lutomski, CA |
| collection | OXFORD |
| description | Understanding the dynamics of membrane protein–ligand interactions within a native lipid bilayer is a major goal for drug discovery. Typically, cell-based assays are used, however, they are often blind to the effects of protein modifications. In this study, using the archetypal G protein-coupled receptor rhodopsin, we found that the receptor and its effectors can be released directly from retina rod disc membranes using infrared irradiation in a mass spectrometer. Subsequent isolation and dissociation by infrared multiphoton dissociation enabled the sequencing of individual retina proteoforms. Specifically, we categorized distinct proteoforms of rhodopsin, localized labile palmitoylations, discovered a Gβγ proteoform that abolishes membrane association and defined lipid modifications on G proteins that influence their assembly. Given reports of undesirable side-effects involving vision, we characterized the off-target drug binding of two phosphodiesterase 5 inhibitors, vardenafil and sildenafil, to the retina rod phosphodiesterase 6 (PDE6). The results demonstrate differential off-target reactivity with PDE6 and an interaction preference for lipidated proteoforms of G proteins. In summary, this study highlights the opportunities for probing proteoform–ligand interactions within natural membrane environments. |
| first_indexed | 2025-02-19T04:37:48Z |
| format | Journal article |
| id | oxford-uuid:57d3bd35-a43b-46d8-b833-389a3932520f |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:37:48Z |
| publishDate | 2025 |
| publisher | Nature Research |
| record_format | dspace |
| spelling | oxford-uuid:57d3bd35-a43b-46d8-b833-389a3932520f2025-02-05T20:13:07ZDefining proteoform-specific interactions for drug targeting in a native cell signalling environmentJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:57d3bd35-a43b-46d8-b833-389a3932520fEnglishJisc Publications RouterNature Research2025Lutomski, CABennett, JLEl-Baba, TJWu, DHinkle, JDBurnap, SALiko, IMullen, CSyka, JEPStruwe, WBRobinson, CVUnderstanding the dynamics of membrane protein–ligand interactions within a native lipid bilayer is a major goal for drug discovery. Typically, cell-based assays are used, however, they are often blind to the effects of protein modifications. In this study, using the archetypal G protein-coupled receptor rhodopsin, we found that the receptor and its effectors can be released directly from retina rod disc membranes using infrared irradiation in a mass spectrometer. Subsequent isolation and dissociation by infrared multiphoton dissociation enabled the sequencing of individual retina proteoforms. Specifically, we categorized distinct proteoforms of rhodopsin, localized labile palmitoylations, discovered a Gβγ proteoform that abolishes membrane association and defined lipid modifications on G proteins that influence their assembly. Given reports of undesirable side-effects involving vision, we characterized the off-target drug binding of two phosphodiesterase 5 inhibitors, vardenafil and sildenafil, to the retina rod phosphodiesterase 6 (PDE6). The results demonstrate differential off-target reactivity with PDE6 and an interaction preference for lipidated proteoforms of G proteins. In summary, this study highlights the opportunities for probing proteoform–ligand interactions within natural membrane environments. |
| spellingShingle | Lutomski, CA Bennett, JL El-Baba, TJ Wu, D Hinkle, JD Burnap, SA Liko, I Mullen, C Syka, JEP Struwe, WB Robinson, CV Defining proteoform-specific interactions for drug targeting in a native cell signalling environment |
| title | Defining proteoform-specific interactions for drug targeting in a native cell signalling environment |
| title_full | Defining proteoform-specific interactions for drug targeting in a native cell signalling environment |
| title_fullStr | Defining proteoform-specific interactions for drug targeting in a native cell signalling environment |
| title_full_unstemmed | Defining proteoform-specific interactions for drug targeting in a native cell signalling environment |
| title_short | Defining proteoform-specific interactions for drug targeting in a native cell signalling environment |
| title_sort | defining proteoform specific interactions for drug targeting in a native cell signalling environment |
| work_keys_str_mv | AT lutomskica definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT bennettjl definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT elbabatj definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT wud definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT hinklejd definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT burnapsa definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT likoi definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT mullenc definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT sykajep definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT struwewb definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment AT robinsoncv definingproteoformspecificinteractionsfordrugtargetinginanativecellsignallingenvironment |