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Identifying the biological targets of a small molecule plays an important role in drug discovery and chemical biology. In the first study, we developed a new target identification strategy that takes advantage of Proteolysis targeting chimeras (PROTACS) which could degrade specific proteins in live cells. This strategy can be applied to intact cells directly and the degraded proteins can be identified from a complex mixture using state-of-the-art mass spectrometry techniques.
\nWe applied this method to oxysterols, which are responsible for regulating cholesterol homeostasis, but whose new functions continue to be discovered. As a proof-of-concept, we generated four sterol-bearing PROTACs (C1-C4) based on pomalidomide, a ligand of the E3 ligase cereblon, and their degradation profiles were determined by TMT-based proteomics. Golgi Integral Membrane Protein 4 (GOLIM4), a known target of cholesterol and a significantly degraded protein by C1-C4 was chosen for further target validation by different in vitro assays.
\nSurprisingly, although C3 led to GOLIM4 degradation in a dose- and time-dependent manner, this degradation was not mediated by the ubiquitin–proteasome system (UPS). Furthermore the post-translational modifications (PTMs) of GOLIM4 were also significantly affected. Target engagement assays confirmed that C3 could not bind to GOLIM4 directly and C3 and further derivatives thereof also led to the degradation of several other Golgi-resident proteins and glycosyltransferases. Finally, a targeted screening campaign employing fluorescence polarization (FP) assays proved C3 and its derivatives can bind to oxysterol binding protein (OSBP), a sterol transport proteins, with potencies in the low nanomolar range.
\nThe second study focused on design, synthesis and biological evaluation of Aster-A PROTACS. We chose Autogramin-2, an established Aster-A selective inhibitor and different E3 ligands and linkers to obtain and optimize Aster-A PROTACS.
\nPrincipal Supervisor:
\nAssociate Professor Luca Laraia, DTU Chemistry
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Co-supervisor:
\nProfessor Mads Clausen, DTU Chemistry
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Examiners:
\nAssociate Professor Erwin Schoof, DTU Bioengineering
\nGroup Leader, Professor Georg Winter, Center for Molecular Medicine, Vienna
\nProfessor Christian Adam Olsen, University of Copenhagen
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\nChairperson:
Associate Professor Sebastian Meier, DTU Chemistry
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Identifying the biological targets of a small molecule plays an important role in drug discovery and chemical biology. In the first study, we developed a new target identification strategy that takes advantage of Proteolysis targeting chimeras (PROTACS) which could degrade specific proteins in live cells. This strategy can be applied to intact cells directly and the degraded proteins can be identified from a complex mixture using state-of-the-art mass spectrometry techniques.
\nWe applied this method to oxysterols, which are responsible for regulating cholesterol homeostasis, but whose new functions continue to be discovered. As a proof-of-concept, we generated four sterol-bearing PROTACs (C1-C4) based on pomalidomide, a ligand of the E3 ligase cereblon, and their degradation profiles were determined by TMT-based proteomics. Golgi Integral Membrane Protein 4 (GOLIM4), a known target of cholesterol and a significantly degraded protein by C1-C4 was chosen for further target validation by different in vitro assays.
\nSurprisingly, although C3 led to GOLIM4 degradation in a dose- and time-dependent manner, this degradation was not mediated by the ubiquitin–proteasome system (UPS). Furthermore the post-translational modifications (PTMs) of GOLIM4 were also significantly affected. Target engagement assays confirmed that C3 could not bind to GOLIM4 directly and C3 and further derivatives thereof also led to the degradation of several other Golgi-resident proteins and glycosyltransferases. Finally, a targeted screening campaign employing fluorescence polarization (FP) assays proved C3 and its derivatives can bind to oxysterol binding protein (OSBP), a sterol transport proteins, with potencies in the low nanomolar range.
\nThe second study focused on design, synthesis and biological evaluation of Aster-A PROTACS. We chose Autogramin-2, an established Aster-A selective inhibitor and different E3 ligands and linkers to obtain and optimize Aster-A PROTACS.
\nPrincipal Supervisor:
\nAssociate Professor Luca Laraia, DTU Chemistry
\n\n
Co-supervisor:
\nProfessor Mads Clausen, DTU Chemistry
\n\n
Examiners:
\nAssociate Professor Erwin Schoof, DTU Bioengineering
\nGroup Leader, Professor Georg Winter, Center for Molecular Medicine, Vienna
\nProfessor Christian Adam Olsen, University of Copenhagen
\n
\nChairperson:
Associate Professor Sebastian Meier, DTU Chemistry
URL:https://www.dtu.dk/da/sitecore/content/Institutter/Kemi/DTU_Kemi_OLD/Forside/Arrangementer/2023/02/PhD-Defence-Nianzhe-He DTSTAMP:20260601T065000Z UID:{848F9FB4-C574-4C8A-9732-3B140F690529}-20230208T080000Z-20230208T080000Z LOCATION: Technical University of Denmark, Building 303A, Aud. 49 END:VEVENT END:VCALENDAR