Lecture 6 (Winter 2025)
Document Details
Uploaded by EliteAmbiguity1280
University of California, Irvine
2025
Prof. Trader
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Summary
This is a lecture on protein degradation processes, covering topics like proteasome and lysosome pathways, and various technologies used in drug discovery. The slides describe different types of protein degradation systems and illustrate the molecules involved, as well as techniques like fluorescence gel imaging and thermal shift assays.
Full Transcript
Lecture 6 Prof. Trader Winter 2025 https://www.nature.com/articles/s41392-022-00966-4 Proteasome Lysosome TPD Proact vs Glue Targeting to Lysosome AUTAC DOIhttps://doi.org/10.1039/D1CC04661F...
Lecture 6 Prof. Trader Winter 2025 https://www.nature.com/articles/s41392-022-00966-4 Proteasome Lysosome TPD Proact vs Glue Targeting to Lysosome AUTAC DOIhttps://doi.org/10.1039/D1CC04661F Molecule Design Degradation Quantitation Target Engagement You have a ligand or an inhibitorà It causes the response you wantà But what is it LITERALLY doing in the cell? Where to start Future Med. Chem. (2019) 11(10), 1195–1224 Anteater Pharmaceutical Discovery Anteater Pharmaceutical Discovery O -Modify this amine to a H 2N O covalent and add an enrichment tag Diazirine Benzophenone O H O O N O H Anteater Pharmaceutical Discovery O -Modify this amine to a H 2N O covalent and add an enrichment tag Alkyne Diazrine H N N F3C O O N O H Pharmacophore Cross-Linkingà Click Chemistry- Azide Cross-Linkingà Click Chemistry- Tetrazine N N F3C O O Trans-cyclooctene N O H Fluorescence Gel Imaging H N N UV Light Lysis F3C O O N O H Targets are cross-linked Cu SDS-PAGE “Click” Soluble protein Fluorescence Gel Imaging Common Fluorophores -BODIPY -TAMRA -AMC Biotin H N N Cu POI F C O O 3 N O H Streptavidin -Dimer H H N -Binds Biotin with Kd 10-14 M S O N POI H H Nearly Covalent! Mo le c N3 u le https://en.wikipedia.org/wiki/Streptavidin Enrichment CETSA Cellular Thermal Shift Assay You need to have an idea of what your target is… Molecules 2021, 26, 6083. https://doi.org/10.3390/molecules26196083 Protein Thermal Stability Use a dye, typically Sypro Orange https://biotium.com/wp-content/uploads/2018/03/Glomelt-flyer.pdf Thermal Shift But this uses purified protein… How do for target engagement?? https://biotium.com/wp-content/uploads/2018/03/Glomelt-flyer.pdf Example Do they bind RIPK1? Activity-Based Protein Profiling ABPPà Commonly used to identify covalent inhibitors of enzymes Methods Enzymol. 2014 ; 538: 151–169 Reactive Groups Fluorophosphonate Serine Fluorosulfonate Lysine ABPP Competition https://www.cravattlab.com/research In Summary For a molecule to become a viable drug you must know what it is doing on a cellular level Non-specific binding leads to off-target effects In order to be FDA approved the MOA typically needs to be well documented.
