Browsing by Author "Brownsey, Duncan Keith"

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    Exploration of Pomalidomide for Heterobifunctional Protein Degraders: From Chemistry to Application
    (2023-04-04) Brownsey, Duncan Keith; Derksen, Darren J.; Van Humbeck, Jeffrey F.; Back, Thomas G.; Ling, Chang-Chun; Williams, Florence J.
    Targeted protein degradation (TPD) has recently come to the forefront of chemical biology and medicinal chemistry as a means to disrupt protein function by selective degradation. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that exemplify TPD technology. One of the most widely used E3 ligase ligands in PROTAC development is pomalidomide which make up 46% of all CRBN-targeting PROTACs. This work explores the chemistry of pomalidomide: first by improving the synthesis of pomalidomide derivatives, second by application in new protein degraders of p300, and then third by characterizing the fluorescent properties of pomalidomide. The improved synthesis of pomalidomide derivatives via an SNAr pathway was began by identification and avoidance of the formation of dimethyl pomalidomide, an undesired byproduct. This led to greatly enhanced yields of compounds that can be used to build new pomalidomide containing PROTACs. As well, differences in the reactivity of amines used to create pomalidomide derivatives were exploited to produce PROTACs in one-pot reactions, which in some instances, exceeded the yields of multistep syntheses used in the literature. Secondly, new degraders of the protein target p300 were prepared using a known p300 ligand A-485, outlining a new strategy to create PROTACs. To verify that linker attachment onto A-485 was amenable to PROTAC function, biotin conjugates were installed and verified using immunoprecipitation blots. A library of A-485-pomalidomide conjugates were then prepared and tested for their p300 degradation capability, which showed a general trend that the PROTACs with longer linkers were more effective degraders. Finally, the fluorescent properties of pomalidomide derivatives were explored. In aqueous solutions, pomalidomide emits visible light with a maximum at ~530 nm upon excitation at 420 nm. This emission was characterized and a library of pomalidomide PROTACs were synthesized and tested in vitro with osteosarcoma cells. In vitro fluorescence microscopy imaging of the pomalidomide library showed trends with linker structure. Relationships between fluorescence, target degradation and calculated partition coefficients were then explored.