Design and Synthesis of Bifunctional Compounds for Targeted Protein Degradation and Phosphorylation
[eng] In this doctoral thesis, the design and synthesis of bifunctional molecules that induce the degradation of a target protein in cells are described. The synthesis of molecules capable of inducing the phosphorylation of a protein has also been investigated. Bifunctional degraders, or Proteolysis...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Recursos: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/202520 |
| Acesso em linha: | https://hdl.handle.net/2445/202520 http://hdl.handle.net/10803/689084 |
| Access Level: | acceso abierto |
| Palavra-chave: | Ciències de la salut Biomolècules Proteïnes Receptors cel·lulars Estrògens Medical sciences Biomolecules Proteins Cell receptors Estrogen |
| Resumo: | [eng] In this doctoral thesis, the design and synthesis of bifunctional molecules that induce the degradation of a target protein in cells are described. The synthesis of molecules capable of inducing the phosphorylation of a protein has also been investigated. Bifunctional degraders, or Proteolysis Targeting Chimeras (PROTACs), are molecules composed of two ligands: one with affinity for the target protein and the other for an E3 ligase, linked by a linker. E3 ligases are proteins responsible for transferring ubiquitin, a cellular marker that induces the degradation of marked proteins. Compounds capable of degrading the estrogen receptor (ER) have been designed and synthesized. These compounds were synthesized based on a derivative ligand of 4-hydroxytamoxifen, a drug with affinity for ER. By combining this ligand with other ligands for E3 ligases CRBN and VHL, along with connectors of different lengths and chemical compositions, several compounds were obtained. Among the 9 synthesized compounds, TAM-VHL-1 and TAM-VHL-2 are the most potent ER degraders (DC50 4.5 nM and 5.3 nM, respectively). These compounds have been used as tools in chemical biology to study the biology of ER. A previously developed p38 degrader in our laboratory, NR-7h, has been optimized to increase its solubility in aqueous medium for intravenous administration. NR-11c is an analog of NR-7h that recruits the VHL E3 ligase instead of CRBN, with comparable potency and higher solubility in aqueous medium. Administration of NR-11c in mice results in exclusive degradation of p38 in the liver, suggesting tissue retention of the compound. To find compounds that do not act selectively in the liver, analogs of NR-11c with different linker compositions were designed. Out of the 16 prepared compounds, NR-13c and NR-15 have similar potency to NR-11c. These compounds will be studied in vivo to determine their distribution in the organism. Phosphorylation Inducing Chimeras (PHICS), bifunctional compounds that recruit a kinase instead of an E3 ligase and induce phosphorylation of the target protein, have been designed and synthesized. A warhead based on a protein kinase C (PCK) activator has been synthesized, and bifunctional molecules with different linker lengths and a warhead with affinity for a tag have been constructed. Two tags have been selected: dTAG, for which our laboratory had the synthesized ligand, and aTAG, the ligand that was synthesized and used to prepare potential PHICS. The ligands of aTAG and dTAG have affinity for a protein fragment that can be expressed fused to the target protein to be studied. Thus, an ERF construct with dTAG or aTAG was overexpressed in cells, and these cells were treated with the potential PHICS. The phosphorylation of the tag-ERF construct was measured, indicating that compounds based on dTAG increased the phosphorylation of the construct to some extent, while compounds based on aTAG showed greater induction of phosphorylation by two out of the four compounds. |
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