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Design and Synthesis of Bifunctional Compounds for Targeted Protein Degradation and Phosphorylation

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Universitat de Barcelona. Facultat de Química

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[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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[spa] En esta tesis doctoral se describe el diseño y la síntesis de moléculas bifuncionales que inducen la degradación de una proteína de interés en células. También se ha investigado la síntesis de moléculas capaces de inducir la fosforilación de una proteína.
Los degradadores bifuncionales, o PROTACs (Proteolysis Targeting Chimaera) son moléculas compuestas por dos ligandos, uno con afinidad por la proteína de interés y el otro por una E3 ligasa, unidos por un linker. Las E3 ligasas son proteínas responsables de transferir ubiquitina, un marcador celular que induce la degradación de las proteínas marcadas.
Se han diseñado y sintetizado compuestos capaces de degradar el receptor de estrógeno (ER). Los compuestos se han sintetizado basándose en un ligando derivatizado de 4-hydroxitamoxifeno, un fármaco con afinidad por ER. Combinando este ligando con otros ligandos para las E3 ligasas CRBN y VHL, y conectores de distinta longitud y composición química, se obtuvieron varios compuestos.  De los 9 compuestos sintetizados, TAM-VHL-1 y TAM-VHL-2 son los degradadores de ER más potentes (DC50 4.5 nM and 5.3 nM respectivamente). Los compuestos han sido usados como herramientas de química biológica, para estudiar la biología de ER.
Se ha optimizado un degradador de p38 previamente desarrollado en nuestro laboratorio, NR-7h, para tal de aumentar su solubilidad en medio acuoso y poder así ser administrado intravenosamente.  Para encontrar compuestos que no actuaran selectivamente en el hígado, se diseñaron análogos de NR-11c con diferente composición en el linker. De los 16 compuestos preparados, NR-13c y NR-15 tienen una potencia similar a NR-11c. Los compuestos se estudiarán in vivo, para determinar cuál es su distribución en el organismo.

# metadata.dc.title[].value
Design and Synthesis of Bifunctional Compounds for Targeted Protein Degradation and Phosphorylation