title: Biological Assessment and Tests of Activity of Two Novel Proteolysis Targeting Chimeras (PROTACs)
creator: Gama Brambila, Rodrigo Antonio
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-570
subject: 570 Life sciences
description: In the last 20 years the Proteolysis Targeting Chimera (PROTAC) technology has revolutionized the field of drug discovery. PORTACs are heterobifunctional molecules consisting of a chemical ligand, named in this context warhead, and an E3 ligase recruiter united by a linker. These molecules bring the Ubiquitin Proteasome System machinery into the proximity of a protein of interest through for temporal and reversible degradation without the need of genetic manipulation. In comparison to classical chemical inhibitors, PROTACs provide a new approach to repress the activity of a pathogenic protein, which is independent of its catalytic or enzymatic activity. Because of lack of chemical binders, just a small fraction of the  proteome has been reported to be targeted by PROTACs.    To increase the number of proteins prone to be targeted by PROTACs, two strategies are followed in this work, exemplified by two new PROTACs. First, the incorporation of a perfluoroaromatic moiety within the structure of the degrader, which can target the sulfhydryl motif in the cysteine residue of the sequence Phe-Cys-Pro-Phe in a nucleophilic aromatic substitution reaction. This tetrapeptide was inserted into the C-terminus of Cas9 (Cas9FCPF), while the perfluoroaromatic moiety was tether to Lenalidomide, a broadly used ligand and recruiter of the E3 ligase cereblon. The resulting PROTAC-FCPF successfully degraded Cas9FCPF in a time- and concentration-dependent manner, with a Ubiquitin Proteasome System (UPS)-driven proved mechanism with a half-degradation concentration (DC50) of around 150 nM over 24 hours treatment. PROTAC-FCPF was also effective against genetically engineered related proteins dCas9FCPF, Cas12FCPF, and Cas13FCPF, all modified to have the Phe-Cys-Pro-Phe sequence inserted in their C-termini.  The second strategy was High-Throughput Screening, out of which the compound O4I2 was identified to chemically induce the expression of the Yamanaka factor Oct4 and, thus, supported the induction of somatic cell reprogramming and maintenance of human induced Pluripotent Stem cells. In a proteomics analysis of cells treated with O4I2, it was observed that the Splicing Factor 3 Subunit 1 (SF3B1) was the main hit, a protein that has been associated to several tumors. To target it, O4I2 was bound to another cereblon recruiter, Thalidomide. The resulting PROTAC-O4I2 successfully degraded SF3B1 in a time- and concentration-dependent manner, driven by the UPS with a DC50 of 244 nM over 24 hours of treatment. Furthermore, the degrader spared other proteins that were also hits during the proteomics analysis, and successfully degraded the mutant SF3B1K700E, present in 55% of patients with myelodysplastic syndromes.
date: 2022
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/31644/1/Rodrigo%20Gama%20Brambila%20doctoral%20thesis.pdf
identifier: DOI:10.11588/heidok.00031644
identifier: urn:nbn:de:bsz:16-heidok-316445
identifier:   Gama Brambila, Rodrigo Antonio  (2022) Biological Assessment and Tests of Activity of Two Novel Proteolysis Targeting Chimeras (PROTACs).  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/31644/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng