Development of Proteolysis Targeting Chimeras Prodrugs Targeting Cancer and Senescence
Publisher
The University of Arizona.Rights
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.Abstract
Proteolysis targeting chimeras (PROTACs) is an emerging therapeutic modality that recruits the cell’s ubiquitin-proteasome system to degrade the target protein by bringing the protein and an E3 ligase together. The technology has been demonstrated in the treatment of various disorders, including virus, immunity defense, and neurological disease. Although PROTACs have become promising therapeutic modalities, important concerns exist about the potential toxicity of the approach owning to uncontrolled degradation of proteins and undesirable ligase-mediated off-target effects. Precision manipulation of degradation activity of PROTACs could minimize potential toxicity and side effects. Toward this end, my research focuses on developing PROTAC prodrugs to control their activities.In the first effort, we developed a novel dual-functional MDM2 E3 ligase-based Bcl-XL PROTAC BMM4. Inhibition or degradation of anti-apoptotic protein BCL-XL is a viable strategy for cancer treatment. Despite the recent development of PROTACs for degradation of BCL-XL, the E3 ligases are confined to the commonly used VHL and CRBN. We achieved the development of MDM2-BCL-XL PROTACs using MDM2 as E3 ligase for degradation of BCL-XL. We found BMM4 exhibited potent, selective degradation activity against BCL-XL and stabilized tumor suppressor p53 in U87, A549 and MV-4-11 cancer cell lines. Moreover, the combination of BMM4 and BCL-2 inhibitor ABT-199 showed synergistic antiproliferative activity. The unique dual-functional PROTACs offers an alternative strategy for targeted protein degradation. In the second study, we developed a bioorthogonal on-demand prodrug strategy (termed click-release “crPROTACs”) that enables on-target activation of PROTAC prodrugs and release of PROTACs in cancer cells selectively. Inactive PROTAC prodrugs TCO-ARV-771 and TCO-DT2216 are rationally designed by conjugating a bioorthogonal trans-cyclooctenes (TCO) group into the ligand of the VHL E3 ubiquitin ligase. The tetrazine (Tz) modified RGD peptide, c(RGDyK)-Tz, which targets integrin αvβ3 biomarker in cancer cells, serves as the activation component for click-release of the PROTAC prodrugs to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. The results of studies assessing the viability of this strategy show that the PROTAC prodrugs are selectively activated in an integrin αvβ3-dependent manner to produce PROTACs, which degrade POIs in cancer cells. The crPROTAC strategy might be a general, abiotic approach to induce selective cancer cell death through the ubiquitin-proteasome pathway. In the third effort, we achieved a senolytic strategy enabled by senescent cell-sensitive bioorthogonal tetrazine ligation. Our design is engineering a galactose (Gal) moiety into dihydrotetrazine, which serves both as recognition moiety for senescence-associated β-galactosidase (SA-β-gal) and a caging group for control of tetrazine activity. We demonstrate that Gal-Tz enables efficient click-release of fluorescent HCA and doxorubicin (Dox) from TCO caged HCA and Dox prodrug to detect and eliminate senescent HeLa and A549 cells over non-senescent counterparts with 16.44 senolytic index. Furthermore, we leverage the strategy for selective activation and delivery of proteolysis-targeting chimeras (PROTACs) as senolytics. PROTAC prodrug TCO-ARV-771 can be selectively activated by Gal-Tz and delivered into senescent HeLa and A549 cells to induce BRD4 degradation. The proof of concept of PROTACs as senolytics may offer an efficient way for intervention of cell senescence due to their unique capacity capable of degrading targeted proteins in a sub-stoichiometric and catalytic fashion. The results of the study establish the bioorthogonal tetrazine ligation approach as a viable strategy for selective removal of senescent cells. Finally, we devised a new strategy for selective degradation of target proteins in senescent cancer cells to eliminate senescent cells. Prodrugs Gal-ARV-771 and Gal-MS99 comprised of the SA-β-gal substrate galactose (galacto) and PROTAC molecules as senolytic agents are designed. The prodrugs were found to display higher senolytic indexes than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to significant inhibition of tumor growth without eliciting significant toxicity.Type
textElectronic Dissertation
Degree Name
Ph.D.Degree Level
doctoralDegree Program
Graduate CollegeBiochemistry