Repurposing MGMT as a kill switch in chemical biology and as a novel mode of action in protein degradation

Vogel, Caspar Heinrich. Repurposing MGMT as a kill switch in chemical biology and as a novel mode of action in protein degradation. 2023, Doctoral Thesis, University of Basel, Faculty of Science.

[img] PDF
Restricted to Repository staff only until 1 January 2025.


Official URL: https://edoc.unibas.ch/95913/

Downloads: Statistics Overview


The manipulation of protein levels in vivo as well as in vitro is an important tool for studying biology and
biochemistry. Genetic techniques are excellent, but they lack precise, tunable, and reversible control.
The selective degradation of a protein with a small molecule offers the possibility to precisely silence
proteins without the drawbacks of genetic methods. Herein, we present our work about repurposing the
DNA repair protein methylguanine methyltransferase (MGMT) as a degradation hub for the selective
depletion of target proteins. MGMT is a suicide protein that removes alkylations from the O6 position
of guanine; once alkylated, MGMT is quickly degraded by the ubiquitin proteasome system. Inspired
by the mode of action, we first show that MGMT can serve as a potent degron in fusion proteins.
Furthermore, mechanistic studies revealed that MGMT can be degraded by at least two E3 ligases. We
demonstrate the functionality of the degron by showing induced degradation of two clinically relevant
targets fused to MGMT: KRASG12C and a chimeric antigen receptor.
The degron technique requires genome editing for protein silencing. To expand the pool of pos-
sible targets, we present an approach where the target protein is cross-linked to native MGMT by a
bifunctional molecule. We first show the degradation of a Halo-GFP fusion by its covalent recruit-
ment to MGMT. With proven functionality of the system, we present degradation of a stably expressed
KRASG12C-GFP fusion in HEK293T cells as a clinically relevant target. Finally, we present the degrada-
tion of native KRASG12C in MiaPaca2 cells by covalent co-localization of the target to MGMT.
Advisors:Gillingham, Dennis
Committee Members:Sparr, Christof and Hausch, Felix
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Organische Chemie (Gillingham)
05 Faculty of Science > Departement Chemie > Chemie > Organische Chemie (Sparr)
UniBasel Contributors:Gillingham, Dennis and Sparr, Christof
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:15148
Thesis status:Complete
Number of Pages:xviii, 127
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss151485
edoc DOI:
Last Modified:27 Oct 2023 04:30
Deposited On:26 Oct 2023 12:32

Repository Staff Only: item control page