Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System

Sascha Roth, Thomas Macartney, Agnieszka Konopacka, Kwok-Ho Chan, Houjiang Zhou, Markus A. Queisser, Gopal Sapkota (Lead / Corresponding author)

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Abstract

K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called “AdPROM.” Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549GFPKRAS) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549GFPKRAS led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.
Original languageEnglish
Number of pages13
JournalCell Chemical Biology
Early online date14 Jul 2020
DOIs
Publication statusE-pub ahead of print - 14 Jul 2020

Keywords

  • cancer targets
  • monobody
  • nanobody
  • oncogene
  • PROTAC
  • protein degradation
  • RAS
  • RAS/MAPK signaling
  • targeted proteolysis
  • ubiquitin proteasome system

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