Exploiting the CRL4-CRBM E3 ubiquitin ligase complex for the development of iTAG: a versatile targeted protein degradation system to enable functional evaluation and target validation

Abstract

Modulating target protein expression to probe for its relevance and function in a disease is critical in the drug discovery process of target validation. Current genetic methods and chemico-biology tools are widely employed for target protein modulation; however, they exhibit several limitations in their use. The work presented in this thesis describes the development of a novel protein degradation method named iTAG (Inducible and TArgeted protein deGradation) based on thalidomide and its analogues [IMiDs (Immunomodulatory imide drugs/CELMoDs (Cereblon E3 ligase modulation drugs)]. These compounds engage CRBN (cereblon), the substrate receptor of CUL4CRBN E3 ubiquitin ligase, and alter the enzyme target specificity leading to the recruitment and subsequent ubiquitination of neosubstrates. Up-to-date examples of neosubstrates include GSPT1, CK1a, Ikaros/IKZF1, Aiolos/IKZF3, and ZFP91. Mechanistically, neosubstrates interact with the IMiD/CELMoD-bound surface of CRBN through recognition motifs known as degrons. Using structural and sequence analysis, native and synthetic degron-containing domains (DCDs) (size ranging from 10 to 197 residues) were systematically explored and evaluated for their degradability when fused to a target protein. An optimal DCD (DCD23; 60 residues) was identified, that induces a rapid, potent, and selective degradation of nuclear, cytoplasmic, and mitochondrial following IMiD/CELMoD treatment. DCD23 was also demonstrated to constitute a modular and versatile tool for degrading proteins when fused to either their N or C terminus. Furthermore, acute iTAG-mediated degradation of cMYC and KRAS[G12V] was shown to enable the exploration of the downstream functions of these classical oncoproteins. In vivo, the iTAG system enables potent and reversible protein degradation in tumours, with loss of target protein by 4h following a single oral administration of the CELMoD CC-220. The iTAG system therefore constitutes a transformational modular and versatile tool to specifically degrade proteins of interest, which will enable systematic exploration of protein function across the human proteome.