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Self-Assembled PROTACs Enable Glycoproteins Degradation in the Living Cells

ABSTRACT: We report here a two-component proteolysis targeting chimeras (PROTACs) strategy selectively targeting O-GalNAcylated and O-GlcNAcylated proteins for proteasomal degradation, which leads to severe toxicity in human cancer cell lines through perturbation of critical metabolic and signaling pathways governed by glycoproteins. Our approach termed as GlyTACs leverages from metabolic incorporation of easily accessible and cell-permeable peracetylated N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) analogues bearing an azido group into glycoproteins. In the living cells, the azido-modified glycoproteins serve as a covalent anchor for the introduction of thalidomide moiety by strain-promoted azide-alkyne cycloaddition (SPAAC) to recruit E3 ligase cereblon (CRBN), resulting in stepwise ubiquitination of ‘sensitized’ proteins and their degradation by proteasome. We show the efficiency of the system in a series of human cancer cell lines and verify the mechanistic pathway by performing control experiments at each stage of the process. Given the characteristic features of cancer cells including fast nutrient turnover, and overall increase of protein glycosylation, as well as the low cytotoxicity of the individual components, our approach may open a feasible strategy in cancer therapy.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Pavel Kielkowski

LAB HEAD: Pavel Kielkowski

PROVIDER: PXD059044 | Pride | 2025-04-04

REPOSITORIES: Pride

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	20240515_HC335_10_1.raw	Raw
	20240515_HC335_10_2.raw	Raw
	20240515_HC335_10_3.raw	Raw
	20240515_HC335_11_1.raw	Raw
	20240515_HC335_11_2.raw	Raw

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Project description:Immunomodulatory imide drugs (IMiDs), such as thalidomide and its analogues, are some of the most commonly utilized E3 ligase ligands for the development of proteolysis targeting chimeras (PROTACs). While the canonical neo-substrates of IMiDs (i.e., Ikaros and Aiolos) are often considered to be unwanted off-targets of PROTACs, maintaining the degradation of these neo-substrates also provides the opportunity to synergistically degrade multiple proteins with a single compound. Here, we report the development of ALV-07-082-03, a CDK4/CDK6/Helios triple degrader that consists of palbociclib, an FDA-approved CDK4/6 inhibitor, conjugated to DKY709, a novel IMiD-based Helios degrader. Pharmacological co-degradation of CDK4/6 and Helios resulted in potent suppression of downstream signaling and proliferation in cancer cells, as well as enhanced de-repression of IL-2 secretion. Thus, not only do we demonstrate the possibility of rationally re-directing the neo-substrate specificity of PROTACs by incorporating alternative molecular glue molecules as E3 ligase ligands, but our findings also suggest that co-targeting CDK4/6 and Helios may have synergistic effects.

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