\"Undruggable\" targets such as KRAS are particularly challenging in the development of drugs. We devised a novel chemical knockdown strategy, CANDDY (Chemical knockdown with Affinity aNd Degradation DYnamics) technology, which promotes protein degradation using small molecules (CANDDY molecules) that are conjugated to a degradation tag (CANDDY tag) modified from proteasome inhibitors. We demonstrated that CANDDY tags allowed for direct proteasomal target degradation independent of ubiquitination. We synthesized a KRAS-degrading CANDDY molecule, TUS-007, which induced degradation in KRAS mutants (G12D and G12V) and wild-type KRAS. We confirmed the tumor suppression effect of TUS-007 in subcutaneous xenograft models of human colon cells (KRAS G12V) with intraperitoneal administrations and in orthotopic xenograft models of human pancreatic cells (KRAS G12D) with oral administrations. Thus, CANDDY technology has the potential to therapeutically target previously undruggable proteins, providing a simpler and more practical drug targeting approach and avoiding the difficulties in matchmaking between the E3 enzyme and the target.

Glycogen synthase kinase 3β (GSK-3β) is a serine/threonine kinase and an attractive therapeutic target for Alzheimer\'s disease. Based on proteolysis-targeting chimera (PROTAC) technology, a small set of novel GSK-3β degraders was designed and synthesized by linking two different GSK-3β inhibitors, SB-216763 and tideglusib, to pomalidomide, as E3 recruiting element, through linkers of different lengths. Compound 1 emerged as the most effective PROTAC being nontoxic up to 20 μM to neuronal cells and already able to degrade GSK-3β starting from 0.5 μM in a dose-dependent manner. PROTAC 1 significantly reduced the neurotoxicity induced by Aβ25-35 peptide and CuSO4 in SH-SY5Y cells in a dose-dependent manner. Based on its encouraging features, PROTAC 1 may serve as a starting point to develop new GSK-3β degraders as potential therapeutic agents.

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules that trigger the poly-ubiquitination of the protein of interest (POI) inducing its degradation via the recruitment of the ubiquitin-proteasome system, thus suppressing the POI\'s intracellular levels and indirectly all its functions. Recently, one of the fields where the protein knockdown induced by PROTACs has demonstrated to serve as a promising biochemical tool and to provide new opportunities for drug discovery is the epigenetics (epi-PROTACs). A full inhibition of the functions of all domains of a specific epigenetic POI (e-POI), rather than just the block of its catalytic/single domain activity, is in fact a new more effective modality to hit an e-POI and, in principle, the complex it belongs to, and potentially to treat the related diseases, first cancer. In this review, we will present the most relevant progresses made, especially in the last two years, in the application of PROTACs technology to the three main classes of e-POIs: \"writers\", \"erasers\" and \"readers\". Emphasis will be devoted to the medicinal chemistry aspects of the epi-PROTACs design, preparation, and optimization and to the comparison with small molecule epi-drugs for both epi-targets functional annotation and potential anticancer therapy purposes.

DNA methylation is a critical epigenetic modification that is established and maintained across the genome by DNA methyltransferase enzymes (Dnmts). Altered patterns of DNA methylation are a frequent occurrence in many tumor genomes, and inhibitors of Dnmts have become important epigenetic drugs. Azacitidine is a cytidine analog that is incorporated into DNA and induces the specific inhibition and proteasomal-mediated degradation of Dnmts. The downstream effects of azacitidine on CpG methylation and on gene transcription have been widely studied in many systems, but how azacitidine impacts the proteome is not well-understood. In addition, with its specific ability to induce the rapid degradation of Dnmts (in particular, the primary maintenance DNA methyltransferase, Dnmt1), it may be employed as a specific chemical knockdown for investigating the Dnmt1-associated functional or physical interactome. In this study, we use quantitative proteomics to analyze the degradation profile of proteins in the nuclear proteome of cells treated with azacitidine. We identify specific proteins as well as multiple pathways and processes that are impacted by azacitidine. The Dnmt1 interaction partner, Uhrf1, exhibits significant azacitidine-induced degradation, and this azacitidine-induced degradation is independent of the levels of Dnmt1 protein. We identify multiple other chromatin- and epigenetic-associated factors, including the bromodomain-containing transcriptional regulator, Brd2. We show that azacitidine induces highly specific perturbations of the Dnmt1-associated proteome, and while interaction partners such as Uhrf1 are sensitive to azacitidine, others such as the Dnmt1 interaction partner and stability regulator, Usp7, are not. In summary, we have conducted the first comprehensive proteomic analysis of the azacitidine-sensitive nuclear proteome, and we show how 5-azacitidine can be used as a specific probe to explore Dnmt- and chromatin-related protein networks.

Protein homeostasis networks are highly regulated systems responsible for maintaining the health and productivity of cells. Whereas therapeutics have been developed to disrupt protein homeostasis, more recently identified techniques have been used to repurpose homeostatic networks to effect degradation of disease-relevant proteins. Here, we review recent advances in the use of small molecules to degrade proteins in a selective manner. First, we highlight all-small-molecule techniques with direct clinical application. Second, we describe techniques that may find broader acceptance in the biomedical research community that require little or no synthetic chemistry. In addition to serving as innovative research tools, these new approaches to control intracellular protein levels offer the potential to develop novel therapeutics targeting proteins that are not currently pharmaceutically vulnerable.