For 17c a DC50 value of 14 nM was determined in MM.1S cells. Overall, our data confirms that 17c is a highly potent, selective, and non-toxic HDAC6 degrader. Thus, this promising tool compound was selected for a more comprehensive evaluation. HDAC6 degradation is dependent on the cyclic imide degron of 17c. To investigate, if the degradation of HDAC6 is mediated by the fluorothalidomide cyclic imide degron of 17c, a methylated, non-degrading control, NC-17c (Figure ), was synthesized (see Scheme S1, Supporting Information) and evaluated head-to-head with 17c. As depicted in Figure , 17c showed potent degradation of HDAC6, but not of HDAC1. NC-17c did not show any degradation of HDAC1 or HDAC6. These results confirm the necessity of the free imide group for HDAC6 degradation, which is essential for binding and recruiting CRBN. Additionally, the hyperacetylation of the HDAC6 substrate α-tubulin was determined by immunoblot analysis (Figure ). Notably, hyperacetylation of the substrate was not found in NC-17c-treated cells. NC-17c was determined to inhibit HDAC6 at 0.389 µM, which is similar to the inhibitory activity of 17c (0.295 µM, Table ). Only by degradation of HDAC6 through 17c, we found significantly hyperacetylated α-tubulin. This effect was more pronounced at 5 µM than at 0.5 µM, which is possibly due to a combination of HDAC6 degradation and inhibition of residual protein. HDAC6 degradation is mediated by the ubiquitin-proteasome system. Proteasomal rescue experiments were performed to confirm that HDAC6 degradation by 17c is mediated via the ubiquitinproteasome pathway. For this purpose, MM1.S cells were pretreated for 30 minutes with 10 M of the binding competitors tubastatin A, pomalidomide or the NEDD8-activating enzyme inhibitor MLN4924 before 17c was added. As illustrated in Figure , HDAC6 levels were almost completely rescued when the cells were pretreated with these compounds. The rescue experiments confirmed that 17c induced HDAC6 degradation is neddylation dependent and requires the interaction with both CRBN and HDAC6. Quantitive chemoproteomic investigation. To investigate the effects of 17c on the global proteome, a chemoproteomic investigation by quantitative diaPASEF-based mass spectrometry in MM.1S cells was conducted. In total, 5738 proteins were identified and among them, seven Zn 2+ -dependent HDAC isoforms were found. The volcano plot in Figure shows that HDAC6 was significantly reduced in 17c-treated cells compared to vehicle-treated cells. Additionally, degradation of IKZF3, a CRBN neosubstrate, as well as collateral degradation of MIER1, a part of an HDAC1/2 multienzyme complex, was observed upon 17c treatment. The degradation of MIER1 has also been observed in previous chemoproteomic investigations of a HDAC-focused degrader library. The degradation of other HDAC isoforms was not observed (Figure ). Thus, the HDAC6-selective degradation over other HDAC isoforms, as shown by immunoblotting, could be confirmed. Ternary complex modeling. To investigate the binding mode of the most active degrader 17c, we conducted docking experiments. To this end, we used a CRBN crystal structure (PDB ID: 4CI1) and a human HDAC6 crystal structure (PDB ID: 5EDU), and performed ternary complex modeling (Figure , Supporting Information). The predicted binding mode of the fluorothalidomide moiety closely resembles that of thalidomide in CRBN. As expected, the two phenylalanines (F620 and F680) in the catalytic tunnel of HDAC6 sandwiched the benzyl linker of compound 17c. Tyrosine Y782 was predicted to donate an H-bond to the carbonyl oxygen of the ethyl hydrazide, while the ethylamino function coordinates the Zn 2+ ion. To compare the two groups of degraders we also investigated the ternary binding mode of 18c with HDAC6 (PDB ID: 5EDU) and VHL (PDB ID: 6GFZ). The docking results confirmed our experimental findings, as 17c exhibited significantly higher docking scores than 18c.