Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.


Table of Content
Compounds with blue circles were not considered in the linear regression.

2-Acetamidothiophene-3-carboxylic acid (56).
Compound 55 (1.20 g, 6.0 mmol) was dissolved in MeOH (20 mL), and KOH (0.60 g, 10.8 mmol) in H2O (10 mL) was added.The mixture was stirred at 50 °C for 16 h.After cooling, it was diluted with H2O (100 mL), and the aqueous layer was washed with EtOAc (100 mL).Subsequently, the aqueous solution was acidified with 2N HCl until pH = 2, and it was extracted with EtOAc (2 × 50 mL).The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated  imputed from a normal distribution with a downshift (-1.8 SD from the mean and the distribution width is 0.3 SD).Comparative analysis of experimental groups was conducted using a two-sided moderated two-sample t-test.The resulting p-values were corrected using the Benjamini-Hochberg method.The data analysis was performed using R (4.3.1).

Figure S3 .
Figure S3.Correlation between physiochemical properties of 8c, 8d and 11 (for structures, see also Figure S2).(A) Logarithm of the solubility measured in mol/L at pH 6.8 by an HPLC-based method.(B) Chromatographic hydrophobicity index values referring to IAM chromatography (CHIIAM values), an estimate for drug-membrane interactions and permeability.(C) Plasma protein binding, experimentally determined percentage of compound bound to human serum albumin.Compounds with blue circles were not considered in the linear regression.(D) Inhibitory concentration referring to a competitive MST experiment using the hTBD and our previously described reporter molecule. 1

Figure S4 .
Figure S4.Neosubstrate modulation by benzamides 11 and established IMiDs.(A) MM.1S cells were treated with 0.1 µM of each compound for 16 h before lysis and blotting for IKZF3, GSPT1, and CK1α.(B) HuH6 cells were treated with 0.1 µM of compound for 16 h before lysis and blotting for SALL4.

Figure S9 .
Figure S9.Proteins regulated by PROTAC 44h which includes 44h targets and downstream effects.Most regulated proteins are connected to MYC as indicated in the network graph obtained from STRING database searches.

Table S2 .
X-ray data collection and processing.Values in parentheses correspond to the outer resolution shell.

Table S3 .
X-ray structure solution and refinement.Values in parentheses correspond to the outer resolution shell.