A novel class of USP7 PROTACs were designed and synthesized. CST967, a CRBN-based degrader, showed potent and selective depletion of USP7 leading to apoptosis in multiple cancer lines.

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.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC₅₀ value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science.

Administration of monoclonal antibodies (mAbs) is currently focused on subcutaneous injection associated with increased patient adherence and reduced treatment cost, leading to sustainable healthcare. The main bottleneck is low volume that can be injected, requiring highly concentrated mAb solutions. The latter results in increased solution viscosity with pronounced mAb aggregation propensity because of intensive protein-protein interactions. Small molecule excipients have been proposed to restrict the protein-protein interactions, contributing to reduced viscosity. The aim of the study was to discover novel compounds that reduce the viscosity of highly concentrated mAb solution. First, the chemical space of proline analogs was explored and 35 compounds were determined. Viscosity measurements revealed that 18 proline analogs reduced the mAb solution viscosity similar to or more than proline. The compounds forming both electrostatic and hydrophobic interactions with mAb reduced the viscosity of the formulation more efficiently without detrimentally effecting mAb physical stability. A correlation between the level of interaction and viscosity-reducing effect was confirmed with molecular dynamic simulations. Structure rigidity of the compounds and aromaticity contributed to their viscosity-reducing effect, dependent on molecule size. The study results highlight the novel proline analogs as an effective approach in viscosity reduction in development of biopharmaceuticals for subcutaneous administration.