Revisiting the antiangiogenic mechanisms of fluorinated thalidomide derivatives.

Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. We investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1α, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics.

A titrant for the SARS-CoV-2 main protease (Mpro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active Mpro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of Mpro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the Mpro-catalyzed cleavage of these substrates were determined.

Correction to "Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity".

[This corrects the article DOI: 10.1021/acsptsci.3c00313.].

Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity.

Cathepsins (Cats) are proteases that mediate the successful entry of SARS-CoV-2 into host cells. We designed and synthesized a tailored series of 21 peptidomimetics and evaluated their inhibitory activity against human cathepsins L, B, and S. Structural diversity was realized by combinations of different C-terminal warhead functions and N-terminal capping groups, while a central Leu-Phe fragment was maintained. Several compounds were identified as promising cathepsin L and S inhibitors with Ki values in the low nanomolar to subnanomolar range, for example, the peptide aldehydes 9a and 9b (9a, 2.67 nM, CatL; 0.455 nM, CatS; 9b, 1.76 nM, CatL; 0.512 nM, CatS). The compounds' inhibitory activity against the main protease of SARS-CoV-2 (Mpro) was additionally investigated. Based on the results at CatL, CatS, and Mpro, selected inhibitors were subjected to investigations of their antiviral activity in cell-based assays. In particular, the peptide nitrile 11e exhibited promising antiviral activity with an EC50 value of 38.4 nM in Calu-3 cells without showing cytotoxicity. High metabolic stability and favorable pharmacokinetic properties make 11e suitable for further preclinical development.

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.

Fluorogenic substrates and pre-column derivatization for monitoring the activity of bile salt hydrolase from Clostridium perfringens.

The bile acid pool has a profound impact on human health and disease. The intestinal microbiota initiates the metabolism of conjugated bile acids through a critical first step catalyzed by bacterial bile salt hydrolase (BSH) and provides unique contributions to the diversity of bile acids. There has been great interest in surveying BSH activity. We compared two substrates with either 2-(7-amino-4-methyl-coumarinyl)acetic acid or 7-amino-4-methyl-coumarin as fluorescent reporters of BSH activity. The BSH-catalyzed conversion of the natural substrate taurocholic acid was followed through an HPLC-based assay by applying 7-nitrobenzo[c][1,2,5]oxadiazole as scavenger for taurine, released in the enzymatic reaction. Hence, a new opportunity to monitor the activity of bile salt hydrolases was introduced.

Targeting the deubiquitinase USP7 for degradation with PROTACs.

Targeting deubiquitinating enzymes (DUBs) has emerged as a promising therapeutic approach in several human cancers and other diseases. DUB inhibitors are exciting pharmacological tools but often exhibit limited cellular potency. Here we report PROTACs based on a ubiquitin-specific protease 7 (USP7) inhibitor scaffold to degrade USP7. By investigating several linker and E3 ligand types, including novel cereblon recruiters, we discovered a highly selective USP7 degrader tool compound that induced apoptosis of USP7-dependent cancer cells. This work represents one of the first DUB degraders and unlocks a new drug target class for protein degradation.