Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses.

Since end of 2019, the global and unprecedented outbreak caused by the coronavirus SARS-CoV-2 led to dramatic numbers of infections and deaths worldwide. SARS-CoV-2 produces two large viral polyproteins which are cleaved by two cysteine proteases encoded by the virus, the 3CL protease (3CLpro) and the papain-like protease, to generate non-structural proteins essential for the virus life cycle. Both proteases are recognized as promising drug targets for the development of anti-coronavirus chemotherapy. Aiming at identifying broad spectrum agents for the treatment of COVID-19 but also to fight emergent coronaviruses, we focused on 3CLpro that is well conserved within this viral family. Here we present a high-throughput screening of more than 89,000 small molecules that led to the identification of a new chemotype, potent inhibitor of the SARS-CoV-2 3CLpro. The mechanism of inhibition, the interaction with the protease using NMR and X-Ray, the specificity against host cysteine proteases and promising antiviral properties in cells are reported.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Identification of indole-based activators of insulin degrading enzyme.

Insulin degrading enzyme (IDE) is a zinc metalloprotease that cleaves numerous substrates among which amyloid-ß and insulin. It has been linked through genetic studies to the risk of type-2 diabetes (T2D) or Alzheimer's disease (AD). Pharmacological activation of IDE is an attractive therapeutic strategy in AD. While IDE inhibition gave paradoxal activity in glucose homeostasis, recent studies, in particular in the liver suggest that IDE activators could be also of interest in diabetes. Here we describe the discovery of an original series of IDE activators by screening and structure-activity relationships. Early cellular studies show that hit 1 decreases glucose-stimulating insulin secretion. Docking studies revealed it has an unprecedented extended binding to the polyanion-binding site of IDE. These indole-based pharmacological tools are activators of both Aß and insulin hydrolysis by IDE and could be helpful to explore the multiple roles of IDE.

Drug Target Engagement Using Coupled Cellular Thermal Shift Assay-Acoustic Reverse-Phase Protein Array.

In the last 5 years, cellular thermal shift assay (CETSA), a technology based on ligand-induced changes in protein thermal stability, has been increasingly used in drug discovery to address the fundamental question of whether drug candidates engage their intended target in a biologically relevant setting. To analyze lysates from cells submitted to increasing temperature, the detection and quantification of the remaining soluble protein can be achieved using quantitative mass spectrometry, Western blotting, or AlphaScreen techniques. Still, these approaches can be time- and cell-consuming. To cope with limitations of throughput and protein amount requirements, we developed a new coupled assay combining the advantages of a nanoacoustic transfer system and reverse-phase protein array technology within CETSA experiments. We validated the technology to assess engagement of inhibitors of insulin-degrading enzyme (IDE), an enzyme involved in diabetes and Alzheimer's disease. CETSA-acoustic reverse-phase protein array (CETSA-aRPPA) allows simultaneous analysis of many conditions and drug-target engagement with a small sample size, in a rapid, cost-effective, and biological material-saving manner.

Identification of ebselen as a potent inhibitor of insulin degrading enzyme by a drug repurposing screening.

Insulin-degrading enzyme, IDE, is a metalloprotease implicated in the metabolism of key peptides such as insulin, glucagon, ß-amyloid peptide. Recent studies have pointed out its broader role in the cell physiology. In order to identify new drug-like inhibitors of IDE with optimal pharmacokinetic properties to probe its multiple roles, we ran a high-throughput drug repurposing screening. Ebselen, cefmetazole and rabeprazole were identified as reversible inhibitors of IDE. Ebselen is the most potent inhibitor (IC50(insulin) = 14 nM). The molecular mode of action of ebselen was investigated by biophysical methods. We show that ebselen induces the disorder of the IDE catalytic cleft, which significantly differs from the previously reported IDE inhibitors. IDE inhibition by ebselen can explain some of its reported activities in metabolism as well as in neuroprotection.