Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations.

Viruses belonging to the Flaviviridae family account for millions of cases of infections worldwide, mainly Dengue (DENV) and Zika (ZIKV) viruses. Both mosquito-borne flaviviruses are transmitted by bites of infected Aedes vectors, which are mainly endemic in tropical and sub-tropical countries. DENV and ZIKV are composed of structural and non-structural (NS) proteins that are initially expressed as a single precursor polyprotein. NS2B/NS3 is a serine protease complex that has a critical role during the viral replication cycle since the polyprotein processing is also accomplished by this enzyme. As an approach for developing novel NS2B/NS3 inhibitors, a virtual fragment-based drug design protocol was developed to select small fragments for new inhibitors. Based on this, we developed a series of α-cyanoacrylates and α-cyanoacrylamides targeting ZIKV and DENV NS2B/NS3 proteases. Among them, LQM467, 471, 472, and 474 were found to be active against both proteases, with K_i values ranging from 7.8 to 20 μM. Dynamics simulations revealed good stability during 200 ns for all enzyme inhibitor-complexes. Further, the MM/BPSA approach revealed that these active compounds present similar energetic profiles, corroborating our experimental data. Additionally, docking analyses showed that the compounds share similar binding poses involving the catalytic residues. Subsequently, these compounds were screened for their cytotoxic effects, showing that only LQM474 is toxic to Vero E6 cells. Thus, LQM467, LQM471, and LQM472 were evaluated on ZIKV-infected cells to determine their antiviral potential. As a result, it was verified that LQM471 is the most promising compound, exhibiting an EC₅₀ value of 35.07 μM. Finally, in order to obtain insights into its mechanism of action, a qPCR was performed, exhibiting that LQM471 is able to reduce the viral RNA copies compared to the untreated ZIKV-infected cells, suggesting that this compound can be a promising inhibitor of virus replication. This study presents an interesting alternative to design new hit compounds against DENV-2 and ZIKV NS2B/NS3 proteases contributing to the development of new therapeutic agents against these flaviviruses.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (M^pro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against M^pro (K_i: 1–10 μM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC₅₀: 1–12 μM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the M^pro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand‐induced conformational changes of the dengue virus (DENV) NS2B‐NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.

Protein‐ligand interactions are crucial for many cellular processes, with details of the binding mechanism being discussed as essential for biological functions. Interestingly, protein binding often involves conformational changes between two or more states, whereby different binding mechanisms are possible even with a simple two‐state description. Two models are widely used to portray protein‐ligand interactions: Induced fit and conformational selection. However, distinguishing them experimentally is challenging. Single‐molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool to resolve structural dynamics at the level of single proteins. Here, we investigated immobilized Zika virus (ZIKV) and dengue virus (DENV2) NS2B‐NS3 proteases using smFRET to compare their conformational changes upon binding to competitive small molecule inhibitors. The analysis of the smFRET data allowed us to distinguish between induced fit and conformational selection models and assign the binding mechanism from the kinetic parameters obtained. Although DENV and ZIKV protease are proteins with high structural similarities, our results reveal that they have opposite binding mechanisms for competitive ligands. While the protein‐ligand interaction in the ZIKV protease follows an induced fit mechanism, the DENV protease follows the conformational selection mechanism.