Stefan Josef Hammerschmidt

Isothermal titration calorimetry (ITC) is the method of choice for thermodynamic binding profiling. As a label-free in-solution technique, ITC experiments do not require modified or immobilized proteins or ligands and hence can be performed under close-to-native environmental conditions. It directly follows the released heat of the molecular interaction and does not need competing reporter molecules. However, advanced experimental ITC setups are frequently necessary to derive thermodynamic parameters for medicinal chemistry applications. Beyond 1:1 protein–ligand binding, complex systems include displacement titrations to determine thermodynamics for ligands with affinities either too high or too low to be derived from direct titrations, the characterization of protonation changes upon binding, or the elucidation of heat capacities. We present hands-on experiments for graduate medicinal chemists and students familiar with standard ITC methods, reliably highlighting these issues and their respective solutions. The setup can be either a biochemical trypsin–ligand system or a more cost-efficient EDTA-based variant. This demonstration of best practice protocols comes together with the freely accessible ITC web calculator ITCcalc (https://itccalc.uni-mainz.de), guiding through the underlying evaluation of the obtained experimental results.

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.

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.

Fluorometric assays are one of the most frequently used methods in medicinal chemistry. Over the last 50 years, the reporter molecules for the detection of protease activity have evolved from first‐generation colorimetricp‐nitroanilides, through FRET substrates, and 7‐amino‐4‐methyl coumarin (AMC)‐based substrates. The aim of further substrate development is to increase sensitivity and reduce vulnerability to assay interferences. Herein, we describe a new generation of substrates for protease assays based on 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl‐amides (NBD‐amides). In this study, we synthesized and tested substrates for 10 different proteases from the serine‐, cysteine‐, and metalloprotease classes. Enzyme‐ and substrate‐specific parameters as well as the inhibitory activity of literature‐known inhibitors confirmed their suitability for application in fluorometric assays. Hence, we were able to present NBD‐based alternatives for common protease substrates. In conclusion, these NBD substrates are not only less susceptible to common assay interference, but they are also able to replace FRET‐based substrates with the requirement of a prime site amino acid residue.

COVID‐19 has caused many deaths since the first outbreak in 2019. The burden on healthcare systems around the world has been reduced by the success of vaccines. However, population adherence and the occurrence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants are still challenging tasks to be affronted. In addition, the newly approved drug presents some limitations in terms of side effects and drug interference, highlighting the importance of searching for new antiviral agents against SARS‐CoV‐2. The SARS‐CoV‐2 main protease (M^pro) represents a versatile target to search for new drug candidates due to its essential role in proteolytic activities responsible for the virus replication. In this work, a series of 190 compounds, composed of 27 natural ones and 163 synthetic compounds, were screened in vitro for their inhibitory effects against SARS‐CoV‐2 M^pro. Twenty‐five compounds inhibited M^pro with inhibitory constant values (K_i) between 23.2 and 241 µM. Among them, a thiosemicarbazone derivative was the most active compound. Molecular docking studies using Protein Data Bank ID 5RG1, 5RG2, and 5RG3 crystal structures of M^pro revealed important interactions identified as hydrophobic, hydrogen bonding and steric interactions with amino acid residues in the active site cavity. Overall, our findings indicate the described thiosemicarbazones as good candidates to be further explored to develop antiviral leads against SARS‐CoV‐2. Moreover, the studies showed the importance of careful evaluation of test results to detect and exclude false‐positive findings.

Highly symmetric ligands can bind in multiple identical binding modes. The improved binding affinity arising from higher entropy was elucidated in this work.

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 exhibited enzyme inhibitory activity against M^pro (Ki: 1-10 micM) and three of them good anti-SARS-CoV-2 infection activity in the low micromolar range (EC₅₀: 1-12 micM) 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. QM/MM computer simulations assisted in the design and in elucidating the way of action. Finally, structural analysis shows, in agreement with the computer predictions, the binding mode of FGA146 and FGA147 to the active site of the protein. Our results illustrate that peptidyl nitroalkenes are potent covalent reversible inhibitors of the M^pro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection becoming promising drugs against SARS-CoV-2.

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.

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.

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.