Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 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 Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

The effects of allosteric and competitive inhibitors on ZIKV protease conformational dynamics explored through smFRET, nanoDSF, DSF, and 19F NMR.

Zika and dengue viruses cause mosquito-borne diseases of high epidemic relevance. The viral NS2B-NS3 proteases play crucial roles in the pathogen replication cycle and are validated drug targets. They can adopt at least two conformations depending on the position of the NS2B cofactor. Recently, we reported ligand-induced conformational changes of dengue virus NS2B-NS3 protease by single-molecule Förster resonance energy transfer (smFRET). Here, we investigated the conformational dynamics of the homologous Zika virus protease through an integrated methodological approach combining smFRET, thermal shift assays (DSF and nanoDSF) and 19F NMR spectroscopy. Our results show that allosteric inhibitors favor the open conformation and competitive inhibitors stabilize the closed conformation of the Zika virus protease.

Next Generation of Fluorometric Protease Assays: 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-Amides) as Class-Spanning Protease Substrates.

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 colorimetric p-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.

Improving binding entropy by higher ligand symmetry? - A case study with human matriptase.

Understanding different contributions to the binding entropy of ligands is of utmost interest to better predict affinity and the thermodynamic binding profiles of protein-ligand interactions and to develop new strategies for ligand optimization. To these means, the largely neglected effects of introducing higher ligand symmetry, thereby reducing the number of energetically distinguishable binding modes on binding entropy using the human matriptase as a model system, were investigated. A set of new trivalent phloroglucinol-based inhibitors that address the roughly symmetric binding site of the enzyme was designed, synthesized, and subjected to isothermal titration calorimetry. These highly symmetric ligands that can adopt multiple indistinguishable binding modes exhibited high entropy-driven affinity in line with affinity-change predictions.

Inhibitory effects of 190 compounds against SARS-CoV-2 Mpr o protein: Molecular docking interactions.

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 (Mpr o ) 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 Mpro . Twenty-five compounds inhibited Mpro with inhibitory constant values (Ki ) 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 Mpro 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.

Investigation of the Compatibility between Warheads and Peptidomimetic Sequences of Protease Inhibitors-A Comprehensive Reactivity and Selectivity Study.

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.

Structure-based lead optimization of peptide-based vinyl methyl ketones as SARS-CoV-2 main protease inhibitors.

Despite several major achievements in the development of vaccines and antivirals, the fight against SARS-CoV-2 and the health problems accompanying COVID-19 are still ongoing. SARS-CoV-2 main protease (Mpro), an essential viral cysteine protease, is a crucial target for the development of antiviral agents. A virtual screening analysis of in-house cysteine protease inhibitors against SARS-CoV-2 Mpro allowed us to identify two hits (i.e., 1 and 2) bearing a methyl vinyl ketone warhead. Starting from these compounds, we herein report the development of Michael acceptors targeting SARS-CoV-2 Mpro, which differ from each other for the warhead and for the amino acids at the P2 site. The most promising vinyl methyl ketone-containing analogs showed sub-micromolar activity against the viral protease. SPR38, SPR39, and SPR41 were fully characterized, and additional inhibitory properties towards hCatL, which plays a key role in the virus entry into host cells, were observed. SPR39 and SPR41 exhibited single-digit micromolar EC50 values in a SARS-CoV-2 infection model in cell culture.

Thermodynamic characterization of a macrocyclic Zika virus NS2B/NS3 protease inhibitor and its acyclic analogs.

Cyclization of small molecules is a widely applied strategy in drug design for ligand optimization to improve affinity, as it eliminates the putative need for structural preorganization of the ligand before binding, or to improve pharmacokinetic properties. In this work, we provide a deeper insight into the binding thermodynamics of a macrocyclic Zika virus NS2B/NS3 protease inhibitor and its linear analogs. Characterization of the thermodynamic binding profiles by isothermal titration calorimetry experiments revealed an unfavorable entropy of the macrocycle compared to the open linear reference ligands. Molecular dynamic simulations and X-ray crystal structure analysis indicated only minor benefits from macrocyclization to fixate a favorable conformation, while linear ligands retained some flexibility even in the protein-bound complex structure, possibly explaining the initially surprising effect of a higher entropic penalty for the macrocyclic ligand.

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease.

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 KD 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.

Nintedanib plus docetaxel after progression on first-line immunochemotherapy in patients with lung adenocarcinoma: Cohort C of the non-interventional study, VARGADO.

Background: Immune checkpoint inhibitors (ICIs) with or without chemotherapy represent first-line standard of care for patients with advanced non-small cell lung cancer (NSCLC) without targetable driver mutations. The most appropriate second-line therapy after failing immunochemotherapy remains an open question. Nintedanib, an oral triple angiokinase inhibitor that targets the vascular endothelial growth factor receptor, fibroblast growth factor receptor, and, platelet-derived growth factor receptor, in combination with docetaxel, is approved for treatment of advanced NSCLC (adenocarcinoma histology) following progression on first-line chemotherapy. Methods: VARGADO (NCT02392455) is an ongoing, prospective, non-interventional study investigating the efficacy and safety of nintedanib plus docetaxel following first-line chemotherapy with or without ICIs in patients with locally advanced, metastatic, or locally recurrent NSCLC of adenocarcinoma histology. This analysis focuses on Cohort C, which enrolled patients who had received prior first line chemotherapy with ICIs. Patients received second-line docetaxel (75 mg/m2) by intravenous infusion on Day 1, plus oral nintedanib (200 mg twice daily) on Days 2-21 of each 21-day cycle during routine clinical care. The primary endpoint is overall survival (OS) rate 1 year after the start of treatment with nintedanib plus docetaxel. Secondary endpoints include progression-free survival (PFS), OS, and disease control rate (DCR). Safety was also assessed. Results: Among 137 patients treated, the median age was 63 years (range, 37-84); 57 patients (41.6%) were female, most patients had Eastern Cooperative Oncology Group performance status of 0 (28.5%) or 1 (43.1%); 118 (86.1%) had stage IV NSCLC and 27 (19.7%) had brain metastases. Most (n=120, 87.6%) patients had received pembrolizumab/pemetrexed/platinum-based chemotherapy as first-line treatment. In 80 patients with available response data, the DCR was 72.5% (complete response: 1.3%; partial response: 36.3%; stable disease: 35.0%). Median progression-free survival was 4.8 months (95% confidence interval: 3.7-6.6). OS data were immature. Grade ≥3 treatment-emergent adverse events (TEAEs), serious TEAEs, and TEAEs leading to treatment discontinuation were reported in 62 (45.3%), 50 (36.5%), and 40 patients (29.2%), respectively. Conclusions: This analysis indicates that nintedanib plus docetaxel represents an effective second-line treatment option in patients with advanced adenocarcinoma NSCLC following progression on first-line immunochemotherapy. The safety profile was manageable with no unexpected signals.

Development of Urea-Bond-Containing Michael Acceptors as Antitrypanosomal Agents Targeting Rhodesain.

Human African Trypanosomiasis (HAT) is a neglected tropical disease widespread in sub-Saharan Africa. Rhodesain, a cysteine protease of Trypanosoma brucei rhodesiense, has been identified as a valid target for the development of anti-HAT agents. Herein, we report a series of urea-bond-containing Michael acceptors, which were demonstrated to be potent rhodesain inhibitors with K i values ranging from 0.15 to 2.51 nM, and five of them showed comparable k 2nd values to that of K11777, a potent antitrypanosomal agent. Moreover, most of the urea derivatives exhibited single-digit micromolar activity against the protozoa, and the presence of substituents at the P3 position appears to be essential for the antitrypanosomal effect. Replacement of Phe with Leu at the P2 site kept unchanged the inhibitory properties. Compound 7 (SPR7) showed the best compromise in terms of rhodesain inhibition, selectivity, and antiparasitic activity, thus representing a new lead compound for future SAR studies.

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments.

Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176 €, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I2S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled high-accuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform high-resolution thermal shift assays (TSA) from exemplary biological applications.

Identification of novel small-molecular inhibitors of Staphylococcus aureus sortase A using hybrid virtual screening.

Staphylococcus aureus is one of the most dangerous pathogens commonly associated with high levels of morbidity and mortality. Sortase A is considered as a promising molecular target for the development of antistaphylococcal agents. Using hybrid virtual screening approach and FRET analysis, we have identified five compounds able to decrease the activity of sortase A by more than 50% at the concentration of 200 µM. The most promising compound was 2-(2-amino-3-chloro-benzoylamino)-benzoic acid which was able to inhibit S. aureus sortase A at the IC50 value of 59.7 µM. This compound was selective toward sortase A compared to other four cysteine proteases - cathepsin L, cathepsin B, rhodesain, and the SARS-CoV2 main protease. Microscale thermophoresis experiments confirmed that this compound bound sortase A with KD value of 189 µM. Antibacterial and antibiofilm assays also confirmed high specificity of the hit compound against two standard and three wild-type, S. aureus hospital infection isolates. The effect of the compound on biofilms produced by two S. aureus ATCC strains was also observed suggesting that the compound reduced biofilm formation by changing the biofilm structure and thickness.

Identification, Characterization, and Synthesis of Natural Parasitic Cysteine Protease Inhibitors: Pentacitidins Are More Potent Falcitidin Analogues.

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogues of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization, and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogues by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogues followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and, additionally, low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

Interfering with Host Proteases in SARS-CoV-2 Entry as a Promising Therapeutic Strategy.

Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since there is currently no causative drug against this viral infection available, science is striving for new drugs and other approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARSCoV- 2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.

SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B/NS3 proteases.

In recent years, dengue virus (DENV) and Zika virus (ZIKV), both mosquito-borne members of the Flaviviridae family, have emerged as intercontinental health issues since their vectors have spread from their tropical origins to temperate climate zones due to climate change and increasing globalization. DENV and ZIKV are positive-sense, single-stranded RNA viruses, whose genomes consist of three structural (capsid, membrane precursor, envelope) and seven non-structural (NS) proteins, all of which are initially expressed as a single precursor polyprotein. For virus maturation, the polyprotein processing is accomplished by host proteases and the viral NS2B/NS3 protease complex, whose inhibitors have been shown to be effective antiviral agents with loss of viral pathogenicity. In this work, we elucidate new structure-activity relationships of benzo[d]thiazole-based allosteric NS2B/NS3 inhibitors. We developed a new series of Y-shaped inhibitors, which, with its larger hydrophobic contact surface, should bind to previously unaddressed regions of the allosteric NS2B/NS3 binding pocket. By scaffold-hopping, we varied the benzo[d]thiazole core and identified benzofuran as a new lead scaffold shifting the selectivity of initially ZIKV-targeting inhibitors to higher activities towards the DENV protease. In addition, we were able to increase the ligand efficiency from 0.27 to 0.41 by subsequent inhibitor truncation and identified N-(5,6-dihydroxybenzo[d]thiazol-2-yl)-4-iodobenzamide as a novel sub-micromolar NS2B/NS3 inhibitor. Utilizing cell-based assays, we could prove the antiviral activity in cellulo. Overall, we report new series of sub-micromolar allosteric DENV and ZIKV inhibitors with good efficacy profile in terms of cytotoxicity and protease inhibition selectivity.

Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET (fluorescence resonance energy transfer) couple. Fluorescence correlation spectroscopy and photon-burst detection were employed to examine FRET pair labeled DENV-PRs freely diffusing in solution. The measurements were performed with two double mutants and with two dye couples. The data provide strong evidence that an equilibrium of at least two conformations of DENV-PR exists in solution. The competitive inhibitor stabilizes the closed state. Because the open and closed conformations appear to coexist in solution, our results support the picture of a conformational selection rather than that of an induced fit mechanism with respect to the inhibitor-induced formation of the closed state.

SARS-CoV-PLpro-Inhibitoren als mögliche Breitspektrum-Virostatika.

The SARS-CoV-encoded papain-like cysteine protease (PLpro) plays crucial roles in viral replication and maturation processes. It is required to cleave the precursor polyproteins into functional proteins. Thus, it is considered to be a promising target for developing specific drugs. For rational optimization of hit compounds, information about the structure-activity relationship (SAR) is fundamental. Herein, we characterize isoindolines as a new class of PLpro inhibitors.

Lead Discovery of SARS-CoV-2 Main Protease Inhibitors through Covalent Docking-Based Virtual Screening.

During almost all 2020, coronavirus disease 2019 (COVID-19) pandemic has constituted the major risk for the worldwide health and economy, propelling unprecedented efforts to discover drugs for its prevention and cure. At the end of the year, these efforts have culminated with the approval of vaccines by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA) giving new hope for the future. On the other hand, clinical data underscore the urgent need for effective drugs to treat COVID-19 patients. In this work, we embarked on a virtual screening campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro chymotrypsin-like cysteine protease employing our in-house database of peptide and non-peptide ligands characterized by different types of warheads acting as Michael acceptors. To this end, we employed the AutoDock4 docking software customized to predict the formation of a covalent adduct with the target protein. In vitro verification of the inhibition properties of the most promising candidates allowed us to identify two new lead inhibitors that will deserve further optimization. From the computational point of view, this work demonstrates the predictive power of AutoDock4 and suggests its application for the in silico screening of large chemical libraries of potential covalent binders against the SARS-CoV-2 Mpro enzyme.

2-Sulfonylpyrimidines as Privileged Warheads for the Development of S. aureus Sortase A Inhibitors.

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar KI values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the S N Ar reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog.