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Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development.Antiviral Research Mar 2021The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of...
The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.
Topics: Antiviral Agents; Binding Sites; Cathepsin L; Coronavirus 3C Proteases; Drug Discovery; Glycoproteins; Humans; Kinetics; Models, Molecular; Protease Inhibitors; Pyrrolidines; Rhinovirus; SARS-CoV-2; Sulfonic Acids
PubMed: 33515606
DOI: 10.1016/j.antiviral.2021.105020 -
Bioorganic & Medicinal Chemistry Letters Jun 2017Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The...
Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives gave SK80 with an IC value of 43μM against SARS CoV 3CL R188I mutant protease.
Topics: Coronavirus 3C Proteases; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Humans; Models, Molecular; Molecular Structure; Protease Inhibitors; Serine; Structure-Activity Relationship; Viral Proteins
PubMed: 28454669
DOI: 10.1016/j.bmcl.2017.04.056 -
Journal of Biomolecular Structure &... May 2022To address coronavirus disease (COVID-19), currently, no effective drug or vaccine is available. In this regard, molecular modeling approaches are highly useful to...
To address coronavirus disease (COVID-19), currently, no effective drug or vaccine is available. In this regard, molecular modeling approaches are highly useful to discover potential inhibitors of the main protease (M) enzyme of SARS-CoV-2. Since, the M enzyme plays key roles in mediating viral replication and transcription; therefore, it is considered as an attractive drug target to control SARS-CoV-2 infection. By using structure-based drug design, pharmacophore modeling, and virtual high throughput drug screening combined with docking and all-atom molecular dynamics simulation approach, we have identified five potential inhibitors of SARS-CoV-2 M. MD simulation studies revealed that compound 54035018 binds to the M with high affinity (Δ -37.40 kcal/mol), and the complex is more stable in comparison with other protein-ligand complexes. We have identified promising leads to fight COVID-19 infection as these compounds fulfill all drug-likeness properties. However, experimental and clinical validations are required for COVID-19 therapy.Communicated by Ramaswamy H. Sarma.
Topics: Coronavirus 3C Proteases; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Protease Inhibitors; SARS-CoV-2; COVID-19 Drug Treatment
PubMed: 33210561
DOI: 10.1080/07391102.2020.1848634 -
Clinical and Applied... Oct 2016The pathological consequences of decreased protein Z (PZ) and/or Z-dependent protease inhibitor (ZPI) levels remain as yet unclear, despite a growing body of evidence...
The pathological consequences of decreased protein Z (PZ) and/or Z-dependent protease inhibitor (ZPI) levels remain as yet unclear, despite a growing body of evidence which supports their involvement in an increased thrombotic risk. The purpose of the present study was 2-fold: to evaluate plasma concentrations of protein Z and ZPI in patients with essential thrombocythemia (ET) and to determine their significance in thrombotic complications. The median (range) plasma concentrations of PZ in our patients with ET were lower, but not significantly, than in healthy individuals: PZ (1.42 µg/mL, 0.36-3.14 µg/mL vs 1.6 µg/mL, 0.75-2.56 µg/mL, P = .08). On the other hand, the median (range) plasma concentrations of ZPI in the said patients with ET were meaningfully lower than in the reference group: ZPI (3.22 µg/mL, 0.85-6.97 µg/mL vs 4.41 µg/mL, 1.63-7.83 µg/mL, P = .0004). More importantly, the study revealed a statistically significant lower concentration of PZ and ZPI in patients with the presence of the JAK2V617F mutation relative to patients without the mutation, for PZ: 1.38 µg/mL, 0.36-2.6 µg/mL versus 1.63 µg/mL, 0.88-3.14 µg/mL, P = .03, and ZPI 2.89 µg/mL, 0.85-5.91 µg/mL versus 3.61 µg/mL, 1.53-6.97 µg/mL, P = .002. Additionally, significant differences between the concentrations of PZ and ZPI were found in patients with venous thrombotic episodes compared to healthy individuals, for PZ: 1.23 µg/mL, 0.82-1.99 µg/mL versus 1.6 µg/mL, 0.75-2.56 µg/mL, P = .043, and ZPI: 2.42 µg/mL, 0.85-4.21 µg/mL versus 4.41 µg/mL, 1.63-7.83 µg/mL, P < .0001. To recapitulate, our results suggest that the deficiency of PZ may increase tendency to thrombosis in patients with ET.
Topics: Adult; Aged; Aged, 80 and over; Blood Proteins; Female; Humans; Male; Middle Aged; Protease Inhibitors; Thrombocythemia, Essential; Young Adult
PubMed: 25783867
DOI: 10.1177/1076029615576741 -
MBio Feb 2023The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of...
The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid . The characteristics of antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.
Topics: Humans; Antiviral Agents; COVID-19; Enzyme Inhibitors; Protease Inhibitors; SARS-CoV-2
PubMed: 36625640
DOI: 10.1128/mbio.02815-22 -
Molecules (Basel, Switzerland) Apr 2020The inhibition of viral protease is an important target in antiviral drug discovery and development. To date, protease inhibitor drugs, especially HIV-1 protease... (Review)
Review
The inhibition of viral protease is an important target in antiviral drug discovery and development. To date, protease inhibitor drugs, especially HIV-1 protease inhibitors, have been available for human clinical use in the treatment of coronaviruses. However, these drugs can have adverse side effects and they can become ineffective due to eventual drug resistance. Thus, the search for natural bioactive compounds that were obtained from bio-resources that exert inhibitory capabilities against HIV-1 protease activity is of great interest. Fungi are a source of natural bioactive compounds that offer therapeutic potential in the prevention of viral diseases and for the improvement of human immunomodulation. Here, we made a brief review of the current findings on fungi as producers of protease inhibitors and studies on the relevant candidate fungal bioactive compounds that can offer immunomodulatory activities as potential therapeutic agents of coronaviruses in the future.
Topics: Antiviral Agents; Biological Products; Coronavirus; Coronavirus Infections; Fungi; Humans; Immunologic Factors; Molecular Structure; Protease Inhibitors; Structure-Activity Relationship
PubMed: 32295300
DOI: 10.3390/molecules25081800 -
Nature Communications May 2022A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory...
A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory mechanism of human A2ML1 and determine the structures of its native and protease-cleaved conformations. The functional inhibitory unit of A2ML1 is a monomer that depends on covalent binding of the protease (mediated by A2ML1's thioester) to achieve inhibition. In contrast to the A2M tetramer which traps proteases in two internal chambers formed by four subunits, in protease-cleaved monomeric A2ML1 disordered regions surround the trapped protease and may prevent substrate access. In native A2ML1, the bait region is threaded through a hydrophobic channel, suggesting that disruption of this arrangement by bait region cleavage triggers the extensive conformational changes that result in protease inhibition. Structural comparisons with complement C3/C4 suggest that the A2M superfamily of proteins share this mechanism for the triggering of conformational change occurring upon proteolytic activation.
Topics: Cryoelectron Microscopy; Endopeptidases; Humans; Protease Inhibitors; alpha-Macroglobulins
PubMed: 35641520
DOI: 10.1038/s41467-022-30758-x -
Biophysical Journal Oct 2022Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed...
Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed that NM was a highly safe drug for the treatment of different cancers, but the precise functions and mechanisms of NM are not clear. In this study, we determined a series of crystal structures of NM and its hydrolysates in complex with a serine protease (urokinase-type plasminogen activator [uPA]). These structures reveal that NM was cleaved by uPA and that a hydrolyzed product (4-guanidinobenzoic acid [GBA]) remained covalently linked to Ser195 of uPA, and the other hydrolyzed product (6-amidino-2-naphthol [6A2N]) released from uPA. Strikingly, in the inactive uPA (uPA-S195A):NM structure, the 6A2N side of intact NM binds to the specific pocket of uPA. Molecular dynamics simulations and end-point binding free-energy calculations show that the conf1 of NM (6A2N as P1 group) in the uPA-S195A:NM complex may be more stable than conf2 of NM (GBA as P1 group). Moreover, in the structure of uPA:NM complex, the imidazole group of His57 flips further away from Ser195 and disrupts the stable canonical catalytic triad conformation. These results not only reveal the inhibitory mechanism of NM as an efficient serine protease inhibitor but also might provide the structural basis for the further development of serine protease inhibitors.
Topics: Urokinase-Type Plasminogen Activator; Serine Proteinase Inhibitors; Serine Proteases; Imidazoles
PubMed: 36039386
DOI: 10.1016/j.bpj.2022.08.034 -
Journal of Medicinal Chemistry Mar 2017θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC = 390 ± 20 nM, K = 365 ± 20 nM) and a weak inhibitor of other mammalian...
θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC = 390 ± 20 nM, K = 365 ± 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNFα converting enzyme (TACE) (K = 4.45 ± 0.48 μM). Using full sequence amino acid scanning in combination with a highly efficient "one-pot" cyclization-folding approach, we obtained an RTD-1-based peptide that was around 10 times more active than wild-type RTD-1 in inhibiting LF protease (IC = 43 ± 3 nM, K = 18 ± 1 nM). The most active peptide was completely symmetrical, rich in Arg and Trp residues, and able to adopt a native RTD-1-like structure. These results show the power of optimized chemical peptide synthesis approaches for the efficient production of libraries of disulfide-rich backbone-cyclized peptides to quickly perform structure-activity relationship studies for optimizing protease inhibitors.
Topics: Amino Acids; Antigens, Bacterial; Bacterial Toxins; Cyclization; Defensins; Protease Inhibitors
PubMed: 28151653
DOI: 10.1021/acs.jmedchem.6b01689 -
Antiviral Research Dec 2022Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (M), which presents...
Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (M), which presents structural similarity with the active site domain of enterovirus 3C protease, is one of the best-characterized drug targets of these viruses. Here we studied the antiviral activity of the orally bioavailable enterovirus protease inhibitor AG7404 against SARS-CoV-1 and SARS-CoV-2 from a structural, biochemical, and cellular perspective, comparing it with the related molecule rupintrivir (AG7800). Crystallographic structures of AG7404 in complex with SARS-CoV-1 M and SARS-CoV-2 M and of rupintrivir in complex with SARS-CoV-2 M were solved, revealing that all protein residues interacting with the inhibitors are conserved between the two proteins. A detailed analysis of protein-inhibitor interactions indicates that AG7404 has a better fit to the active site of the target protease than rupintrivir. This observation was further confirmed by biochemical FRET assays showing IC values of 47 μM and 101 μM for AG7404 and rupintrivir, respectively, in the case of SARS-CoV-2 M. Equivalent IC values for SARS-CoV-1 also revealed greater inhibitory capacity of AG7404, with a value of 29 μM vs. 66 μM for rupintrivir. Finally, the antiviral activity of the two inhibitors against SARS-CoV-2 was confirmed in a human cell culture model of SARS-CoV-2 infection, although rupintrivir showed a higher potency and selectivity index in this assay.
Topics: Humans; SARS-CoV-2; Antiviral Agents; Cysteine Endopeptidases; Protease Inhibitors; Molecular Docking Simulation; COVID-19 Drug Treatment
PubMed: 36336176
DOI: 10.1016/j.antiviral.2022.105458