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1.
ACS Chem Biol ; 16(9): 1692-1700, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1366786

ABSTRACT

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin's active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin's OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.


Subject(s)
Antiviral Agents/metabolism , Furin/antagonists & inhibitors , Guanidines/metabolism , Hydrazones/metabolism , Serine Proteinase Inhibitors/metabolism , Antiviral Agents/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzyme Assays , Furin/chemistry , Furin/metabolism , Guanidines/chemistry , HEK293 Cells , Humans , Hydrazones/chemistry , Kinetics , Proprotein Convertase 5/antagonists & inhibitors , Proprotein Convertase 5/chemistry , Protein Binding , Protein Conformation , Serine Proteinase Inhibitors/chemistry , Subtilisins/antagonists & inhibitors , Subtilisins/chemistry
2.
Int J Mol Sci ; 22(13)2021 Jun 30.
Article in English | MEDLINE | ID: covidwho-1288905

ABSTRACT

Positively charged groups that mimic arginine or lysine in a natural substrate of trypsin are necessary for drugs to inhibit the trypsin-like serine protease TMPRSS2 that is involved in the viral entry and spread of coronaviruses, including SARS-CoV-2. Based on this assumption, we identified a set of 13 approved or clinically investigational drugs with positively charged guanidinobenzoyl and/or aminidinobenzoyl groups, including the experimentally verified TMPRSS2 inhibitors Camostat and Nafamostat. Molecular docking using the C-I-TASSER-predicted TMPRSS2 catalytic domain model suggested that the guanidinobenzoyl or aminidinobenzoyl group in all the drugs could form putative salt bridge interactions with the side-chain carboxyl group of Asp435 located in the S1 pocket of TMPRSS2. Molecular dynamics simulations further revealed the high stability of the putative salt bridge interactions over long-time (100 ns) simulations. The molecular mechanics/generalized Born surface area-binding free energy assessment and per-residue energy decomposition analysis also supported the strong binding interactions between TMPRSS2 and the proposed drugs. These results suggest that the proposed compounds, in addition to Camostat and Nafamostat, could be effective TMPRSS2 inhibitors for COVID-19 treatment by occupying the S1 pocket with the hallmark positively charged groups.


Subject(s)
Antiviral Agents/chemistry , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Benzamidines/chemistry , Benzamidines/metabolism , Binding Sites , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Esters/chemistry , Esters/metabolism , Guanidines/chemistry , Guanidines/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/metabolism , Serine Proteinase Inhibitors/therapeutic use , Thermodynamics
3.
Chem Biol Interact ; 338: 109428, 2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1103757

ABSTRACT

Camostat mesylate, a potent inhibitor of the human transmembrane protease, serine 2 (TMPRSS2), is currently under investigation for its effectiveness in COVID-19 patients. For its safe application, the risks of camostat mesylate to induce pharmacokinetic drug-drug interactions with co-administered drugs should be known. We therefore tested in vitro the potential inhibition of important efflux (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2)), and uptake transporters (organic anion transporting polypeptides OATP1B1, OATP1B3, OATP2B1) by camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA). Transporter inhibition was evaluated using fluorescent probe substrates in transporter over-expressing cell lines and compared to the respective parental cell lines. Moreover, possible mRNA induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) was analysed in LS180 cells by quantitative real-time PCR. The results of our study for the first time demonstrated that camostat mesylate and GBPA do not relevantly inhibit P-gp, BCRP, OATP1B1 or OATP1B3. Only OATP2B1 was profoundly inhibited by GBPA with an IC50 of 11 µM. Induction experiments in LS180 cells excluded induction of PXR-regulated genes such as cytochrome P450 3A4 (CYP3A4) and ABCB1 and AhR-regulated genes such as CYP1A1 and CYP1A2 by camostat mesylate and GBPA. Together with the summary of product characteristics of camostat mesylate indicating no inhibition of CYP1A2, 2C9, 2C19, 2D6, and 3A4 in vitro, our data suggest a low potential of camostat mesylate to act as a perpetrator in pharmacokinetic drug-drug interactions. Only inhibition of OATP2B1 by GBPA warrants further investigation.


Subject(s)
Drug Interactions , Esters/metabolism , Guanidines/metabolism , Serine Proteinase Inhibitors/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Cell Line , Cell Survival/drug effects , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Esters/chemistry , Esters/pharmacology , Guanidines/chemistry , Guanidines/pharmacology , Humans , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters/metabolism , Pregnane X Receptor/genetics , Pregnane X Receptor/metabolism , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology
4.
Adv Drug Deliv Rev ; 167: 47-65, 2020 12.
Article in English | MEDLINE | ID: covidwho-921794

ABSTRACT

To date, no effective vaccines or therapies are available against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pandemic agent of the coronavirus disease 2019 (COVID-19). Due to their safety, efficacy and specificity, peptide inhibitors hold great promise for the treatment of newly emerging viral pathogens. Based on the known structures of viral proteins and their cellular targets, antiviral peptides can be rationally designed and optimized. The resulting peptides may be highly specific for their respective targets and particular viral pathogens or exert broad antiviral activity. Here, we summarize the current status of peptides inhibiting SARS-CoV-2 entry and outline the strategies used to design peptides targeting the ACE2 receptor or the viral spike protein and its activating proteases furin, transmembrane serine protease 2 (TMPRSS2), or cathepsin L. In addition, we present approaches used against related viruses such as SARS-CoV-1 that might be implemented for inhibition of SARS-CoV-2 infection.


Subject(s)
Antiviral Agents/administration & dosage , COVID-19/metabolism , Peptide Fragments/administration & dosage , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Drug Delivery Systems/methods , Humans , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding/drug effects , Protein Binding/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/administration & dosage , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/metabolism
5.
J Phys Chem Lett ; 11(16): 6655-6663, 2020 Aug 20.
Article in English | MEDLINE | ID: covidwho-678528

ABSTRACT

The COVID-19 pandemic is an urgent global health emergency, and the presence of Furin site in the SARS-CoV-2 spike glycoprotein alters virulence and warrants further molecular, structural, and biophysical studies. Here we report the structure of Furin in complex with SARS-CoV-2 spike glycoprotein, demonstrating how Furin binds to the S1/S2 region of spike glycoprotein and eventually cleaves the viral protein using experimental functional studies, molecular dynamics, and docking. The structural studies underline the mechanism and mode of action of Furin, which is a key process in host cell entry and a hallmark of enhanced virulence. Our whole-exome sequencing analysis shows the genetic variants/alleles in Furin were found to alter the binding affinity for viral spike glycoprotein and could vary in infectivity in humans. Unravelling the mechanisms of Furin action, binding dynamics, and the genetic variants opens the growing arena of bona fide antibodies and development of potential therapeutics targeting the blockage of Furin cleavage.


Subject(s)
Betacoronavirus/chemistry , Furin/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virulence/physiology , Amino Acid Sequence , Animals , Betacoronavirus/pathogenicity , CHO Cells , Catalytic Domain , Cricetulus , Furin/chemistry , Furin/genetics , Gene Expression/physiology , Hexosamines/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Proteolysis , SARS-CoV-2 , Serine Proteinase Inhibitors/metabolism , Spike Glycoprotein, Coronavirus/chemistry
6.
FEBS Open Bio ; 10(6): 995-1004, 2020 06.
Article in English | MEDLINE | ID: covidwho-186395

ABSTRACT

A novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or 2019 novel coronavirus] has been identified as the pathogen of coronavirus disease 2019. The main protease (Mpro , also called 3-chymotrypsin-like protease) of SARS-CoV-2 is a potential target for treatment of COVID-19. A Mpro homodimer structure suitable for docking simulations was prepared using a crystal structure (PDB ID: 6Y2G; resolution 2.20 Å). Structural refinement was performed in the presence of peptidomimetic α-ketoamide inhibitors, which were previously disconnected from each Cys145 of the Mpro homodimer, and energy calculations were performed. Structure-based virtual screenings were performed using the ChEMBL database. Through a total of 1 485 144 screenings, 64 potential drugs (11 approved, 14 clinical, and 39 preclinical drugs) were predicted to show high binding affinity with Mpro . Additional docking simulations for predicted compounds with high binding affinity with Mpro suggested that 28 bioactive compounds may have potential as effective anti-SARS-CoV-2 drug candidates. The procedure used in this study is a possible strategy for discovering anti-SARS-CoV-2 drugs from drug libraries that may significantly shorten the clinical development period with regard to drug repositioning.


Subject(s)
Betacoronavirus/enzymology , Chymases/metabolism , Coronavirus Infections/metabolism , Drug Discovery/methods , Drug Repositioning/methods , Pharmaceutical Preparations/metabolism , Pneumonia, Viral/metabolism , Serine Proteinase Inhibitors/metabolism , Viral Proteins/metabolism , Betacoronavirus/drug effects , COVID-19 , Catalytic Domain , Chymases/antagonists & inhibitors , Chymases/chemistry , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Crystallization , Databases, Chemical , Humans , Models, Molecular , Molecular Docking Simulation , Pandemics , Pharmaceutical Preparations/chemistry , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , SARS-CoV-2 , Serine Proteinase Inhibitors/chemistry , Viral Proteins/chemistry
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