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1.
Int J Mol Sci ; 23(14)2022 Jul 15.
Article in English | MEDLINE | ID: covidwho-1964008

ABSTRACT

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a pivotal enzyme in tocopherol and plastoquinone synthesis and a potential target for novel herbicides. Thirty-five pyridine derivatives were selected to establish a Topomer comparative molecular field analysis (Topomer CoMFA) model to obtain correlation information between HPPD inhibitory activity and the molecular structure. A credible and predictive Topomer CoMFA model was established by "split in two R-groups" cutting methods and fragment combinations (q2 = 0.703, r2 = 0.957, ONC = 6). The established model was used to screen out more active compounds and was optimized through the auto in silico ligand directing evolution (AILDE) platform to obtain potential HPPD inhibitors. Twenty-two new compounds with theoretically good HPPD inhibition were obtained by combining the high-activity contribution substituents in the existing molecules with the R-group search via Topomer search. Molecular docking results revealed that most of the 22 fresh compounds could form stable π-π interactions. The absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction and drug-like properties made 9 compounds potential HPPD inhibitors. Molecular dynamics simulation indicated that Compounds Y12 and Y14 showed good root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values and stability. According to the AILDE online verification, 5 new compounds with potential HPPD inhibition were discovered as HPPD inhibitor candidates. This study provides beneficial insights for subsequent HPPD inhibitor design.


Subject(s)
4-Hydroxyphenylpyruvate Dioxygenase , Herbicides , Computers , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Herbicides/chemistry , Herbicides/pharmacology , Hydrolases/metabolism , Ligands , Molecular Docking Simulation , Molecular Structure
2.
Molecules ; 27(7)2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1785838

ABSTRACT

Obesity is the most common nutritional disorder in the developed world and is associated with important comorbidities. Pancreatic lipase (PL) inhibitors play a key role in the metabolism of human fat. A series of novel epoxyketones peptide derivatives were investigated for their pancreatic lipase inhibitory activity. The epoxyketone moiety is a well-known reactive electrophile group that has been used as part of proteasome inhibitors in cancer therapy, and it is widely believed that these are very selective for targeting the proteasome active site. Here we investigated various peptide derivatives with an epoxide warhead for their anti-lipase activity. The assessment of these novel epoxyketones was performed by an in-house method that we developed for rapid screening and identification of lipase inhibitors using GC-FID. Herein, we present a novel anti-lipase pharmacophore based on epoxyketone peptide derivatives that showed potent anti-lipase activity. Many of these derivatives had comparable or more potent activity than the clinically used lipase inhibitors such as orlistat. In addition, the lipase appears to be inhibited by a wide range of epoxyketone analogues regardless of the configuration of the epoxide in the epoxyketone moiety. The presented data in this study shows the first example of the use of epoxyketone peptides as novel lipase inhibitors.


Subject(s)
Peptides , Proteasome Inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Epoxy Compounds/pharmacology , Humans , Lipase , Peptides/chemistry , Peptides/pharmacology , Proteasome Endopeptidase Complex/chemistry , Proteasome Inhibitors/chemistry
3.
Nat Commun ; 13(1): 621, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1671551

ABSTRACT

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Guanosine Monophosphate/analogs & derivatives , Phosphoramides/chemistry , Phosphoramides/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , COVID-19/virology , Cryoelectron Microscopy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/pharmacology , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Proteins/genetics
4.
Science ; 372(6547): 1169-1175, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1583231

ABSTRACT

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cystathionine gamma-Lyase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hydrogen Sulfide/metabolism , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Biofilms , Crystallography, X-Ray , Cystathionine gamma-Lyase/chemistry , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/metabolism , Drug Discovery , Drug Resistance, Bacterial , Drug Synergism , Drug Tolerance , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Docking Simulation , Molecular Structure , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development
5.
Pharmacol Res ; 172: 105820, 2021 10.
Article in English | MEDLINE | ID: covidwho-1531713

ABSTRACT

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enter the host cells through the interaction between its receptor binding domain (RBD) of spike glycoprotein with angiotensin-converting enzyme 2 (ACE2) receptor on the plasma membrane of host cell. Neutralizing antibodies and peptide binders of RBD can block viral infection, however, the concern of accessibility and affordability of viral infection inhibitors has been raised. Here, we report the identification of natural compounds as potential SARS-CoV-2 entry inhibitors using the molecular docking-based virtual screening coupled with bilayer interferometry (BLI). From a library of 1871 natural compounds, epigallocatechin gallate (EGCG), 20(R)-ginsenoside Rg3 (RRg3), 20(S)-ginsenoside Rg3 (SRg3), isobavachalcone (Ibvc), isochlorogenic A (IscA) and bakuchiol (Bkc) effectively inhibited pseudovirus entry at concentrations up to 100 µM. Among these compounds, four compounds, EGCG, Ibvc, salvianolic acid A (SalA), and isoliensinine (Isl), were effective in inhibiting SARS-CoV-2-induced cytopathic effect and plaque formation in Vero E6 cells. The EGCG was further validated with no observable animal toxicity and certain antiviral effect against SARS-CoV-2 pseudovirus mutants (D614G, N501Y, N439K & Y453F). Interestingly, EGCG, Bkc and Ibvc bind to ACE2 receptor in BLI assay, suggesting a dual binding to RBD and ACE2. Current findings shed some insight into identifications and validations of SARS-CoV-2 entry inhibitors from natural compounds.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Antiviral Agents/chemistry , Biological Products/chemistry , COVID-19/drug therapy , Enzyme Inhibitors/chemistry , SARS-CoV-2/enzymology , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antiviral Agents/pharmacology , Binding, Competitive , Biological Products/pharmacology , Catechin/analogs & derivatives , Catechin/pharmacology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Chalcones/pharmacology , Chlorogenic Acid/analogs & derivatives , Chlorogenic Acid/pharmacology , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Ginsenosides/pharmacology , Humans , Interferometry , Mice, Inbred C57BL , Molecular Dynamics Simulation , Phenols/pharmacology , Protein Binding
6.
Int J Mol Sci ; 22(20)2021 Oct 15.
Article in English | MEDLINE | ID: covidwho-1470891

ABSTRACT

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new pathogen from the family of Coronaviridae that caused a global pandemic of COVID-19 disease. In the absence of effective antiviral drugs, research of novel therapeutic targets such as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) becomes essential. This viral protein is without a human counterpart and thus represents a unique prospective drug target. However, in vitro biological evaluation testing on RdRp remains difficult and is not widely available. Therefore, we prepared a database of commercial small-molecule compounds and performed an in silico high-throughput virtual screening on the active site of the SARS-CoV-2 RdRp using ensemble docking. We identified a novel thioether-amide or guanidine-linker class of potential RdRp inhibitors and calculated favorable binding free energies of representative hits by molecular dynamics simulations coupled with Linear Interaction Energy calculations. This innovative procedure maximized the respective phase-space sampling and yielded non-covalent inhibitors representing small optimizable molecules that are synthetically readily accessible, commercially available as well as suitable for further biological evaluation and mode of action studies.


Subject(s)
Antiviral Agents/chemistry , Enzyme Inhibitors/chemistry , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , Amides/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Catalytic Domain , Databases, Chemical , Drug Design , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Guanidine/chemistry , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/isolation & purification , Structure-Activity Relationship , Sulfides/chemistry , Thermodynamics , Viral Proteins/metabolism
7.
Bioorg Med Chem ; 46: 116366, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1437407

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, which started in late 2019, drove the scientific community to conduct innovative research to contain the spread of the pandemic and to care for those already affected. Since then, the search for new drugs that are effective against the virus has been strengthened. Featuring a relatively low cost of production under well-defined methods of cultivation, fungi have been providing a diversity of antiviral metabolites with unprecedented chemical structures. In this review, we present viral RNA infections highlighting SARS-CoV-2 morphogenesis and the infectious cycle, the targets of known antiviral drugs, and current developments in this area such as drug repurposing. We also explored the metabolic adaptability of fungi during fermentation to produce metabolites active against RNA viruses, along with their chemical structures, and mechanisms of action. Finally, the state of the art of research on SARS-CoV-2 inhibitors of fungal origin is reported, highlighting the metabolites selected by docking studies.


Subject(s)
Antiviral Agents/pharmacology , Biological Products/pharmacology , COVID-19/drug therapy , Fungi/chemistry , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , Biological Products/chemistry , COVID-19/epidemiology , Cell Line , Drug Repositioning , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Pandemics , SARS-CoV-2/physiology
8.
Drug Res (Stuttg) ; 71(8): 462-472, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1404894

ABSTRACT

BACKGROUND: Replication of SARS-CoV-2 depends on viral RNA-dependent RNA-polymerase (RdRp). Remdesivir, the broad-spectrum RdRp inhibitor acts as nucleoside-analogues (NAs). Remdesivir has initially been repurposed as a promising drug against SARS-CoV-2 infection with some health hazards like liver damage, allergic reaction, low blood-pressure, and breathing-shortness, throat-swelling. In comparison, theaflavin-3'-O-gallate (TFMG), the abundant black tea component has gained importance in controlling viral infection. TFMG is a non-toxic, non-invasive, antioxidant, anticancer and antiviral molecule. RESULTS: Here, we analyzed the inhibitory effect of theaflavin-3'-O-gallate on SARS CoV-2 RdRp in comparison with remdesivir by molecular-docking study. TFMG has been shown more potent in terms of lower Atomic-Contact-Energy (ACE) and higher occupancy of surface area; -393.97 Kcal/mol and 771.90 respectively, favoured with lower desolvation-energy; -9.2: Kcal/mol. TFMG forms more rigid electrostatic and H-bond than remdesivir. TFMG showed strong affinity to RNA primer and template and RNA passage-site of RdRp. CONCLUSIONS: TFMG can block the catalytic residue, NTP entry site, cation binding site, nsp7-nsp12 junction with binding energy of -6. 72 Kcal/mol with Ki value of 11.79, and interface domain with binding energy of -7.72 and -6.16 Kcal/mol with Ki value of 2.21 and 30.71 µM. And most importantly, TFMG shows antioxidant/anti-inflammatory/antiviral effect on human studies.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Biflavonoids/pharmacology , COVID-19/drug therapy , Catechin/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Gallic Acid/analogs & derivatives , Molecular Docking Simulation , SARS-CoV-2/drug effects , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/chemistry , Biflavonoids/chemistry , COVID-19/virology , Catalytic Domain , Catechin/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/chemistry , Gallic Acid/chemistry , Gallic Acid/pharmacology , Protein Conformation , SARS-CoV-2/enzymology , Structure-Activity Relationship
9.
Phys Chem Chem Phys ; 23(36): 20117-20128, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1404891

ABSTRACT

The ongoing pandemic caused by SARS-CoV-2 emphasizes the need for effective therapeutics. Inhibition of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by nucleotide analogs provides a promising antiviral strategy. One common group of RdRp inhibitors, 2'-modified nucleotides, are reported to exhibit different behaviors in the SARS-CoV-2 RdRp transcription assay. Three of these analogs, 2'-O-methyl UTP, Sofosbuvir, and 2'-methyl CTP, act as effective inhibitors in previous biochemical experiments, while Gemcitabine and ara-UTP show no inhibitory activity. To understand the impact of the 2'-modification on their inhibitory effects, we conducted extensive molecular dynamics simulations and relative binding free energy calculations using the free energy perturbation method on SARS-CoV-2 replication-transcription complex (RTC) with these five nucleotide analogs. Our results reveal that the five nucleotide analogs display comparable binding affinities to SARS-CoV-2 RdRp and they can all be added to the nascent RNA chain. Moreover, we examine how the incorporation of these nucleotide triphosphate (NTP) analogs will impact the addition of the next nucleotide. Our results indicate that 2'-O-methyl UTP can weaken the binding of the subsequent NTP and consequently lead to partial chain termination. Additionally, Sofosbuvir and 2'-methyl CTP can cause immediate termination due to the strong steric hindrance introduced by their bulky 2'-methyl groups. In contrast, nucleotide analogs with smaller substitutions, such as the fluorine atoms and the ara-hydroxyl group in Gemcitabine and ara-UTP, have a marginal impact on the polymerization process. Our findings are consistent with experimental observations, and more importantly, shed light on the detailed molecular mechanism of SARS-CoV-2 RdRp inhibition by 2'-substituted nucleotide analogs, and may facilitate the rational design of antiviral agents to inhibit SARS-CoV-2 RdRp.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nucleotides/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Models, Molecular , Nucleic Acid Conformation , Nucleotides/chemistry , SARS-CoV-2/enzymology
10.
ChemMedChem ; 16(23): 3548-3552, 2021 12 06.
Article in English | MEDLINE | ID: covidwho-1400781

ABSTRACT

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIß, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIß block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIß inhibition, the role of PI4KIIIß in SARS-CoV-2 entry/replication is debated.


Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Antiviral Agents/pharmacology , Enzyme Inhibitors/chemistry , Rhinovirus/physiology , SARS-CoV-2/physiology , Thiazoles/chemistry , Virus Replication/drug effects , Zika Virus/physiology , 1-Phosphatidylinositol 4-Kinase/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/pathology , COVID-19/virology , Cell Line , Drug Stability , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , SARS-CoV-2/isolation & purification , Thiazoles/metabolism , Zika Virus/isolation & purification , Zika Virus Infection/pathology
11.
Nat Commun ; 12(1): 4848, 2021 08 11.
Article in English | MEDLINE | ID: covidwho-1354102

ABSTRACT

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Subject(s)
Methyltransferases/chemistry , RNA Helicases/chemistry , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Models, Molecular , Phosphates/chemistry , Phosphates/metabolism , Protein Conformation , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/enzymology , Structure-Activity Relationship , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
12.
Chem Commun (Camb) ; 57(67): 8352-8355, 2021 Aug 28.
Article in English | MEDLINE | ID: covidwho-1337131

ABSTRACT

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(µ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-ß-d-glucose tetraacetate) and TG-RRE, [(µ-S-TG)Fe(NO)2]2 (TG = 1-thio-ß-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 µM for TGTA-RRE and 33 ± 2 µM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.


Subject(s)
Enzyme Inhibitors/pharmacology , Iron/pharmacology , Nitrogen Oxides/pharmacology , Peptide Hydrolases/metabolism , SARS-CoV-2/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Humans , Iron/chemistry , Models, Molecular , Molecular Structure , Nitrogen Oxides/chemistry , SARS-CoV-2/enzymology
13.
Int J Mol Sci ; 21(19)2020 Sep 27.
Article in English | MEDLINE | ID: covidwho-1299427

ABSTRACT

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.


Subject(s)
Enzyme Inhibitors/chemistry , Membrane Proteins , Molecular Docking Simulation , Nucleotidyltransferases , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Drug Evaluation, Preclinical , Endoplasmic Reticulum Chaperone BiP , Fluorescence Polarization , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/chemistry
14.
J Pharmacol Sci ; 147(1): 62-71, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1240460

ABSTRACT

Owing to the urgent need for therapeutic interventions against the SARS-coronavirus 2 (SARS-CoV-2) pandemic, we employed an in silico approach to evaluate the SARS-CoV-2 inhibitory potential of newly synthesized imidazoles. The inhibitory potentials of the compounds against SARS-CoV-2 drug targets - main protease (Mpro), spike protein (Spro) and RNA-dependent RNA polymerase (RdRp) were investigated through molecular docking analysis. The binding free energy of the protein-ligand complexes were estimated, pharmacophore models were generated and the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the compounds were determined. The compounds displayed various levels of binding affinities for the SARS-CoV-2 drug targets. Bisimidazole C2 scored highest against all the targets, with its aromatic rings including the two imidazole groups contributing to the binding. Among the phenyl-substituted 1H-imidazoles, C9 scored highest against all targets. C11 scored highest against Spro and C12 against Mpro and RdRp among the thiophene-imidazoles. The compounds interacted with HIS 41 - CYS 145 and GLU 288 - ASP 289 - GLU 290 of Mpro, ASN 501 of Spro receptor binding motif and some active site amino acids of RdRp. These novel imidazole compounds could be further developed as drug candidates against SARS-CoV-2 following lead optimization and experimental studies.


Subject(s)
Computational Biology/methods , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Molecular Docking Simulation/methods , SARS-CoV-2/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Protein Binding/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
15.
PLoS One ; 16(5): e0251801, 2021.
Article in English | MEDLINE | ID: covidwho-1226905

ABSTRACT

Drug repurposing studies targeting inhibition of RNA dependent RNA polymerase (RdRP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have exhibited the potential effect of small molecules. In the present work a detailed interaction study between the phytochemicals from Indian medicinal plants and the RdRP of SARS-CoV-2 has been performed. The top four phytochemicals obtained through molecular docking were, swertiapuniside, cordifolide A, sitoindoside IX, and amarogentin belonging to Swertia chirayita, Tinospora cordifolia and Withania somnifera. These ligands bound to the RdRP were further studied using molecular dynamics simulations. The principal component analysis of these systems showed significant conformational changes in the finger and thumb subdomain of the RdRP. Hydrogen bonding, salt-bridge and water mediated interactions supported by MM-GBSA free energy of binding revealed strong binding of cordifolide A and sitoindoside IX to RdRP. The ligand-interacting residues belonged to either of the seven conserved motifs of the RdRP. These residues were polar and charged amino acids, namely, ARG 553, ARG 555, ASP 618, ASP 760, ASP 761, GLU 811, and SER 814. The glycosidic moieties of the phytochemicals were observed to form favourable interactions with these residues. Hence, these phytochemicals may hold the potential to act as RdRP inhibitors owing to their stability in binding to the druggable site.


Subject(s)
COVID-19/drug therapy , Enzyme Inhibitors/pharmacology , Phytochemicals/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Biological Products/chemistry , Biological Products/pharmacology , Drug Discovery , Enzyme Inhibitors/chemistry , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Phytochemicals/chemistry , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects
16.
J Enzyme Inhib Med Chem ; 36(1): 1016-1028, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1226495

ABSTRACT

Elastase is a proteolytic enzyme belonging to the family of hydrolases produced by human neutrophils, monocytes, macrophages, and endothelial cells. Human neutrophil elastase is known to play multiple roles in the human body, but an increase in its activity may cause a variety of diseases. Elastase inhibitors may prevent the development of psoriasis, chronic kidney disease, respiratory disorders (including COVID-19), immune disorders, and even cancers. Among polyphenolic compounds, some flavonoids and their derivatives, which are mostly found in herbal plants, have been revealed to influence elastase release and its action on human cells. This review focuses on elastase inhibitors that have been discovered from natural sources and are biochemically characterised as flavonoids. The inhibitory activity on elastase is a characteristic of flavonoid aglycones and their glycoside and methylated, acetylated and hydroxylated derivatives. The presented analysis of structure-activity relationship (SAR) enables the determination of the chemical groups responsible for evoking an inhibitory effect on elastase. Further study especially of the in vivo efficacy and safety of the described natural compounds is of interest in order to gain better understanding of their health-promoting potential.


Subject(s)
Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Leukocyte Elastase/antagonists & inhibitors , Neutrophils/enzymology , COVID-19/drug therapy , COVID-19/metabolism , Enzyme Inhibitors/chemistry , Flavonoids/chemistry , Humans , Leukocyte Elastase/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Neutrophils/drug effects , Structure-Activity Relationship
17.
SLAS Discov ; 26(6): 757-765, 2021 07.
Article in English | MEDLINE | ID: covidwho-1194439

ABSTRACT

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Subject(s)
Adenosine/analogs & derivatives , High-Throughput Screening Assays , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/chemistry , Adenosine/pharmacology , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Methylation , Methyltransferases , Models, Molecular , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Tritium , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism
18.
J Med Chem ; 64(9): 5632-5644, 2021 05 13.
Article in English | MEDLINE | ID: covidwho-1193564

ABSTRACT

To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 µM) of HCoV-229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/enzymology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Oleanolic Acid/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Virus Replication/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Line , Dose-Response Relationship, Drug , Endoribonucleases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Models, Molecular , Oleanolic Acid/chemical synthesis , Oleanolic Acid/chemistry , Oleanolic Acid/pharmacology , Viral Nonstructural Proteins/metabolism
19.
SLAS Discov ; 26(6): 766-774, 2021 07.
Article in English | MEDLINE | ID: covidwho-1192708

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.


Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Exonucleases/antagonists & inhibitors , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
20.
SLAS Discov ; 26(6): 749-756, 2021 07.
Article in English | MEDLINE | ID: covidwho-1136206

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , Nitro Compounds/pharmacology , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiparasitic Agents/chemistry , Antiparasitic Agents/pharmacology , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Drug Repositioning , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Mass Spectrometry/methods , Methylation , Nitro Compounds/chemistry , Prescription Drugs/chemistry , Prescription Drugs/pharmacology , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Thiazoles/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
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