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1.
Anal Chem ; 94(31): 10949-10958, 2022 08 09.
Article in English | MEDLINE | ID: covidwho-1960210

ABSTRACT

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins' exposed surface, hindered accessibility, and exchange processes occurring on a µs-ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.


Subject(s)
COVID-19 , Protein Aggregates , Humans , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular/methods , SARS-CoV-2
2.
Biomolecules ; 12(7)2022 07 10.
Article in English | MEDLINE | ID: covidwho-1928473

ABSTRACT

In this study, we combined all-atom MD simulations, the ensemble-based mutational scanning of protein stability and binding, and perturbation-based network profiling of allosteric interactions in the SARS-CoV-2 spike complexes with a panel of cross-reactive and ultra-potent single antibodies (B1-182.1 and A23-58.1) as well as antibody combinations (A19-61.1/B1-182.1 and A19-46.1/B1-182.1). Using this approach, we quantify the local and global effects of mutations in the complexes, identify protein stability centers, characterize binding energy hotspots, and predict the allosteric control points of long-range interactions and communications. Conformational dynamics and distance fluctuation analysis revealed the antibody-specific signatures of protein stability and flexibility of the spike complexes that can affect the pattern of mutational escape. A network-based perturbation approach for mutational profiling of allosteric residue potentials revealed how antibody binding can modulate allosteric interactions and identified allosteric control points that can form vulnerable sites for mutational escape. The results show that the protein stability and binding energetics of the SARS-CoV-2 spike complexes with the panel of ultrapotent antibodies are tolerant to the effect of Omicron mutations, which may be related to their neutralization efficiency. By employing an integrated analysis of conformational dynamics, binding energetics, and allosteric interactions, we found that the antibodies that neutralize the Omicron spike variant mediate the dominant binding energy hotpots in the conserved stability centers and allosteric control points in which mutations may be restricted by the requirements of the protein folding stability and binding to the host receptor. This study suggested a mechanism in which the patterns of escape mutants for the ultrapotent antibodies may not be solely determined by the binding interaction changes but are associated with the balance and tradeoffs of multiple local and global factors, including protein stability, binding affinity, and long-range interactions.


Subject(s)
COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , COVID-19/genetics , Humans , Molecular Conformation , Mutation , Protein Binding , Protein Stability , SARS-CoV-2/genetics
3.
Sci Rep ; 12(1): 2505, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1747189

ABSTRACT

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , Binding Sites , COVID-19/pathology , COVID-19/virology , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Docking Simulation , Nitriles/chemistry , Nitriles/metabolism , Nitriles/pharmacology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/metabolism , Quinazolines/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Virus Replication/drug effects
4.
Int J Mol Sci ; 22(21)2021 Nov 02.
Article in English | MEDLINE | ID: covidwho-1745034

ABSTRACT

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.


Subject(s)
DNA/chemistry , DNA/metabolism , Dendrimers/chemistry , Phenols/chemistry , Sulfides/chemistry , Animals , Male , Molecular Conformation , Salmon , Spermatozoa/metabolism
5.
Molecules ; 27(5)2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1715570

ABSTRACT

A new flavonoid, Jusanin, (1) has been isolated from the aerial parts of Artemisia commutata. The chemical structure of Jusanin has been elucidated using 1D, 2D NMR, and HR-Ms spectroscopic methods to be 5,2',4'-trihydroxy-6,7,5'-trimethoxyflavone. Being new in nature, the inhibition potential of 1 has been estimated against SARS-CoV-2 using different in silico techniques. Firstly, molecular similarity and fingerprint studies have been conducted for Jusanin against co-crystallized ligands of eight different SARS-CoV-2 essential proteins. The studies indicated the similarity between 1 and X77, the co-crystallized ligand SARS-CoV-2 main protease (PDB ID: 6W63). To confirm the obtained results, a DFT study was carried out and indicated the similarity of (total energy, HOMO, LUMO, gap energy, and dipole moment) between 1 and X77. Accordingly, molecular docking studies of 1 against the target enzyme have been achieved and showed that 1 bonded correctly in the protein's active site with a binding energy of -19.54 Kcal/mol. Additionally, in silico ADMET in addition to the toxicity evaluation of Jusanin against seven models have been preceded and indicated the general safety and the likeness of Jusanin to be a drug. Finally, molecular dynamics simulation studies were applied to investigate the dynamic behavior of the Mpro-Jusanin complex and confirmed the correct binding at 100 ns. In addition to 1, three other metabolites have been isolated and identified to be сapillartemisin A (2), methyl-3-[S-hydroxyprenyl]-cumarate (3), and ß-sitosterol (4).


Subject(s)
Artemisia/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Flavonoids/chemistry , SARS-CoV-2/enzymology , Animals , Artemisia/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/metabolism , Density Functional Theory , Flavonoids/isolation & purification , Flavonoids/metabolism , Flavonoids/pharmacology , Humans , Lethal Dose 50 , Male , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Rats , SARS-CoV-2/isolation & purification , Skin/drug effects , Skin/pathology
6.
Int J Mol Sci ; 23(4)2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1700048

ABSTRACT

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against the SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with single nanobodies Nb6, VHH E, and complex with VHH E/VHH V nanobody combination. We combined coarse-grained and all-atom molecular simulations and collective dynamics analysis with binding free energy scanning, perturbation-response scanning, and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein might exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH E/VHH V nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.


Subject(s)
SARS-CoV-2/genetics , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Allosteric Regulation , Cryoelectron Microscopy , Molecular Conformation , Molecular Dynamics Simulation , Protein Stability , Spike Glycoprotein, Coronavirus/genetics
7.
Int J Mol Sci ; 23(3)2022 Jan 31.
Article in English | MEDLINE | ID: covidwho-1667196

ABSTRACT

Structural and functional studies of the SARS-CoV-2 spike proteins have recently determined distinct functional states of the B.1.1.7 and B.1.351 spike variants, providing a molecular framework for understanding the mechanisms that link the effect of mutations with the enhanced virus infectivity and transmissibility. A detailed dynamic and energetic analysis of these variants was undertaken in the present work to quantify the effects of different mutations on functional conformational changes and stability of the SARS-CoV-2 spike protein. We employed the efficient and accurate coarse-grained (CG) simulations of multiple functional states of the D614G mutant, B.1.1.7 and B.1.351 spike variants to characterize conformational dynamics of the SARS-CoV-2 spike proteins and identify dynamic signatures of the functional regions that regulate transitions between the closed and open forms. By combining molecular simulations with full atomistic reconstruction of the trajectories and the ensemble-based mutational frustration analysis, we characterized how the intrinsic flexibility of specific spike regions can control functional conformational changes required for binding with the host-cell receptor. Using the residue-based mutational scanning of protein stability, we determined protein stability hotspots and identified potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. The results suggested that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites and the inter-protomer hinges of functional motions. The proposed mechanism of mutation-induced energetic frustration may result in greater adaptability and the emergence of multiple conformational states in the open form. This study suggested that SARS-CoV-2 B.1.1.7 and B.1.351 variants may leverage the intrinsic plasticity of functional regions in the spike protein for mutation-induced modulation of protein dynamics and allosteric regulation to control binding with the host cell receptor.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/ultrastructure , Allosteric Regulation , Binding Sites , COVID-19/pathology , Humans , Molecular Conformation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Stability , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics
8.
Nucleic Acids Res ; 50(2): 635-650, 2022 01 25.
Article in English | MEDLINE | ID: covidwho-1621653

ABSTRACT

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus/drug effects , Coronavirus/enzymology , Methyltransferases/antagonists & inhibitors , Methyltransferases/chemistry , Methyltransferases/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Binding Sites , Coronavirus/genetics , Drug Discovery , Humans , Methylation , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , Structure-Activity Relationship
9.
Chem Biodivers ; 18(11): e2100674, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1615945

ABSTRACT

Chemical investigation on a Streptomyces sp. strain MS180069 isolated from a sediment sample collected from the South China Sea, yielded the new benzo[f]isoindole-dione alkaloid, bhimamycin J (1). The structure was determined by extensive spectroscopic analysis, including HRMS, 1D, 2D NMR, and X-ray diffraction techniques. A molecular docking study revealed 1 as a new molecular motif that binds with human angiotensin converting enzyme2 (ACE2), recently described as the cell surface receptor responsible for uptake of 2019-CoV-2. Using enzyme assays we confirm that 1 inhibits human ACE2 79.7 % at 25 µg/mL.


Subject(s)
Alkaloids/chemistry , Geologic Sediments/microbiology , Isoindoles/chemistry , Streptomyces/chemistry , Alkaloids/metabolism , Alkaloids/pharmacology , Alkaloids/therapeutic use , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Fungi/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Isoindoles/isolation & purification , Isoindoles/metabolism , Isoindoles/pharmacology , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Docking Simulation , SARS-CoV-2/isolation & purification , Streptomyces/isolation & purification , Streptomyces/metabolism
10.
Chem Biodivers ; 19(1): e202100668, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1611203

ABSTRACT

Forsyqinlingines C (1) and D (2), two C9 -monoterpenoid alkaloids bearing a rare skeleton, were isolated from the ripe fruits of Forsythia suspensa. Their structures, including absolute configurations, were fully elucidated by extensive spectroscopic data and ECD experiments. The plausible biogenetic pathway for compounds 1 and 2 was also proposed. In vitro, two C9 -monoterpenoid alkaloids showed anti-inflammatory activity performed by the inhibitory effect on the release of ß-glucuronidase in rat polymorphonuclear leukocytes (PMNs), as well as antiviral activity against influenza A (H1N1) virus and respiratory syncytial virus (RSV).


Subject(s)
Alkaloids/chemistry , Anti-Inflammatory Agents/chemistry , Antiviral Agents/chemistry , Forsythia/chemistry , Monoterpenes/chemistry , Alkaloids/isolation & purification , Alkaloids/pharmacology , Animals , Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Forsythia/metabolism , Fruit/chemistry , Fruit/metabolism , Glucuronidase/metabolism , Influenza A Virus, H1N1 Subtype/drug effects , Magnetic Resonance Spectroscopy , Molecular Conformation , Neutrophils/cytology , Neutrophils/drug effects , Neutrophils/metabolism , Platelet Activating Factor/pharmacology , Rats , Respiratory Syncytial Viruses/drug effects
11.
Nat Commun ; 12(1): 7345, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1585860

ABSTRACT

The emergence of SARS-CoV-2 Kappa and Beta variants with enhanced transmissibility and resistance to neutralizing antibodies has created new challenges for the control of the ongoing COVID-19 pandemic. Understanding the structural nature of Kappa and Beta spike (S) proteins and their association with ACE2 is of significant importance. Here we present two cryo-EM structures for each of the Kappa and Beta spikes in the open and open-prone transition states. Compared with wild-type (WT) or G614 spikes, the two variant spikes appear more untwisted/open especially for Beta, and display a considerable population shift towards the open state as well as more pronounced conformational dynamics. Moreover, we capture four conformational states of the S-trimer/ACE2 complex for each of the two variants, revealing an enlarged conformational landscape for the Kappa and Beta S-ACE2 complexes and pronounced population shift towards the three RBDs up conformation. These results implicate that the mutations in Kappa and Beta may modify the kinetics of receptor binding and viral fusion to improve virus fitness. Combined with biochemical analysis, our structural study shows that the two variants are enabled to efficiently interact with ACE2 receptor despite their sensitive ACE2 binding surface is modified to escape recognition by some potent neutralizing MAbs. Our findings shed new light on the pathogenicity and immune evasion mechanism of the Beta and Kappa variants.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Cryoelectron Microscopy , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19 , Humans , Kinetics , Molecular Conformation , Mutation , Protein Binding
12.
Molecules ; 26(21)2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1512506

ABSTRACT

Three silver(I) dipeptide complexes [Ag(GlyGly)]n(NO3)n (AgGlyGly), [Ag2(GlyAla)(NO3)2]n (AgGlyAla) and [Ag2(HGlyAsp)(NO3)]n (AgGlyAsp) were prepared, investigated and characterized by vibrational spectroscopy (mid-IR), elemental and thermogravimetric analysis and mass spectrometry. For AgGlyGly, X-ray crystallography was also performed. Their stability in biological testing media was verified by time-dependent NMR measurements. Their in vitro antimicrobial activity was evaluated against selected pathogenic microorganisms. Moreover, the influence of silver(I) dipeptide complexes on microbial film formation was described. Further, the cytotoxicity of the complexes against selected cancer cells (BLM, MDA-MB-231, HeLa, HCT116, MCF-7 and Jurkat) and fibroblasts (BJ-5ta) using a colorimetric MTS assay was tested, and the selectivity index (SI) was identified. The mechanism of action of Ag(I) dipeptide complexes was elucidated and discussed by the study in terms of their binding affinity toward the CT DNA, the ability to cleave the DNA and the ability to influence numbers of cells within each cell cycle phase. The new silver(I) dipeptide complexes are able to bind into DNA by noncovalent interaction, and the topoisomerase I inhibition study showed that the studied complexes inhibit its activity at a concentration of 15 µM.


Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Dipeptides/chemistry , Silver/chemistry , Anti-Infective Agents/chemical synthesis , Antineoplastic Agents/chemical synthesis , Cell Cycle/drug effects , Cell Line, Tumor , Chemical Phenomena , Chemistry Techniques, Synthetic , Coordination Complexes/chemical synthesis , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Stability , Humans , Molecular Conformation , Molecular Dynamics Simulation , Spectrum Analysis , Structure-Activity Relationship , Thermogravimetry
13.
J Am Chem Soc ; 143(46): 19306-19310, 2021 11 24.
Article in English | MEDLINE | ID: covidwho-1510556

ABSTRACT

The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N-1HN and 13C'-1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.


Subject(s)
Coronavirus 3C Proteases/chemistry , Crystallography, X-Ray/methods , SARS-CoV-2 , COVID-19 , Catalytic Domain , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Dynamics Simulation , Protein Conformation , Protein Folding , Software , X-Rays
14.
Sci Rep ; 11(1): 21735, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1504063

ABSTRACT

The COVID19 pandemic, caused by SARS-CoV-2, has infected more than 200 million people worldwide. Due to the rapid spreading of SARS-CoV-2 and its impact, it is paramount to find effective treatments against it. Human neutralizing antibodies are an effective method to fight viral infection. However, the recent discovery of new strains that substantially change the S-protein sequence has raised concern about vaccines and antibodies' effectiveness. Here, using molecular simulations, we investigated the binding mechanisms between the S-protein and several antibodies. Multiple mutations were included to understand the strategies for antibody escape in new variants. We found that the combination of mutations K417N, E484K, L452R, and T478K produced higher binding energy to ACE2 than the wild type, suggesting higher efficiency to enter host cells. The mutations' effect depends on the antibody class. While Class I enhances the binding avidity in the presence of N501Y mutation, class II antibodies showed a sharp decline in the binding affinity. Our simulations suggest that Class I antibodies will remain effective against the new strains. In contrast, Class II antibodies will have less affinity to the S-protein, potentially affecting these antibodies' efficiency.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , COVID-19/immunology , COVID-19/virology , Mutation , SARS-CoV-2/genetics , Antibodies, Viral/immunology , Cluster Analysis , Computational Biology , Computer Simulation , Humans , Hydrogen Bonding , Molecular Conformation , Molecular Dynamics Simulation , Protein Binding , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism
15.
J Med Chem ; 65(4): 2956-2970, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1500413

ABSTRACT

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Antiviral Agents/pharmacology , Biological Products/pharmacology , COVID-19/drug therapy , Cathepsin L/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Animals , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , COVID-19/metabolism , Cathepsin L/metabolism , Chlorocebus aethiops , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Conformation , Proteomics , Structure-Activity Relationship , Vero Cells
16.
Int J Mol Sci ; 22(19)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1463709

ABSTRACT

Cancer persists as a global challenge due to the extent to which conventional anticancer therapies pose high risks counterbalanced with their therapeutic benefit. Naturally occurring substances stand as an important safer alternative source for anticancer drug development. In the current study, a series of modified lupane and ursane derivatives was subjected to in vitro screening on the NCI-60 cancer cell line panel. Compounds 6 and 7 have been identified as highly active with GI50 values ranging from 0.03 µM to 5.9 µM (compound 6) and 0.18-1.53 µM (compound 7). Thus, these two compounds were further assessed in detail in order to identify a possible antiproliferative mechanism of action. DAPI (4',6-diamidino-2-phenylindole) staining revealed that both compounds induced nuclei condensation and overall cell morphological changes consistent with apoptotic cell death. rtPCR analysis showed that both compounds induced upregulation of proapoptotic Bak and Bad genes while downregulating Bcl-XL and Bcl-2 antiapoptotic genes. Molecular docking analysis revealed that both compounds exhibited high scores for Bcl-XL inhibition, while compound 7 showed higher in silico Bcl-XL inhibition potential as compared to the native inhibitor ATB-737, suggesting that compounds may induce apoptotic cell death through targeted antiapoptotic protein inhibition, as well.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Biological Products/pharmacology , Triterpenes/pharmacology , Angiogenesis Inhibitors , Antineoplastic Agents/chemistry , Binding Sites , Biological Products/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Structure-Activity Relationship , Triterpenes/chemistry
17.
J Phys Chem Lett ; 11(19): 8084-8093, 2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-1387116

ABSTRACT

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.


Subject(s)
Epitopes/chemistry , Molecular Dynamics Simulation , Spike Glycoprotein, Coronavirus/chemistry , Algorithms , Betacoronavirus/chemistry , Binding Sites, Antibody , Glycosylation , Humans , Models, Molecular , Molecular Conformation , Peptides/chemistry , Polysaccharides/chemistry , SARS-CoV-2
18.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: covidwho-1370748

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K i] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (K i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Drug Discovery/methods , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Animals , COVID-19/drug therapy , COVID-19/virology , Cells, Cultured , Coronavirus 3C Proteases/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Genetic Engineering , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , SARS-CoV-2/metabolism , Structure-Activity Relationship , Virus Replication
19.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1368372

ABSTRACT

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Subject(s)
Coronavirus/physiology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Liquid , Computational Biology/methods , Coronavirus/drug effects , Coronavirus Infections/veterinary , Gene Expression Regulation, Viral , Glycosylation/drug effects , Infectious bronchitis virus/physiology , Models, Molecular , Molecular Conformation , Molecular Weight , Neutralization Tests , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Poultry Diseases/virology , Protein Transport , Spectrometry, Mass, Electrospray Ionization , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Virus Replication/drug effects
20.
Cell Rep ; 36(9): 109650, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1363915

ABSTRACT

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.


Subject(s)
COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/physiology , Nucleotides/metabolism , RNA, Viral/biosynthesis , SARS-CoV-2/physiology , Coronavirus RNA-Dependent RNA Polymerase/chemistry , High-Throughput Screening Assays , Humans , Models, Molecular , Molecular Conformation , Nucleotides/chemistry , RNA, Viral/chemistry , Single Molecule Imaging , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/physiology
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