Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
J Am Chem Soc ; 143(48): 20095-20108, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1531986

ABSTRACT

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/chemistry , Fluorescent Dyes/chemistry , Ligands , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/metabolism , Agammaglobulinaemia Tyrosine Kinase/metabolism , Catalytic Domain , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Half-Life , Humans , Piperidines/chemistry , Piperidines/metabolism , Proteolysis , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , SARS-CoV-2/enzymology
2.
Int J Mol Sci ; 22(13)2021 Jun 30.
Article in English | MEDLINE | ID: covidwho-1288906

ABSTRACT

Coronavirus disease (COVID)-19 is the leading global health threat to date caused by a severe acute respiratory syndrome coronavirus (SARS-CoV-2). Recent clinical trials reported that the use of Bruton's tyrosine kinase (BTK) inhibitors to treat COVID-19 patients could reduce dyspnea and hypoxia, thromboinflammation, hypercoagulability and improve oxygenation. However, the mechanism of action remains unclear. Thus, this study employs structure-based virtual screening (SBVS) to repurpose BTK inhibitors acalabrutinib, dasatinib, evobrutinib, fostamatinib, ibrutinib, inositol 1,3,4,5-tetrakisphosphate, spebrutinib, XL418 and zanubrutinib against SARS-CoV-2. Molecular docking is conducted with BTK inhibitors against structural and nonstructural proteins of SARS-CoV-2 and host targets (ACE2, TMPRSS2 and BTK). Molecular mechanics-generalized Born surface area (MM/GBSA) calculations and molecular dynamics (MD) simulations are then carried out on the selected complexes with high binding energy. Ibrutinib and zanubrutinib are found to be the most potent of the drugs screened based on the results of computational studies. Results further show that ibrutinib and zanubrutinib could exploit different mechanisms at the viral entry and replication stage and could be repurposed as potential inhibitors of SARS-CoV-2 pathogenesis.


Subject(s)
Adenine/analogs & derivatives , Drug Repositioning , Molecular Dynamics Simulation , Piperidines/chemistry , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Pyrimidines/chemistry , Adenine/chemistry , Adenine/metabolism , Adenine/therapeutic use , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Agammaglobulinaemia Tyrosine Kinase/metabolism , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , Humans , Molecular Docking Simulation , Piperidines/metabolism , Piperidines/therapeutic use , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/metabolism , Pyrazoles/therapeutic use , Pyrimidines/metabolism , Pyrimidines/therapeutic use , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Thermodynamics , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
3.
Elife ; 92020 11 23.
Article in English | MEDLINE | ID: covidwho-940328

ABSTRACT

Bruton's tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here, we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19.


Treatments for blood cancers, such as leukemia and lymphoma, rely heavily on chemotherapy, using drugs that target a vulnerable aspect of the cancer cells. B-cells, a type of white blood cell that produces antibodies, require a protein called Bruton's tyrosine kinase, or BTK for short, to survive. The drug ibrutinib (Imbruvica) is used to treat B-cell cancers by blocking BTK. The BTK protein consists of several regions. One of them, known as the kinase domain, is responsible for its activity as an enzyme (which allows it to modify other proteins by adding a 'tag' known as a phosphate group). The other regions of BTK, known as regulatory modules, control this activity. In BTK's inactive form, the regulatory modules attach to the kinase domain, blocking the regulatory modules from interacting with other proteins. When BTK is activated, it changes its conformation so the regulatory regions detach and become available for interactions with other proteins, at the same time exposing the active kinase domain. Ibrutinib and other BTK drugs in development bind to the kinase domain to block its activity. However, it is not known how this binding affects the regulatory modules. Previous efforts to study how drugs bind to BTK have used a version of the protein that only had the kinase domain, instead of the full-length protein. Now, Joseph et al. have studied full-length BTK and how it binds to five different drugs. The results reveal that ibrutinib and another drug called dasatinib both indirectly disrupt the normal position of the regulatory domains pushing BTK toward a conformation that resembles the activated state. By contrast, the three other compounds studied do not affect the inactive structure. Joseph et al. also examined a mutation in BTK that confers resistance against ibrutinib. This mutation increases the activity of BTK by disrupting the inactive structure, leading to B cells surviving better. Understanding how drug resistance mechanisms can work will lead to better drug treatment strategies for cancer. BTK is also a target in other diseases such as allergies or asthma and even COVID-19. If interactions between partner proteins and the regulatory domain are important in these diseases, then they may be better treated with drugs that maintain the regulatory modules in their inactive state. This research will help to design drugs that are better able to control BTK activity.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Catalytic Domain , Protein Conformation/drug effects , Protein Kinase Inhibitors/pharmacology , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/metabolism , Adenine/pharmacology , Agammaglobulinaemia Tyrosine Kinase/chemistry , Agammaglobulinaemia Tyrosine Kinase/genetics , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/virology , Dasatinib/chemistry , Dasatinib/metabolism , Dasatinib/pharmacology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/prevention & control , Models, Molecular , Molecular Structure , Mutation , Piperidines/chemistry , Piperidines/metabolism , Piperidines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , SARS-CoV-2/physiology , src Homology Domains/genetics
4.
J Nanosci Nanotechnol ; 20(12): 7311-7323, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-680345

ABSTRACT

We started a study on the molecular docking of six potential pharmacologically active inhibitors compounds that can be used clinically against the COVID-19 virus, in this case, remdesivir, ribavirin, favipiravir, galidesivir, hydroxychloroquine and chloroquine interacting with the main COVID-19 protease in complex with a COVID-19 N3 protease inhibitor. The highest values of affinity energy found in order from highest to lowest were chloroquine (CHL), hydroxychloroquine (HYC), favipiravir (FAV), galidesivir (GAL), remdesivir (REM) and ribavirin (RIB). The possible formation of hydrogen bonds, associations through London forces and permanent electric dipole were analyzed. The values of affinity energy obtained for the hydroxychloroquine ligands was -9.9 kcal/mol and for the chloroquine of -10.8 kcal/mol which indicate that the coupling contributes to an effective improvement of the affinity energies with the protease. Indicating that, the position chosen to make the substitutions may be a pharmacophoric group, and cause changes in the protease.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cysteine Endopeptidases/chemistry , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Adenine/administration & dosage , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/pharmacology , Adenosine/analogs & derivatives , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/administration & dosage , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Amides/administration & dosage , Amides/chemistry , Amides/pharmacology , Antiviral Agents/administration & dosage , Binding Sites , COVID-19 , Chloroquine/administration & dosage , Chloroquine/chemistry , Chloroquine/pharmacology , Coronavirus 3C Proteases , Drug Interactions , Humans , Hydrogen Bonding , Hydroxychloroquine/administration & dosage , Hydroxychloroquine/chemistry , Hydroxychloroquine/pharmacology , Ligands , Molecular Docking Simulation , Nanotechnology , Pandemics , Protease Inhibitors/administration & dosage , Pyrazines/administration & dosage , Pyrazines/chemistry , Pyrazines/pharmacology , Pyrrolidines/administration & dosage , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Ribavirin/administration & dosage , Ribavirin/chemistry , Ribavirin/pharmacology , SARS-CoV-2 , Static Electricity
5.
Eur J Med Chem ; 201: 112557, 2020 Sep 01.
Article in English | MEDLINE | ID: covidwho-597389

ABSTRACT

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.


Subject(s)
Coronavirus Infections/drug therapy , Exoribonucleases/antagonists & inhibitors , Methyltransferases/antagonists & inhibitors , Nucleosides/chemistry , Pneumonia, Viral/drug therapy , RNA Caps/metabolism , S-Adenosylmethionine/analogs & derivatives , S-Adenosylmethionine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Adenine/chemistry , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Exoribonucleases/metabolism , Humans , Methylation , Methyltransferases/metabolism , Molecular Docking Simulation , Pandemics , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , RNA Caps/chemistry , RNA Caps/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
6.
J Phys Chem Lett ; 11(11): 4430-4435, 2020 Jun 04.
Article in English | MEDLINE | ID: covidwho-233085

ABSTRACT

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.


Subject(s)
Betacoronavirus/enzymology , Nucleotidyltransferases/chemistry , RNA-Dependent RNA Polymerase/chemistry , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/metabolism , Adenosine/analogs & derivatives , Amides/chemistry , Amides/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Coronavirus Infections/epidemiology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Molecular Docking Simulation , Nucleotidyltransferases/metabolism , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Structure, Tertiary , Pyrazines/chemistry , Pyrazines/metabolism , Pyrrolidines/chemistry , Pyrrolidines/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2
SELECTION OF CITATIONS
SEARCH DETAIL