scholarly journals Acrylamide Fragment Inhibitors that Induce Unprecedented Conformational Distortions in Enterovirus 71 3C and SARS-CoV-2 Main Protease

2020 ◽  
Author(s):  
Bo Qin ◽  
Gregory B. Craven ◽  
Pengjiao Hou ◽  
Xinran Lu ◽  
Emma S. Child ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Catalytic Triad ◽  
Active Site Cysteine ◽  
Dimeric Unit ◽  
Main Protease ◽  
Selective Agents ◽  
Catalytic Cysteine

ABSTRACTRNA viruses are critically dependent upon virally encoded proteases that cleave the viral polyproteins into functional mature proteins. Many of these proteases are structurally conserved with an essential catalytic cysteine and this offers the opportunity to irreversibly inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that selectively target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we effectively re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold as well as similar catalytic-triad. We demonstrate that the hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. In addition, we provide the first demonstration that targeting the active site cysteine of Mpro can also have profound allosteric effects, distorting secondary structures required for formation of the active dimeric unit of Mpro. These new data provide novel mechanistic insights into the design of EV71 3Cpro and SARS-CoV-2 Mpro inhibitors and identify acrylamide-tagged pharmacophores for elaboration into more selective agents of therapeutic potential.

scholarly journals Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains

2017 ◽  
Author(s):  
Marie Morrow ◽  
Michael Morgan ◽  
Marcello Clerici ◽  
Katerina Growkova ◽  
Ming Yan ◽  
...  
Keyword(s):  
Active Site ◽  
Tight Binding ◽  
Dominant Negative ◽  
Structural Basis ◽  
Deubiquitinating Enzymes ◽  
Active Site Cysteine ◽  
Binding Domains ◽  
Common Strategy ◽  
Catalytic Cysteine

ABSTRACTA common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

scholarly journals Using serpins cysteine protease cross-specificity to possibly trap SARS-CoV-2 Mpro with reactive center loop chimera

2020 ◽  
Vol 134 (17) ◽  
pp. 2235-2241
Author(s):  
Mohamad Aman Jairajpuri ◽  
Shoyab Ansari
Keyword(s):  
Cysteine Protease ◽  
Active Site ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Therapeutic Potential ◽  
Cysteine Proteases ◽  
Protease Inhibition ◽  
Reactive Center Loop ◽  
Main Protease ◽  
Reactive Center

Abstract Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin–Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.

scholarly journals MPI8 is Potent Against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L

2021 ◽  
Author(s):  
Xinyu R Ma ◽  
Yugendar R Alugubelli ◽  
Yuying Ma ◽  
Erol Can Vatansever ◽  
Danielle A Scott ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin L ◽  
Critical Role ◽  
Cathepsin K ◽  
Host Cells ◽  
High Potency ◽  
Active Site Cysteine ◽  
Cellular Analysis ◽  
Main Protease ◽  
Ic50 Values

A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10-3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules

2014 ◽  
Vol 70 (5) ◽  
pp. 1311-1320 ◽  
Author(s):  
Antje Schäfer ◽  
Monika Kuhn ◽  
Hermann Schindelin
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Covalent Attachment ◽  
Adenylation Domain ◽  
Active Site Cysteine ◽  
The Status ◽  
Catalytic Cysteine ◽  
Covalently Linked ◽  
First Time

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1–ubiquitin2arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.

scholarly journals Response of Severe EV71-Infected Patients to Hyperimmune Plasma Treatment: A Pilot Study

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 625
Author(s):  
Chonnamet Techasaensiri ◽  
Artit Wongsa ◽  
Thanyawee Puthanakit ◽  
Kulkanya Chokephaibulkit ◽  
Tawee Chotpitayasunondh ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Antibody Therapy ◽  
Foot And Mouth Disease ◽  
Neurological Complications ◽  
Neurological Involvement ◽  
Healthy Donors ◽  
Enterovirus A71 ◽  
Number Of Patients ◽  
Causative Agents ◽  
East And Southeast Asia

Hand, foot, and mouth disease (HFMD) is highly prevalent in East and Southeast Asia. It particularly affects children under five years of age. The most common causative agents are coxsackieviruses A6 and A16, and enterovirus A71 (EV71). The clinical presentation is usually mild and self-limited, but, in some cases, severe and fatal complications develop. To date, no specific therapy or worldwide vaccine is available. In general, viral infection invokes both antibody and cell-mediated immune responses. Passive immunity transfer can ameliorate the severe symptoms of diseases such as COVID-19, influenza, MERS, and SARS. Hyperimmune plasma (HIP) from healthy donors with high anti-EV71 neutralizing titer were used to transfuse confirmed EV71-infected children with neurological involvement (n = 6). It resulted in recovery within three days, with no neurological sequelae apparent upon examination 14 days later. Following HIP treatment, plasma chemokines were decreased, whereas anti-inflammatory and pro-inflammatory cytokines gradually increased. Interestingly, IL-6 and G-CSF levels in cerebrospinal fluid declined sharply within three days. These findings indicate that HIP has therapeutic potential for HFMD with neurological complications. However, given the small number of patients who have been treated, a larger cohort study should be undertaken. Successful outcomes would stimulate the development of anti-EV71 monoclonal antibody therapy.

scholarly journals Toward the Identification of Potential α-Ketoamide Covalent Inhibitors for SARS-CoV-2 Main Protease: Fragment-Based Drug Design and MM-PBSA Calculations

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1004
Author(s):  
Mahmoud A. El Hassab ◽  
Mohamed Fares ◽  
Mohammed K. Abdel-Hamid Amin ◽  
Sara T. Al-Rashood ◽  
Amal Alharbi ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Binding Free Energy ◽  
Stable Complex ◽  
Active Site Residues ◽  
Structure Based Drug Design ◽  
Enzyme Active Site ◽  
Potential Inhibitor ◽  
Main Protease ◽  
Covalent Docking

Since December 2019, the world has been facing the outbreak of the SARS-CoV-2 pandemic that has infected more than 149 million and killed 3.1 million people by 27 April 2021, according to WHO statistics. Safety measures and precautions taken by many countries seem insufficient, especially with no specific approved drugs against the virus. This has created an urgent need to fast track the development of new medication against the virus in order to alleviate the problem and meet public expectations. The SARS-CoV-2 3CL main protease (Mpro) is one of the most attractive targets in the virus life cycle, which is responsible for the processing of the viral polyprotein and is a key for the ribosomal translation of the SARS-CoV-2 genome. In this work, we targeted this enzyme through a structure-based drug design (SBDD) protocol, which aimed at the design of a new potential inhibitor for Mpro. The protocol involves three major steps: fragment-based drug design (FBDD), covalent docking and molecular dynamics (MD) simulation with the calculation of the designed molecule binding free energy at a high level of theory. The FBDD step identified five molecular fragments, which were linked via a suitable carbon linker, to construct our designed compound RMH148. The mode of binding and initial interactions between RMH148 and the enzyme active site was established in the second step of our protocol via covalent docking. The final step involved the use of MD simulations to test for the stability of the docked RMH148 into the Mpro active site and included precise calculations for potential interactions with active site residues and binding free energies. The results introduced RMH148 as a potential inhibitor for the SARS-CoV-2 Mpro enzyme, which was able to achieve various interactions with the enzyme and forms a highly stable complex at the active site even better than the co-crystalized reference.

Sign in / Sign up

Export Citation Format

Share Document