Molecular mechanism of inhibiting the SARS-CoV-2 cell entry facilitator TMPRSS2 with camostat and nafamostat

The entry of the coronavirus SARS-CoV-2 into human lung cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations. In vitro assays confirm that both drugs inhibit the human protein TMPRSS2, a SARS-Cov-2 spike protein activator. As no experimental structure is available, we provide a model of the TMPRSS2 equilibrium structure and its fluctuations by relaxing an initial homology structure with extensive 330 microseconds of all-atom molecular dynamics (MD) and Markov modeling. Through Markov modeling, we describe the binding process of both drugs and a metabolic product of camostat (GBPA) to TMPRSS2, reaching a Michaelis complex (MC) state, which precedes the formation of a long-lived covalent inhibitory state. We find that nafamostat has a higher MC population than camostat and GBPA, suggesting that nafamostat is more readily available to form the stable covalent enzyme–substrate intermediate, effectively explaining its high potency. This model is backed by our in vitro experiments and consistent with previous virus cell entry assays. Our TMPRSS2–drug structures are made public to guide the design of more potent and specific inhibitors.

The authors unravel the molecular action principle of nafamostat and camostat, two potential COVID-19 drugs targeting the human protein TMPRSS2.     

Translating daily COVID-19 screening into a simple glucose test: a proof of concept study

Home testing is an attractive emerging strategy to combat the COVID-19 pandemic and prevent overloading of healthcare resources through at-home isolation, screening and monitoring of symptoms. However, current diagnostic technologies of SARS-CoV-2 still suffer from some drawbacks because of the tradeoffs between sensitivity, usability and costs, making the test unaffordable to most users at home. To address these limitations, taking advantage of clustered regularly interspaced short palindromic repeats (CRISPRs) and a portable glucose meter (PGM), we present a proof-of-concept demonstration of a target-responsive CRISPR-PGM system for translating SARS-CoV-2 detection into a glucose test. Using this system, a specific N gene, N protein, and pseudo-viruses of SARS-CoV-2 have been detected quantitatively with a PGM. Given the facile integration of various bioreceptors into the CRISPR-PGM system, the proposed method provides a starting point to provide patients with a single-device solution that can quantitatively monitor multiple COVID-19 biomarkers at home.

COVID-19 glucose test: translating SARS-CoV-2 detection into a glucose test is achieved by incorporating target-responsive rolling circle amplification and a CRISPR-based collateral cleavage module with a portable glucose meter.     

Selective covalent targeting of SARS-CoV-2 main protease by enantiopure chlorofluoroacetamide

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (M^pro) is a promising target for COVID-19 treatment. Here, we report an irreversible SARS-CoV-2 M^pro inhibitor possessing chlorofluoroacetamide (CFA) as a warhead for the covalent modification of M^pro. Ugi multicomponent reaction using chlorofluoroacetic acid enabled the rapid synthesis of dipeptidic CFA derivatives that identified 18 as a potent inhibitor of SARS-CoV-2 M^pro. Among the four stereoisomers, (R,R)-18 exhibited a markedly higher inhibitory activity against M^pro than the other isomers. Reaction kinetics and computational docking studies suggest that the R configuration of the CFA warhead is crucial for the rapid covalent inhibition of M^pro. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of proteinous cysteines and provide the basis for improving the potency and selectivity of CFA-based covalent inhibitors.

Chlorofluoroacetamide (CFA) was used as the warhead for covalent targeting of SARS-CoV-2 M^pro. The chirality at CFA showed marked influence on inhibitory activity, suggesting stereospecific activation of CFA for cysteine modification in the protein.     

Multiplex metal-detection based assay (MMDA) for COVID-19 diagnosis and identification of disease severity biomarkers

The ongoing COVID-19 pandemic caused by SARS-CoV-2 highlights the urgent need to develop sensitive methods for diagnosis and prognosis. To achieve this, multidimensional detection of SARS-CoV-2 related parameters including virus loads, immune response, and inflammation factors is crucial. Herein, by using metal-tagged antibodies as reporting probes, we developed a multiplex metal-detection based assay (MMDA) method as a general multiplex assay strategy for biofluids. This strategy provides extremely high multiplexing capability (theoretically over 100) compared with other reported biofluid assay methods. As a proof-of-concept, MMDA was used for serologic profiling of anti-SARS-CoV-2 antibodies. The MMDA exhibits significantly higher sensitivity and specificity than ELISA for the detection of anti-SARS-CoV-2 antibodies. By integrating the high dimensional data exploration/visualization tool (tSNE) and machine learning algorithms with in-depth analysis of multiplex data, we classified COVID-19 patients into different subgroups based on their distinct antibody landscape. We unbiasedly identified anti-SARS-CoV-2-nucleocapsid IgG and IgA as the most potently induced types of antibodies for COVID-19 diagnosis, and anti-SARS-CoV-2-spike IgA as a biomarker for disease severity stratification. MMDA represents a more accurate method for the diagnosis and disease severity stratification of the ongoing COVID-19 pandemic, as well as for biomarker discovery of other diseases.

A MMDA platform is developed by using metal-tagged antibodies as reporting probes combined with machine learning algorithms, as a general strategy for highly multiplexed biofluid assay.     

Platycodin D,a natural component of Platycodon grandiflorum,prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann–Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.Subject terms: Viral infection, Lipid signalling     

A Review of Human Coronaviruses’ Receptors: The Host-Cell Targets for the Crown Bearing Viruses

A novel human coronavirus prompted considerable worry at the end of the year 2019. Now, it represents a significant global health and economic burden. The newly emerged coronavirus disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the primary reason for the COVID-19 global pandemic. According to recent global figures, COVID-19 has caused approximately 243.3 million illnesses and 4.9 million deaths. Several human cell receptors are involved in the virus identification of the host cells and entering them. Hence, understanding how the virus binds to host-cell receptors is crucial for developing antiviral treatments and vaccines. The current work aimed to determine the multiple host-cell receptors that bind with SARS-CoV-2 and other human coronaviruses for the purpose of cell entry. Extensive research is needed using neutralizing antibodies, natural chemicals, and therapeutic peptides to target those host-cell receptors in extremely susceptible individuals. More research is needed to map SARS-CoV-2 cell entry pathways in order to identify potential viral inhibitors.     

Medicinal Plants,Phytochemicals, and Herbs to Combat Viral Pathogens Including SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome corona virus-2 (SARS-CoV-2), is the most important health issue, internationally. With no specific and effective antiviral therapy for COVID-19, new or repurposed antiviral are urgently needed. Phytochemicals pose a ray of hope for human health during this pandemic, and a great deal of research is concentrated on it. Phytochemicals have been used as antiviral agents against several viruses since they could inhibit several viruses via different mechanisms of direct inhibition either at the viral entry point or the replication stages and via immunomodulation potentials. Recent evidence also suggests that some plants and its components have shown promising antiviral properties against SARS-CoV-2. This review summarizes certain phytochemical agents along with their mode of actions and potential antiviral activities against important viral pathogens. A special focus has been given on medicinal plants and their extracts as well as herbs which have shown promising results to combat SARS-CoV-2 infection and can be useful in treating patients with COVID-19 as alternatives for treatment under phytotherapy approaches during this devastating pandemic situation.     

Molecular recognition of SARS-CoV-2 spike glycoprotein: quantum chemical hot spot and epitope analyses

Due to the COVID-19 pandemic, researchers have attempted to identify complex structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S-protein) with angiotensin-converting enzyme 2 (ACE2) or a blocking antibody. However, the molecular recognition mechanism—critical information for drug and antibody design—has not been fully clarified at the amino acid residue level. Elucidating such a microscopic mechanism in detail requires a more accurate molecular interpretation that includes quantum mechanics to quantitatively evaluate hydrogen bonds, XH/π interactions (X = N, O, and C), and salt bridges. In this study, we applied the fragment molecular orbital (FMO) method to characterize the SARS-CoV-2 S-protein binding interactions with not only ACE2 but also the B38 Fab antibody involved in ACE2-inhibitory binding. By analyzing FMO-based interaction energies along a wide range of binding interfaces carefully, we identified amino acid residues critical for molecular recognition between S-protein and ACE2 or B38 Fab antibody. Importantly, hydrophobic residues that are involved in weak interactions such as CH–O hydrogen bond and XH/π interactions, as well as polar residues that construct conspicuous hydrogen bonds, play important roles in molecular recognition and binding ability. Moreover, through these FMO-based analyses, we also clarified novel hot spots and epitopes that had been overlooked in previous studies by structural and molecular mechanical approaches. Altogether, these hot spots/epitopes identified between S-protein and ACE2/B38 Fab antibody may provide useful information for future antibody design, evaluation of the binding property of the SARS-CoV-2 variants including its N501Y, and small or medium drug design against the SARS-CoV-2.

Quantum chemical calculations investigated molecular recognition of SARS-CoV-2 spike glycoproteins including its N501Y variant for ACE2 and antibody. Hot spot and epitope analyses revealed key residues to design drugs and antibodies against COVID-19.     

Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 Mpro by QM/MM computational methods

SARS-CoV-2 M^pro is one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic. This work is focused on exploring its proteolysis reaction by means of QM/MM methods. The resulting free energy landscape of the process provides valuable information on the species appearing along the reaction path and suggests that the mechanism of action of this enzyme, taking place in four steps, slightly differs from that of other cysteine proteases. Our predictions, which are in agreement with some recently published experimental data, can be used to guide the design of COVID-19 antiviral compounds with clinical potential.

The molecular mechanism of the proteolysis reaction catalyzed by SARS-CoV-2 M^pro, one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic, is described using computational QM/MM methods.     

Coronavirus Infection-Associated Cell Death Signaling and Potential Therapeutic Targets

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus–host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.     

Screening of Natural Products Inhibitors of SARS-CoV-2 Entry

The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC₅₀) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC₅₀) of 91.65 µg/mL, a CC₅₀ of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors.     

Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity

The SARS-CoV-2 main protease (M^pro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 M^pro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 M^pro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of M^pro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

QM/MM simulations identify the mechanism of reaction of N3, a covalent peptidyl inhibitor of SARS-CoV-2 main protease. Modelling of two novel proposed compounds, B1 and B2, suggests that reversibility of covalent inhibition could be tailored.     

Does SARS-CoV-2 affect neurodegenerative disorders? TLR2, a potential receptor for SARS-CoV-2 in the CNS

The coronavirus (COVID-19) pandemic, caused by severe acute respiratory system coronavirus 2 (SARS-CoV-2), has created significant challenges for scientists seeking to understand the pathogenic mechanisms of SARS-CoV-2 infection and to identify the best therapies for infected patients. Although ACE2 is a known receptor for the virus and has been shown to mediate viral entry into the lungs, accumulating reports highlight the presence of neurological symptoms resulting from infection. As ACE2 expression is low in the central nervous system (CNS), these neurological symptoms are unlikely to be caused by ACE2-virus binding. In this review, we will discuss a proposed interaction between SARS-CoV-2 and Toll-like receptor 2 (TLR2) in the CNS. TLR2 is an innate immune receptor that recognizes exogenous microbial components but has also been shown to interact with multiple viral components, including the envelope (E) protein of SARS-CoV-2. In addition, TLR2 plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Based on these observations, we hypothesize that TLR2 may play a critical role in the response to SARS-CoV-2 infiltration in the CNS, thereby resulting in the induction or acceleration of AD and PD pathologies in patients.Subject terms: Neurodegeneration, Neurodegenerative diseases     

Determination of intermediate state structures in the opening pathway of SARS-CoV-2 spike using cryo-electron microscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19, a highly infectious disease that is severely affecting our society and welfare systems. In order to develop therapeutic interventions against this condition, one promising strategy is to target spike, the trimeric transmembrane glycoprotein that the virus uses to recognise and bind its host cells. Here we use a metainference cryo-electron microscopy approach to determine the opening pathway that brings spike from its inactive (closed) conformation to its active (open) one. The knowledge of the structures of the intermediate states of spike along this opening pathway enables us to identify a cryptic pocket that is not exposed in the open and closed states. These results underline the opportunities offered by the determination of the structures of the transient intermediate states populated during the dynamics of proteins to allow the therapeutic targeting of otherwise invisible cryptic binding sites.

A structural ensemble derived from cryo-electron microscopy reveals a cryptic pocket site in intermediate states along the opening pathway of the SARS-CoV-2 spike protein.     

In silico study of novel niclosamide derivatives,SARS-CoV-2 nonstructural proteins catalytic residue-targeting small molecules drug candidates

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated coronavirus disease 2019 (COVID-19) infection remains a global pandemic and health emergency with overwhelming social and economic impacts throughout the world. Therapeutics for COVID-19 are limited to only remdesivir; therefore, there is a need for combined, multidisciplinary efforts to develop new therapeutic molecules and explore the effectiveness of existing drugs against SARS-CoV-2. In the present study, we reported eight (SCOV-L-02, SCOV-L-09, SCOV-L-10, SCOV-L-11, SCOV-L-15, SCOV-L-18, SCOV-L-22, and SCOV-L-23) novel structurally related small-molecule derivatives of niclosamide (SCOV-L series) for their targeting potential against angiotensin-converting enzyme-2 (ACE2), type II transmembrane serine protease (TMPRSS2), and SARS-COV-2 nonstructural proteins (NSPs) including NSP5 (3CLpro), NSP3 (PLpro), and RdRp. Our correlation analysis suggested that ACE2 and TMPRSS2 modulate host immune response via regulation of immune-infiltrating cells at the site of tissue/organs entries. In addition, we identified some TMPRSS2 and ACE2 microRNAs target regulatory networks in SARS-CoV-2 infection and thus open up a new window for microRNAs-based therapy for the treatment of SARS-CoV-2 infection. Our in vitro study revealed that with the exception of SCOV-L-11 and SCOV-L-23 which were non-active, the SCOV-L series exhibited strict antiproliferative activities and non-cytotoxic effects against ACE2- and TMPRSS2-expressing cells. Our molecular docking for the analysis of receptor-ligand interactions revealed that SCOV-L series demonstrated high ligand binding efficacies (at higher levels than clinical drugs) against the ACE2, TMPRSS2, and SARS-COV-2 NSPs. SCOV-L-18, SCOV-L-15, and SCOV-L-09 were particularly found to exhibit strong binding affinities with three key SARS-CoV-2’s proteins: 3CLpro, PLpro, and RdRp. These compounds bind to the several catalytic residues of the proteins, and satisfied the criteria of drug-like candidates, having good adsorption, distribution, metabolism, excretion, and toxicity (ADMET) pharmacokinetic profile. Altogether, the present study suggests the therapeutic potential of SCOV-L series for preventing and managing SARs-COV-2 infection and are currently under detailed investigation in our lab.     

Trends in biosensing platforms for SARS-CoV-2 detection: A critical appraisal against standard detection tools

In this ongoing theme of coronavirus disease 2019 (COVID-19) pandemic, highly sensitive analytical testing platforms are extremely necessary to detect SARS-CoV-2 RNA and antiviral antibodies. To limit the viral spread, prompt and precise diagnosis is crucial to facilitate treatment and ensure effective isolation. Accurate detection of antibodies (IgG and IgM) is imperative to understand the prevalence of SARS-CoV-2 in public and to inspect the proportion of immune individuals. In this review, we demonstrate and evaluate some tests that have been used commonly to detect SARS-CoV-2. These include nucleic acid and serological tests for the detection of SARS-CoV-2 RNA and specific antibodies in infected people. Moreover, the vitality of biosensing technologies emphasizing on optical and electrochemical biosensors toward the detection of SARS-CoV-2 has also been discussed here. The early diagnosis of COVID-19 based on detection of reactive oxygen species overproduction because of virus-induced dysfunctioning of lung cells has also been highlighted.