Structural Analysis of the OC43 Coronavirus 2'-O-RNA Methyltransferase.

The OC43 coronavirus is a human pathogen that usually causes only the common cold. One of its key enzymes, similar to other coronaviruses, is the 2'-O-RNA methyltransferase (MTase), which is essential for viral RNA stability and expression. Here, we report the crystal structure of the 2'-O-RNA MTase in a complex with the pan-methyltransferase inhibitor sinefungin solved at 2.2-Å resolution. The structure reveals an overall fold consistent with the fold observed in other coronaviral MTases. The major differences are in the conformation of the C terminus of the nsp16 subunit and an additional helix in the N terminus of the nsp10 subunits. The structural analysis also revealed very high conservation of the S-adenosyl methionine (SAM) binding pocket, suggesting that the SAM pocket is a suitable spot for the design of antivirals effective against all human coronaviruses. IMPORTANCE Some coronaviruses are dangerous pathogens, while some cause only common colds. The reasons are not understood, although the spike proteins probably play an important role. However, to understand the coronaviral biology in sufficient detail, we need to compare the key enzymes from different coronaviruses. We solved the crystal structure of 2'-O-RNA methyltransferase of the OC43 coronavirus, a virus that usually causes mild colds. The structure revealed some differences in the overall fold but also revealed that the SAM binding site is conserved, suggesting that development of antivirals against multiple coronaviruses is feasible.

Quantum chemical insight into molecular structure, NBO analysis of the hydrogen-bonded interactions, spectroscopic (FT-IR, FT-Raman), drug likeness and molecular docking of the novel anti COVID-19 molecule 2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluorophenyl)acetamide - dimer.

Novel antiviral active molecule 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro- phenyl)acetamide has been synthesised and characterized by FT-IR and FT-Raman spectra. The equilibrium geometry, natural bond orbital calculations and vibrational assignments have been carried out using density functional B3LYP method with the 6-311G++(d,p) basis set. The complete vibrational assignments for all the vibrational modes have been supported by normal coordinate analysis, force constants and potential energy distributions. A detailed analysis of the intermolecular interactions has been performed based on the Hirshfeld surfaces. Drug likeness has been carried out based on Lipinski's rule and the absorption, distribution, metabolism, excretion and toxicity of the title molecule has been calculated. Antiviral potency of 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro-phenyl) acetamide has been investigated by docking against SARS-CoV-2 protein. The optimized geometry shows near-planarity between the phenyl ring and the pyrimidine ring. Differences in the geometries due to the substitution of the most electronegative fluorine atom and intermolecular contacts due to amino pyrimidine were analyzed. NBO analysis reveals the formation of two strong stable hydrogen bonded N-H···N intermolecular interactions and weak intramolecular interactions C-H···O and N-H···O. The Hirshfeld surfaces and consequently the 2D-fingerprint confirm the nature of intermolecular interactions and their quantitative contributions towards the crystal packing. The red shift in N-H stretching frequency exposed from IR substantiate the formation of N-H···N intermolecular hydrogen bond. Drug likeness and absorption, distribution, metabolism, excretion and toxicity properties analysis gives an idea about the pharmacokinetic properties of the title molecule. The binding energy -8.7 kcal/mol of the nonbonding interaction present a clear view that 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro- phenyl) acetamide can irreversibly interact with SARS-CoV-2 protease.

SARS-CoV-2 receptor is co-expressed with elements of the kinin-kallikrein, renin-angiotensin and coagulation systems in alveolar cells.

SARS-CoV-2, the pathogenic agent of COVID-19, employs angiotensin converting enzyme-2 (ACE2) as its cell entry receptor. Clinical data reveal that in severe COVID-19, SARS-CoV-2 infects the lung, leading to a frequently lethal triad of respiratory insufficiency, acute cardiovascular failure, and coagulopathy. Physiologically, ACE2 plays a role in the regulation of three systems that could potentially be involved in the pathogenesis of severe COVID-19: the kinin-kallikrein system, resulting in acute lung inflammatory edema; the renin-angiotensin system, promoting cardiovascular instability; and the coagulation system, leading to thromboembolism. Here we assembled a healthy human lung cell atlas meta-analysis with ~ 130,000 public single-cell transcriptomes and show that key elements of the bradykinin, angiotensin and coagulation systems are co-expressed with ACE2 in alveolar cells and associated with their differentiation dynamics, which could explain how changes in ACE2 promoted by SARS-CoV-2 cell entry result in the development of the three most severe clinical components of COVID-19.

Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on Mpro inhibition.

Modelling the structural and reactivity landscapes of tucatinib with special reference to its wavefunction-dependent properties and screening for potential antiviral activity.

HER-2 type breast cancer is one of the most aggressive malignancies found in women. Tucatinib is recently developed and approved as a potential medicine to fight this disease. In this manuscript, we present the gross structural features of this compound and its reactivity and wave function properties using computational simulations. Density functional theory was used to optimise the ground state geometry of the molecule and molecular docking was used to predict biological activity. As the electrons interact with electromagnetic radiations, electronic excitations between different energy levels are analysed in detail using time-dependent density functional theory. Various intermolecular and intermolecular interactions are analysed and reaction sites for attacking electrophiles and nucleophiles identified. Information entropy calculations show that the compound is inherently stable. Docking with COVID-19 proteins show docking score of - 9.42, - 8.93, - 8.45 and - 8.32 kcal/mol respectively indicating high interaction between the drug and proteins. Hence, this is an ideal candidate to study repurposing of existing drugs to combat the pandemic.

Targeting SARS-CoV-2 RNA-dependent RNA polymerase: An in silico drug repurposing for COVID-19.

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), took more lives than combined epidemics of SARS, MERS, H1N1, and Ebola. Currently, the prevention and control of spread are the goals in COVID-19 management as there are no specific drugs to cure or vaccines available for prevention. Hence, the drug repurposing was explored by many research groups, and many target proteins have been examined. The major protease (M pro), and RNA-dependent RNA polymerase (RdRp) are two target proteins in SARS-CoV-2 that have been validated and extensively studied for drug development in COVID-19. The RdRp shares a high degree of homology between those of two previously known coronaviruses, SARS-CoV and MERS-CoV. Methods: In this study, the FDA approved library of drugs were docked against the active site of RdRp using Schrodinger's computer-aided drug discovery tools for in silico drug-repurposing. Results: We have shortlisted 14 drugs from the Standard Precision docking and interaction-wise study of drug-binding with the active site on the enzyme. These drugs are antibiotics, NSAIDs, hypolipidemic, coagulant, thrombolytic, and anti-allergics. In molecular dynamics simulations, pitavastatin, ridogrel and rosoxacin displayed superior binding with the active site through ARG555 and divalent magnesium. Conclusion: Pitavastatin, ridogrel and rosoxacin can be further optimized in preclinical and clinical studies to determine their possible role in COVID-19 treatment.

Identification of saquinavir as a potent inhibitor of dimeric SARS-CoV2 main protease through MM/GBSA.

Among targets selected for studies aimed at identifying potential inhibitors against COVID-19, SARS-CoV2 main proteinase (Mpro) is highlighted. Mpro is indispensable for virus replication and is a promising target of potential inhibitors of COVID-19. Recently, monomeric SARS-CoV2 Mpro, drug repurposing, and docking methods have facilitated the identification of several potential inhibitors. Results were refined through the assessment of dimeric SARS-CoV2 Mpro, which represents the functional state of enzyme. Docking and molecular dynamics (MD) simulations combined with molecular mechanics/generalized Born surface area (MM/GBSA) studies indicated that dimeric Mpro most significantly impacts binding affinity tendency compared with the monomeric state, which suggests that dimeric state is most useful when performing studies aimed at identifying drugs targeting Mpro. In this study, we extend previous research by performing docking and MD simulation studies coupled with an MM/GBSA approach to assess binding of dimeric SARS-CoV2 Mpro to 12 FDA-approved drugs (darunavir, indinavir, saquinavir, tipranavir, diosmin, hesperidin, rutin, raltegravir, velpatasvir, ledipasvir, rosuvastatin, and bortezomib), which were identified as the best candidates for the treatment of COVID-19 in some previous dockings studies involving monomeric SARS-CoV2 Mpro. This analysis identified saquinavir as a potent inhibitor of dimeric SARS-CoV2 Mpro; therefore, the compound may have clinical utility against COVID-19. Graphical abstract.

Structure-based lead optimization of herbal medicine rutin for inhibiting SARS-CoV-2's main protease.

Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic with very limited specific treatments. To fight COVID-19, various traditional antiviral medicines have been prescribed in China to infected patients with mild to moderate symptoms and received unexpected success in controlling the disease. However, the molecular mechanisms of how these herbal medicines interact with the SARS-CoV-2 virus that causes COVID-19 have remained elusive. It is well known that the main protease (Mpro) of SARS-CoV-2 plays an important role in maturation of many viral proteins such as the RNA-dependent RNA polymerase. Here, we explore the underlying molecular mechanisms of the computationally determined top candidate, namely, rutin which is a key component in many traditional antiviral medicines such as Lianhuaqinwen and Shuanghuanlian, for inhibiting the viral target-Mpro. Using in silico methods (docking and molecular dynamics simulations), we revealed the dynamics and energetics of rutin when interacting with the Mpro of SARS-CoV-2, suggesting that the highly hydrophilic rutin molecule can be bound inside the Mpro's pocket (active site) and possibly inhibit its biological functions. In addition, we optimized the structure of rutin and designed two more hydrophobic analogs, M1 and M2, which satisfy the rule of five for western medicines and demonstrated that they (M2 in particular) possess much stronger binding affinities to the SARS-COV-2s Mpro than rutin, due to the enhanced hydrophobic interaction as well as more hydrogen bonds. Therefore, our results provide invaluable insights into the mechanism of a ligand's binding inside the Mpro and shed light on future structure-based designs of high-potent inhibitors for SARS-CoV-2 Mpro.

Biochemical screening for SARS-CoV-2 main protease inhibitors.

The Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) pandemic represents a global challenge. SARS-CoV-2's ability to replicate in host cells relies on the action of its non-structural proteins, like its main protease (Mpro). This cysteine protease acts by processing the viruses' precursor polyproteins. As proteases, together with polymerases, are main targets of antiviral drug design, we here have performed biochemical high throughput screening (HTS) with recombinantly expressed SARS-CoV-2 Mpro. A fluorescent assay was used to identify inhibitors in a compound library containing known drugs, bioactive molecules and natural products. These screens led to the identification of 13 inhibitors with IC50 values ranging from 0.2 µM to 23 µM. The screens confirmed several known SARS-CoV Mpro inhibitors as inhibitors of SARS-CoV-2 Mpro, such as the organo-mercuric compounds thimerosal and phenylmercuric acetate. Benzophenone derivatives could also be identified among the most potent screening hits. Additionally, Evans blue, a sulfonic acid-containing dye, could be identified as an Mpro inhibitor. The obtained compounds could be of interest as lead compounds for the development of future SARS-CoV-2 drugs.

Room-temperature neutron and X-ray data collection of 3CL Mpro from SARS-CoV-2.

The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro) into a series of smaller functional proteins. At the heart of 3CL Mpro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-binding cavity, and to visualize the hydrogen-bonding networks throughout the enzyme, room-temperature neutron and X-ray data were collected from a large H/D-exchanged crystal of ligand-free (apo) 3CL Mpro.

Nucleotide analogues as inhibitors of SARS-CoV Polymerase.

SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate, which are the active forms of two other anti-viral agents, Alovudine and AZT (an FDA-approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.

Virtual Double-System Single-Box: A Nonequilibrium Alchemical Technique for Absolute Binding Free Energy Calculations: Application to Ligands of the SARS-CoV-2 Main Protease.

In the context of drug-receptor binding affinity calculations using molecular dynamics techniques, we implemented a combination of Hamiltonian replica exchange (HREM) and a novel nonequilibrium alchemical methodology, called virtual double-system single-box, with increased accuracy, precision, and efficiency with respect to the standard nonequilibrium approaches. The method has been applied for the determination of absolute binding free energies of 16 newly designed noncovalent ligands of the main protease (3CLpro) of SARS-CoV-2. The core structures of 3CLpro ligands were previously identified using a multimodal structure-based ligand design in combination with docking techniques. The calculated binding free energies for four additional ligands with known activity (either for SARS-CoV or SARS-CoV-2 main protease) are also reported. The nature of binding in the 3CLpro active site and the involved residues besides the CYS-HYS catalytic dyad have been thoroughly characterized by enhanced sampling simulations of the bound state. We have identified several noncongeneric compounds with predicted low micromolar activity for 3CLpro inhibition, which may constitute possible lead compounds for the development of antiviral agents in Covid-19 treatment.

Targeting Proteases for Treating COVID-19.

The unprecedented pandemic of coronavirus disease 2019 (COVID-19) demands effective treatment for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The infection of SARS-CoV-2 critically depends on diverse viral or host proteases, which mediate viral entry, viral protein maturation, as well as the pathogenesis of the viral infection. Endogenous and exogenous agents targeting for proteases have been proved to be effective toward a variety of viral infections ranging from HIV to influenza virus, suggesting protease inhibitors as a promising antiviral treatment for COVID-19. In this Review, we discuss how host and viral proteases participated in the pathogenesis of COVID-19 as well as the prospects and ongoing clinical trials of protease inhibitors as treatments.

Exploring RdRp-remdesivir interactions to screen RdRp inhibitors for the management of novel coronavirus 2019-nCoV.

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has resulted in an increasing number of patients globally, and has become a highly lethal pathogenic member of the coronavirus family affecting humans. 2019-nCoV has established itself as one of the most threatening pandemics that human beings have faced, and therefore analysis and evaluation of all possible responses against infection is required. One such strategy includes utilizing the knowledge gained from the SARS and MERS outbreaks regarding existing antivirals. Indicating a potential for success, one of the drugs, remdesivir, under repurposing studies, has shown positive results in initial clinical studies. Therefore, in the current work, the authors have attempted to utilize the remdesivir-RdRp complex - RdRp (RNA-dependent RNA polymerase) being the putative target for remdesivir - to screen a library of the already reported RdRp inhibitor database. Further clustering on the basis of structural features and scoring refinement was performed to filter out false positive hits. Finally, molecular dynamics simulation was carried out to validate the identification of hits as RdRp inhibitors against novel coronavirus 2019-nCoV. The results yielded two putative hits which can inhibit RdRp with better potency than remdesivir, subject to further biological evaluation.

The inhibitory effect of some natural bioactive compounds against SARS-CoV-2 main protease: insights from molecular docking analysis and molecular dynamic simulation.

This work aimed at evaluating the inhibitory effect of ten natural bioactive compounds (1-10) as potential inhibitors of SARS-CoV-2-3CL main protease (PDB ID: 6LU7) and SARS-CoV main proteases (PDB IDs: 2GTB and 3TNT) by molecular docking analysis. The inhibitory effect of all studied compounds was studied with compared to some proposed antiviral drugs which currently used in COVID-19 treatment such as chloroquine, hydroxychloroquine, azithromycin, remdesivir, baloxvir, lopinavir, and favipiravir. Homology modeling and sequence alignment was computed to evaluate the similarity between the SARS-CoV-2-3CL main protease and other SARS-CoV receptors. ADMET properties of all studied compounds were computed and reported. Also, molecular dynamic (MD) simulation was performed on the compound which has the highest binding affinity inside 6LU7 obtained from molecular docking analysis to study it is stability inside receptor in explicit water solvent. Based on molecular docking analysis, we found that caulerpin has the highest binding affinity inside all studied receptors compared to other bioactive compounds and studied drugs. Our homology modeling and sequence alignment showed that SARS-CoV main protease (PDB ID: 3TNT) shares high similarity with 3CLpro (96.00%). Also, ADMET properties confirmed that caulerpin obeys Lipinski's rule and passes ADMET property, which make it a promising compound to act as a new safe natural drug against SARS-CoV-2-3CL main protease. Finally, MD simulation confirmed that the complex formed between caulerpin and 3CLpro is stable in water explicit and had no major effect on the flexibility of the protein throughout the simulations and provided a suitable basis for our study. Also, binding free energy between caulerpin and 6LU7 confirmed the efficacy of the caulerpin molecule against SARS-CoV-2 main protease. So, this study suggested that caulerpin could be used as a potential candidate in COVID-19 treatment.

Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir.

SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.

Discovery of potent inhibitors for SARS-CoV-2's main protease by ligand-based/structure-based virtual screening, MD simulations, and binding energy calculations.

COVID-19 has caused lockdowns all over the world in early 2020, as a global pandemic. Both theoretical and experimental efforts are seeking to find an effective treatment to suppress the virus. In silico drug design can play a vital role in identifying promising drug candidates against COVID-19. Herein, we focused on the main protease of SARS-CoV-2 that has crucial biological functions in the virus. We performed a ligand-based virtual screening followed by a docking screening for testing approved drugs and bioactive compounds listed in the DrugBank and ChEMBL databases. The top 8 docking results were advanced to all-atom MD simulations to study the relative stability of the protein-ligand interactions. MD simulations support that the catalytic residue, His41, has a neutral side chain with a protonated delta position. An absolute binding energy (ΔG) of -42 kJ mol-1 for the protein-ligand (Mpro-N3) complex has been calculated using the potential-of-mean-force (geometrical) approach. Furthermore, the relative binding energies were computed for the top docking results. Our results suggest several promising approved and bioactive inhibitors of SARS-CoV-2 Mpro as follows: a bioactive compound, ChEMBL275592, which has the best MM/GBSA binding energy; the second-best compound, montelukast, is an approved drug used in the treatment of asthma and allergic rhinitis; the third-best compound, ChEMBL288347, is a bioactive compound. Bromocriptine and saquinavir are other approved drugs that also demonstrate stability in the active site of Mpro, albeit their relative binding energies are low compared to the N3 inhibitor. This study provides useful insights into de novo protein design and novel inhibitor development, which could reduce the cost and time required for the discovery of a potent drug to combat SARS-CoV-2.

Combining fragment docking with graph theory to improve ligand docking for homology model structures.

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease.

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Alkaloids from Cryptolepis sanguinolenta as Potential Inhibitors of SARS-CoV-2 Viral Proteins: An In Silico Study.

The ongoing global pandemic caused by the human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions of people and claimed hundreds of thousands of lives. The absence of approved therapeutics to combat this disease threatens the health of all persons on earth and could cause catastrophic damage to society. New drugs are therefore urgently required to bring relief to people everywhere. In addition to repurposing existing drugs, natural products provide an interesting alternative due to their widespread use in all cultures of the world. In this study, alkaloids from Cryptolepis sanguinolenta have been investigated for their ability to inhibit two of the main proteins in SARS-CoV-2, the main protease and the RNA-dependent RNA polymerase, using in silico methods. Molecular docking was used to assess binding potential of the alkaloids to the viral proteins whereas molecular dynamics was used to evaluate stability of the binding event. The results of the study indicate that all 13 alkaloids bind strongly to the main protease and RNA-dependent RNA polymerase with binding energies ranging from -6.7 to -10.6 kcal/mol. In particular, cryptomisrine, cryptospirolepine, cryptoquindoline, and biscryptolepine exhibited very strong inhibitory potential towards both proteins. Results from the molecular dynamics study revealed that a stable protein-ligand complex is formed upon binding. Alkaloids from Cryptolepis sanguinolenta therefore represent a promising class of compounds that could serve as lead compounds in the search for a cure for the corona virus disease.