Repurposing drugs and identification of inhibitors of integral proteins (spike protein and main protease) of SARS-CoV-2

COVID-19, the global pandemic caused by SARS-Corona virus 2 (SARS-CoV-2), recently ravaged the World with various efforts charged towards finding therapeutic drug targets for this novel virus. The identification of COVID-19 main protease (Mpro) opened the possibility of testing new families of inhibitors as potential anti-coronaviral drugs. Protein-drug interaction is of pivotal importance to understanding the structural features essential for any ligand affinity. This study evaluated the efficacy of an isolated bioactive plant compound and synthetic tetraazamacrocycles against COVID-19 Mpro by molecular docking. Molecular docking investigations were performed using PyRx, Auto Dock vina and Discovery Studio (DS) to analyze the inhibition probability of these compounds against COVID-19. COVID-19 Mpro (PDB ID: 6LU7: Resolution 2.16 Å) was docked with 1 flavonoid and 3 tetraaza-macrocyclic compounds comparatively with known anti-viral drugs (Remdesivir (REMD) and Nelfinavir (NELF)) and hydroxychloroquine (HCQ). Docking studies showed H-TEAD, 5 interacting with 5 residues having the highest binding affinity of −9.4 kcal/mol, followed by TEAD with 5 residue interactions and a binding affinity value of −9.4 kcal/mol, HA-TEAD, 7 has 5 interactions with a binding affinity of −9.3 kcal/mol, and Siam1 has 6 interactions with a binding energy of −7.8 kcal/mol. All the docked potential drugs have binding energies higher than the reference drugs HCQ, 1 and REMD, 2 connoting greater activity except NELF, 3 whose value is only lower than the 3 macrocycles (HA-TEAD, 7 and H-TEAD, 5 and TEA1, 6). They are bound through hydrogen bonds, arene-anion and arene-cation interactions. The trend of binding affinity show H-TEAD (−9.4 kcal/mol) = TEAD1 (−9.4 kcal/mol) > HA-TEAD (−9.3 kcal/mol) > NELF (−8.7 kcal/mol) > Siamone (−8.8 kcal/mol) > HCQ (−7.2 kcal/mol) > REMD (−6.2 kcal/mol) while the number of interactions shows REMD > HA-TEAD = HCQ > Siamone > NELF > H-TEAD > TEAD1. This study, hence, validates the activity of HCQ against COVID-19 and provides a foundation for advanced experimental research, to evaluate the real pharmaceutical potentials of these compounds, towards finding a cure for COVID-19 and other related diseases.

Background: COVID‐19, a disease caused by a novel coronavirus, is a major global human threat that has turned into a pandemic. Identification and development of drugs/vaccines is urgently needed for prevention and therapy of the COVID‐19 virus (SARS‐CoV‐2) infection. SARS‐CoV‐2 main protease (Mpro,) plays a key role in viral protein processing for its replication and inhibition of Mpro leads to prevention of viral replication. The SARS‐CoV‐2 Mpro is considered a promising drug target, as it is dissimilar to human proteases. Several peptidic protease inhibitors have been developed as SARS‐CoV‐2 Mpro inhibitors for COVID‐19 treatment. In this study, we screened naturally occurring peptidic α‐hydroxy amides (PHAs) (phebestin, probestin, bestatin) for SARS‐CoV‐2 Mpro inhibition.Materials and methods: In vitro COVID‐19 Mpro fluorometric assay was performed to evaluate its inhibitory efficacy by PHAs. Isothermal titration calorimetry technique was used to demonstrate the binding affinity between COVID‐19 Mpro and PHAs. Circular dichroism technique was used to determine the structural changes in secondary structure of COVID‐19 Mpro induced by PHAs. Molecular docking studies were performed to demonstrate the molecular level mechanism behind COVID‐19 Mpro inhibitory efficacy of PHAs. In addition, cytotoxic effects of PHAs were evaluated on lung epithelial cells of mice for its therapeutic applications for COVID‐19 through nasal delivery.Results and discussion: Phebestin (IC50 = 17.98 µM) Probestin (IC50 = 48.31 µM) and Bestatin (IC50 > 100 µM) significantly inhibited the COVID‐19 Mpro activity. The KD (dissociation constant) values of phebestin and probestin for COVID‐19 Mpro were 31.23 and 43.51 µM, respectively. CD spectrum analysis demonstrated that phebestin and probestin (10 µM) induced changes in the secondary structure of COVID‐19 Mpro. Molecular docking studies demonstrated that Interactions of phebestin and probestin with active site amino acids. Phebestin and probestin did not show any cytotoxicity on normal mouse lung epithelial cells up to 100 µM.Conclusion: Preliminary in vitro studies demonstrated that naturally occurring PHAs derivatives phebestin and probestin are SARS‐CoV‐2 Mpro inhibitors and warranted further studies to develop as drugs for COVID‐19.

В статье на основании литературных источников представлены современные данные об основных патогенетических особенностях коронавирусной инфекции, связанной с вирусом SARS-CoV-2, вызвавшим в 2019 году, по определению Всемирной организации здравоохранения, пандемию. В литературном обзоре подробно освещены процессы связывания вируса SARS-CoV-2 с рецептором клеток человека, которые экспрессируют ангиотензинпревращающий фермент 2 (АСЕ2), а также интернализация, репликация вируса и высвобождение новых вирионов из инфицированной клетки, которые поражают таргетные органы (легкие, пищеварительный тракт, сердце, центральную нервную систему и почки) и индуцируют развитие местного и системного воспалительного ответа. Описаны существующие способы медикаментозного воздействия, препятствующие инфицированию человека вирусом SARS-CoV-2. Выделены основные эпидемиологические моменты инфицирования вирусом SARS-CoV-2, указывающие на преимущественное поражение пожилых людей и чаще лиц мужского пола в связи с более высоким уровнем экспрессии АСЕ2, в большей степени в альвеолоцитах, чем у лиц женского пола. Продемонстрированы механизмы развития ответной реакции врожденной и адаптивной иммунной системы макроорганизма на инфицирование вирусом SARS-CoV-2. Представлены терапевтические стратегии, связанные с влиянием на различные этапы жизнедеятельности вируса SARS-CoV-2: интернализацию — использование солютабных доменов S-белка, антител против S-белка, одноцепочечного вариабельного фрагмента антител к АСЕ2 или ингибирования гликозилирования клеточных рецепторов, блокирования взаимодействия S-протеина вируса SARS-CoV-2 с протеином ACE2 и подавления интернализации вируса за счет назначения препаратов хлорохин и гидроксихлорохин; репликацию — ингибирование вирусной РНК-зависимой РНК-полимеразой и применение фавипиравира, ненуклеозидного противовирусного препарата триазавирина, антиретровирусных препаратов (лопинавира в сочетании с ритонавиром), нелфинавира, рибавирина, галидесивира, умифеновира, ингибиторов химотрипсиноподобной протеазы (цинансерина, флавоноидов) и папаиноподобной протеазы. Вышеперечисленные терапевтические методы в ближайшем будущем будут направлены на предупреждение развития и лечение как острого респираторного дистресс-синдрома, так и состояний, обусловленных поражением других таргетных органов при COVID-19.

The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape.

As the COVID-19 pandemic has progressed, new variants of the virus SARS-CoV-2 have emerged that are more infectious than the original form. The variants known as Alpha, Beta and Gamma have mutations in a protein on the virus’s surface that is vital for attaching to cells and infecting them. This protein, called Spike, carries out its role by binding to ACE2, a protein on the surface of human cells. Mutations on Spike are found on the region where it binds to ACE2. The interaction between these two proteins appears to be important to the behaviour of SARS-CoV-2, but the impact of individual mutations in Spike is unknown. In addition, some people have different variants of ACE2 with mutations in the region that interacts with Spike, but it is not known whether this affects these people’s risk of contracting COVID-19. To answer these questions, Barton et al. measured the precise effect of mutations in Spike and ACE2 on the strength of the interaction between the two proteins. The experiments showed that three of the five common Spike mutations in the Alpha, Beta and Gamma SARS-CoV-2 variants strengthened binding to ACE2. The two mutations that weakened binding were only found together with other mutations that strengthened binding. This meant that the Spike proteins in all three of these SARS-CoV-2 variants bind to ACE2 more strongly than the original form. The experiments also showed that two common variants of ACE2 also increased the strength of binding to Spike. Interestingly, one of these ACE2 variants reversed the effect of a specific SARS-CoV-2 mutation, suggesting that carriers would be resistant to SARS-CoV-2 variants with this mutation. Identifying the precise effects of Spike mutations on ACE2 binding helps understand why new variants of SARS-CoV-2 spread more rapidly. This could help to identify concerning new variants before they spread widely and inform the response by health authorities. The finding that two common ACE2 variants bind more strongly to Spike suggests that people with these mutations could be more susceptible to SARS-CoV-2.

Antibodies induced by receptor-binding domain in spike protein of SARS-CoV do not cross-neutralize the novel human coronavirus hCoV-EMC

Hydroxychloroquine (HCQ) and its derivatives have recently gained tremendous attention as a probable medicinal agent in the COVID-19 outbreak caused by SARS-CoV-2. An efficient agent to act directly in inhibiting the SARS-CoV-2 replication is yet to be achieved. Thus, the goal is to investigate the dynamic nature of HCQ derivatives against SARS-CoV-2 main protease and spike proteins. Molecular docking studies were also performed to understand their binding affinity in silico methods using the vital protein domains and enzymes involved in replicating and multiplying SARS-CoV-2, which were the main protease and spike protein. Molecular Dynamic simulations integrated with MM-PBSA calculations have identified In silico potential inhibitors ZINC05135012 and ZINC59378113 against the main protease with −185.171 ± 16.388, −130.759 ± 15.741 kJ/mol respectively, ZINC16638693 and ZINC59378113 against spike protein −141.425 ± 22.447, −129.149 ± 11.449 kJ/mol. Identified Hit molecules had demonstrated Drug Likeliness features, PASS values and ADMET predictions with no violations. Communicated by Ramaswamy H. Sarma

Lung cancer patients are more vulnerable to COVID-19 infection. Treatment of patients with lung cancer during the current COVID-19 pandemic is challenging and development of drugs for COVID-19 and lung cancer is urgently needed. Cathepsin L plays key role in lung cancer progression, invasion and more so, endocytosis of SARS-Cov-2 virus into the lung epithelial cells. Importantly, patients with KrasG12V mutation show agreessive disease and high-resistance to chemotherapy due to over-expression of Cathepsin L. Thus, Cathepsin L is a common target for lung cancer and COVID-19 infection. Chymostatin is a known cathepsin L inhibitor, here we screened it for dual inhibition of COVID-19 Mpro and lung cancer patients with COVID-19. In vitro COVID-19 Mpro fluorometric assay was performed to evaluate its inhibitory efficacy by chymostatin. Chymostatin showed dose-dependent inhibition of COVID-19 Mpro activity with IC50 of 15.81 µM (P<0.0001). Isothermal titration calorimetry based binding studies determined that chymostatin strongly bound to COVID-19 Mpro (KD=22.45 µM). CD spectrum analysis demonstrated that chymostatin (10 µM)-induced changes in the secondary structure of COVID-19 Mpro. A unit cell crystal parameter COVID-19 Mpro co-crystalized with chymostatin (a = 49.926 Å, b = 108.71 Å, c = 56.49 Å, α = 90.0°, β = 102.7553°, γ = 90.0°; unit cell volume: 299.03 Ao) was determined using X-ray diffractometer. Molecular docking studies demonstrated that chymostatin interacts with active site amino acids (Cys145). Pro-apoptotic effect of chymostatin was evaluated on human H441 lung cancer cells and mouse lung normal epithelial cells using Annexin V/propidium iodide staining. Chymostatin did not show any cytotoxicity on normal mouse lung epithelial cells up to 100 µM, where as it inhibited proliferation of human H441 lung cancer cells (KrasG12V mutant) with IC50 of 1.2 µM. In conclusion, preliminary in vitro studies demonstrated that naturally occurring cathepsin L inhibitor chymostatin is a SARS-CoV-2 Mpro inhibitor and exhibited anticancer activity against lung cancer cells. The results of the study warranted detailed studies to develop as drugs for lung cancer patients during COVID-19 pandemic (supported by VPR-PHF, the University of Oklahoma Health Sciences Centre for financial support through COVID-19 seed grant program). Citation Format: Nagendra Sastry Yarla, Gopal Pathuri, Simon Terzyan, Yuting Zhang, Anil Singh, Marcus T. Scotti, Venkateshwar Madka, Chinthalapally V. Rao. Chymostatin, a cathepsin L inhibitor, inhibits lung cancer cell proliferation and COVID-19 Mpro in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB074.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Two key therapeutic target proteins of SARS-CoV-2, the Spike (S) protein and the main protease (Mpro), facilitate the entry of virus and its replication inside the host cell, respectively. Notably, several pointmutations in the receptor-binding domain (RBD) of the S-protein have led to the origin of different SARS-CoV-2 Variants of Concern (VOCs) including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), Gamma (P.1), and Omicron (B.1.1.529). The low efficacy of currently available antiviral drugs against these VOCs highlights the need for screening and discovery of novel natural compounds against COVID-19. This study performed molecular docking of marine natural products from soft coral-derived microbes against the Mpro and the RBD of the S-protein from wild type (WT) and five VOCs. Many of the test compounds [e.g., Cottoquinazoline B and D (CQB/D), Tetraorcinol A (TOA), Versicoloritide A and C (VCA/C), Fumiquinazoline K, and Pencillanthranin A) showed stronger binding affinities compared to control antiviral drugs (nelfinavir and remdesivir) and formed favorable interactions with both Mpro and the RBD of S-protein. ADMET analysis revealed that most of the best-docked compounds obey Lipinski rule of five. Molecular dynamics (MD) simulation (200 ns) analysis further revealed stable binding conformations of the top docked complexes of (1) CQB with Mpro, (2) CQB with the RBD of WT S-protein, (3) TOA with the RBD of S-protein from beta variant (4) TOA with the RBD of S-protein from Omicron variant, (5) TOA with the RBD of S-protein from Delta variant, (6) TOA with the RBD of S-protein from Gamma variant, and (7) VCA with the RBD of alpha variant. However, future in vitro and in vivo studies are still required to validate efficacy of these compounds.

Background: Molecular docking has been used recently in pharma industry for drug designing, it’s a powerful tool to find ligand-substrate interactions at molecules level. Since urgent need to develop anti-viral drug that target new coronavirus main proteins, in silico docking has been used to achieve this purpose.
 Materials and Methods: Thirteen herbs are known for their antioxidants and antiviral properties have been selected to investigate their abilities in inhibiting SARS-COV2 spike protein and main protease Mpro. pdb files for RBD (Receptor Binding Domain) region of spike protein and for Mpro and mol2 files for all herbs understudy were uploaded for swiss dock online server, the docking results were analyzed using chimera software. Full fitness energy and hydrogens bonds interactions were considered for docking evaluation. Pharma kinetic properties for compounds have good binding results were evaluated through AMES and ADMET tests.
 Results: All compounds showed negative full fitness energy that means they are able to complex with both SARS-COV2 spike protein and main protease, however some of the herbs form very powerful hydrogen bonding with the RBD site of the spike protein and the catalytic site of Mpro such as coumarylquinic acid, while stigmasterol has strong binding with the spike protein only. Both compounds appear to be safe drugs for human according to AMES test results.
 Conclusion: Coumarylquinic acid and stigmasterol have powerful binding in silico, further in vitro studies include using viral infected human lung cells and testing above compounds ability for inhibiting viral entry and replication should be proceed to confirm the study results.

The lack of any effective cure for the infectious COVID-19 disease has created a sense of urgency and motivated the search for effective antiviral drugs. Abyssomicins are actinomyces-derived spirotetronates polyketides antibiotics known for their promising antibacterial, antitumor, and antiviral activities. In this study, computational approaches were used to investigate the binding mechanism and the inhibitory ability of 38 abyssomicins against the main protease (Mpro) and the spike protein receptor-binding domain (RBD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The results identified abyssomicins C, J, W, atrop-O-benzyl abyssomicin C, and atrop-O-benzyl desmethyl abyssomicin C as the most potential inhibitors of Mpro and RBD with binding energy ranges between −8.1 and −9.9 kcal mol−1; and between −6.9 and −8.2 kcal mol−1, respectively. Further analyses of physicochemical properties and drug-likeness suggested that all selected active abyssomicins, with the exception of abyssomicin J, obeyed Lipinski’s rule of five. The stability of protein–ligand complexes was confirmed by performing molecular dynamics simulation for 100 ns and evaluating parameters such as such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), total number of contacts, and secondary structure. Prime/MM-GBSA (Molecular Mechanics-General Born Surface Area) and principal component analysis (PCA) analyses also confirmed the stable nature of protein–ligand complexes. Overall, the results showed that the studied abyssomicins have significant interactions with the selected protein targets; therefore, they were deemed viable candidates for further in vitro and in vivo evaluation. Communicated by Ramaswamy H. Sarma

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Facing the challenge of viral mutations in the age of pandemic: Developing highly potent, broad-spectrum, and safe COVID-19 vaccines and therapeutics.

Potent and biostable inhibitors of the main protease of SARS-CoV-2

To cure SARS-CoV-2 infection, the repurposing of conventional antiviral drugs is currently advocated by researchers, though their action is not very effective. The present study, based on in silico methods, was intended to increase the therapeutic potential of conventional drugs: hydroxychloroquine (HCQ), favipiravir (FAV), and remdesivir (REM) by using curcuminoids like curcumin (CUR), bisdemethoxycurcumin (BDMC), and demethoxycurcumin (DMC) as adjunct drugs against SARS-CoV-2 receptor proteins, namely main protease (Mpro) and the S1 receptor-binding domain (RBD). The curcuminoids exhibited similar pharmacokinetic properties to the conventional drugs. The webserver (ANCHOR) predicted greater protein stability for both receptors with a disordered score (<0.5). The molecular docking study showed that the binding energy was highest (−27.47 kcal/mol) for BDMC toward Mpro receptors, while the binding energy of CUR (−20.47 kcal/mol) and DMC (−20.58 kcal/mol) was lower than that of HCQ (−24.58 kcal/mol), FAV (−22.87 kcal/mol), and REM (−23.48 kcal/mol). In the case of S1-RBD, CUR had the highest binding energy (−38.84 kcal/mol) and the lowest was in FAV (−23.77 kcal/mol), whereas HCQ (−35.87 kcal/mol) and REM (−38.44 kcal/mol) had greater binding energy than BDMC (−28.07 kcal/mol) and DMC (−30.29 kcal/mol). Hence, this study envisages that these curcuminoids could be employed in combination therapy with conventional drugs to disrupt the stability of SARS-CoV-2 receptor proteins.