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      In silico identification of potential inhibitors of key SARS-CoV-2 3CL hydrolase (Mpro) via molecular docking, MMGBSA predictive binding energy calculations, and molecular dynamics simulation

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          Abstract

          The incidence of 2019 novel corona virus (SARS-CoV-2) has created a medical emergency throughout the world. Various efforts have been made to develop the vaccine or effective treatments against the disease. The discovery of crystal structure of SARS-CoV-2 main protease has made the in silico identification of its inhibitors possible. Based on its critical role in viral replication, the viral protease can prove to be a promising “target” for antiviral drug therapy. We have systematically screened an in-house library of 15,754 natural and synthetic compounds, established at International Center for Chemical and Biological Sciences, University of Karachi. The in silico search for potential viral protease inhibitors resulted in nine top ranked ligands (compounds 19) against SARS-CoV-2 main protease (PDB ID: 6LU7) based on docking scores, and predictive binding energies. The in silico studies were updated via carrying out the docking, and predictive binding energy estimation, with a recently reported crystal structure of main protease (PDB ID: 6Y2F) at a better resolution i. e., 1.95 Å. Compound 2 (molecular bank code AAA396) was found to have highest negative binding energy of −71.63 kcal/mol for 6LU7. While compound 3 (molecular bank code AAD146) exhibited highest negative binding energy of -81.92 kcal/mol for 6Y2F. The stability of the compounds- in complex with viral protease was analyzed by Molecular Dynamics simulation studies, and was found to be stable over the course of 20 ns simulation time. Compound 2, and 3 were predicted to be the significant inhibitors of SARS-CoV-2 3CL hydrolase (Mpro) among the nine short listed compounds.

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          Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

          Summary Background A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.
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            A Novel Coronavirus from Patients with Pneumonia in China, 2019

            Summary In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.)
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              The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health — The latest 2019 novel coronavirus outbreak in Wuhan, China

              The city of Wuhan in China is the focus of global attention due to an outbreak of a febrile respiratory illness due to a coronavirus 2019-nCoV. In December 2019, there was an outbreak of pneumonia of unknown cause in Wuhan, Hubei province in China, with an epidemiological link to the Huanan Seafood Wholesale Market where there was also sale of live animals. Notification of the WHO on 31 Dec 2019 by the Chinese Health Authorities has prompted health authorities in Hong Kong, Macau, and Taiwan to step up border surveillance, and generated concern and fears that it could mark the emergence of a novel and serious threat to public health (WHO, 2020a, Parr, 2020). The Chinese health authorities have taken prompt public health measures including intensive surveillance, epidemiological investigations, and closure of the market on 1 Jan 2020. SARS-CoV, MERS-CoV, avian influenza, influenza and other common respiratory viruses were ruled out. The Chinese scientists were able to isolate a 2019-nCoV from a patient within a short time on 7 Jan 2020 and perform genome sequencing of the 2019-nCoV. The genetic sequence of the 2019-nCoV has become available to the WHO on 12 Jan 2020 and this has facilitated the laboratories in different countries to produce specific diagnostic PCR tests for detecting the novel infection (WHO, 2020b). The 2019-nCoV is a β CoV of group 2B with at least 70% similarity in genetic sequence to SARS-CoV and has been named 2019-nCoV by the WHO. SARS is a zoonosis caused by SARS-CoV, which first emerged in China in 2002 before spreading to 29 countries/regions in 2003 through a travel-related global outbreak with 8,098 cases with a case fatality rate of 9.6%. Nosocomial transmission of SARS-CoV was common while the primary reservoir was putatively bats, although unproven as the actual source and the intermediary source was civet cats in the wet markets in Guangdong (Hui and Zumla, 2019). MERS is a novel lethal zoonotic disease of humans endemic to the Middle East, caused by MERS-CoV. Humans are thought to acquire MERS-CoV infection though contact with camels or camel products with a case fatality rate close to 35% while nosocomial transmission is also a hallmark (Azhar et al., 2019). The recent outbreak of clusters of viral pneumonia due to a 2019-nCoV in the Wuhan market poses significant threats to international health and may be related to sale of bush meat derived from wild or captive sources at the seafood market. As of 10 Jan 2020, 41 patients have been diagnosed to have infection by the 2019-nCoV animals. The onset of illness of the 41 cases ranges from 8 December 2019 to 2 January 2020. Symptoms include fever (>90% cases), malaise, dry cough (80%), shortness of breath (20%) and respiratory distress (15%). The vital signs were stable in most of the cases while leucopenia and lymphopenia were common. Among the 41 cases, six patients have been discharged, seven patients are in critical care and one died, while the remaining patients are in stable condition. The fatal case involved a 61 year-old man with an abdominal tumour and cirrhosis who was admitted to a hospital due to respiratory failure and severe pneumonia. The diagnoses included severe pneumonia, acute respiratory distress syndrome, septic shock and multi-organ failure. The 2019-nCoV infection in Wuhan appears clinically milder than SARS or MERS overall in terms of severity, case fatality rate and transmissibility, which increases the risk of cases remaining undetected. There is currently no clear evidence of human to human transmission. At present, 739 close contacts including 419 healthcare workers are being quarantined and monitored for any development of symptoms (WHO, 2020b, Center for Health Protection and HKSAR, 2020). No new cases have been detected in Wuhan since 3 January 2020. However the first case outside China was reported on 13th January 2020 in a Chinese tourist in Thailand with no epidemiological linkage to the Huanan Seafood Wholesale Market. The Chinese Health Authorities have carried out very appropriate and prompt response measures including active case finding, and retrospective investigations of the current cluster of patients which have been completed; The Huanan Seafood Wholesale Market has been temporarily closed to carry out investigation, environmental sanitation and disinfection; Public risk communication activities have been carried out to improve public awareness and adoption of self-protection measures. Technical guidance on novel coronavirus has been developed and will continue to be updated as additional information becomes available. However, many questions about the new coronavirus remain. While it appears to be transmitted to humans via animals, the specific animals and other reservoirs need to be identified, the transmission route, the incubation period and characteristics of the susceptible population and survival rates. At present, there is however very limited clinical information of the 2019-nCoV infection and data are missing in regard to the age range, animal source of the virus, incubation period, epidemic curve, viral kinetics, transmission route, pathogenesis, autopsy findings and any treatment response to antivirals among the severe cases. Once there is any clue to the source of animals being responsible for this outbreak, global public health authorities should examine the trading route and source of movement of animals or products taken from the wild or captive conditions from other parts to Wuhan and consider appropriate trading restrictions or other control measures to limit. The rapid identification and containment of a novel coronavirus virus in a short period of time is a re-assuring and a commendable achievement by China’s public health authorities and reflects the increasing global capacity to detect, identify, define and contain new outbreaks. The latest analysis show that the Wuhan CoV cluster with the SARS CoV.10 (Novel coronavirus - China (01): (HU) WHO, phylogenetic tree Archive Number: 20200112.6885385). This outbreak brings back memories of the novel coronavirus outbreak in China, the severe acute respiratory syndrome (SARS) in China in 2003, caused by a novel SARS-CoV-coronavirus (World Health Organization, 2019a). SARS-CoV rapidly spread from southern China in 2003 and infected more than 3000 people, killing 774 by 2004, and then disappeared – never to be seen again. However, The Middle East Respiratory Syndrome (MERS) Coronavirus (MERS-CoV) (World Health Organization, 2019b), a lethal zoonotic pathogen that was first identified in humans in the Kingdom of Saudi Arabia (KSA) in 2012 continues to emerge and re-emerge through intermittent sporadic cases, community clusters and nosocomial outbreaks. Between 2012 and December 2019, a total of 2465 laboratory-confirmed cases of MERS-CoV infection, including 850 deaths (34.4% mortality) were reported from 27 countries to WHO, the majority of which were reported by KSA (2073 cases, 772 deaths. Whilst several important aspects of MERS-CoV epidemiology, virology, mode of transmission, pathogenesis, diagnosis, clinical features, have been defined, there remain many unanswered questions, including source, transmission and epidemic potential. The Wuhan outbreak is a stark reminder of the continuing threat of zoonotic diseases to global health security. More significant and better targeted investments are required for a more concerted and collaborative global effort, learning from experiences from all geographical regions, through a ‘ONE-HUMAN-ENIVRONMENTAL-ANIMAL-HEALTH’ global consortium to reduce the global threat of zoonotic diseases (Zumla et al., 2016). Sharing experience and learning from all geographical regions and across disciplines will be key to sustaining and further developing the progress being made. Author declarations All authors have a specialist interest in emerging and re-emerging pathogens. FN, RK, OD, GI, TDMc, CD and AZ are members of the Pan-African Network on Emerging and Re-emerging Infections (PANDORA-ID-NET) funded by the European and Developing Countries Clinical Trials Partnership the EU Horizon 2020 Framework Programme for Research and Innovation. AZ is a National Institutes of Health Research senior investigator. All authors declare no conflicts of interest.
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                Contributors
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draft
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 July 2020
                2020
                24 July 2020
                : 15
                : 7
                : e0235030
                Affiliations
                [1 ] Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
                [2 ] H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
                University of Calgary, CANADA
                Author notes

                Competing Interests: The study has financial support of the Searle Company, Pakistan through a research project funding. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0001-5356-3585
                Article
                PONE-D-20-07387
                10.1371/journal.pone.0235030
                7380638
                32706783
                ebf95b8d-d06d-42ce-8b3a-551f83bfc38c
                © 2020 Choudhary et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 March 2020
                : 7 June 2020
                Page count
                Figures: 7, Tables: 2, Pages: 15
                Funding
                The study has financial support of the Searle Company, Pakistan through a research project funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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