Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug Carmofur

Abstract

25COVID-19 virus is the cause of a debilitating and life-threatening infectious 26 pulmonary disease that is now responsible for a global pandemic. Currently, there are 27 no specific drugs or vaccines to contain this virus. The main protease (M pro ) of COVID-28 19 virus is a key enzyme, which plays an essential role in viral replication and 29 transcription, making it an ideal drug target. An FDA-approved antineoplastic drug, 30 carmofur, has been identified as an inhibitor that targets COVID-19 virus M pro . 31However, its inhibitory mechanism is unknown. Here, we report the 1.6-Å crystal 32 structure of COVID-19 virus M pro in complex with carmofur. The crystal structure 33 shows that carmofur contains an electrophilic carbonyl reactive group, which 34 covalently binds to C145, a member of the catalytic dyad. As a result, its fatty acid tail 35 occupies the hydrophobic S2 subsite of M pro whilst its 5-fluorouracil head is cleaved as 36 product of the new covalent bond that has formed. Carmofur is active in a cell based 37 antiviral assay with an EC50 of 24.87 μM. It is therefore a promising lead compound 38 for the development of new antivirals to target COVID-19. : bioRxiv preprint 41 Starting in December 2019, a highly infectious viral disease has now spread and 42 reached over 200 countries leading to a global public health emergency and pandemic. 43The etiological agent of the disease is a coronavirus (identified as . 44According to the WHO COVID-2019 Situation Report-77, there were 1,210,956 45 confirmed cases and 67,594 deaths, with a mortality rate at 5.58%. The number of 46 confirmed cases worldwide continues to grow at a rapid rate and is far from peaking. 47However, there are no specific drugs or vaccines available to control symptoms or the 48 spread of this disease. 49The COVID-19 virus has a ~30,000 nt RNA genome encoded with two translation 50 products, polyproteins 1a and 1ab (pp1a and pp1ab) which are crucial for replication 51 and transcription 1,2 . These polyproteins become matured non-structural and structural 52 proteins through auto-cleavage by the main protease (M pro ) and by a papain-like 53 protease 3 . Because of this, M pro is an excellent target for anti-coronavirus (CoV) drug 54 development 4-6 . In order to rapidly discover new drug leads that target COVID-19 virus 55 M pro , our group screened over 10,000 compounds from a library that consisted of 56 approved drugs, drug candidates in clinical trials, and other pharmacologically active 57 compounds. Amongst these we identified carmofur as compound that can inhibit M pro 58 with an IC50 of 1.82 μM 7 . 59Carmofur is an FDA-approved antineoplastic drug, and a derivative of 5-60 fluorouracil (5-FU) a widely drug used against solid cancers. 5-FU is especially 61 efficient for controlling head, neck, and gastrointestinal tumors 8 . Carmofur (Figure 1A) 62 is a derivative of this compound and has been used in colon cancer therapy since 1981 9 . 63Clinical research has also shown that carmofur has a curative effect on breast, gastri...

A computational approach to aid clinicians in selecting anti-viral drugs for COVID-19 trials

“…All the associations corresponding to anti-viral drugs clinically shown to act against human host viruses have been assembled from standard DrugBank database [55] (https://www.drugbank.ca/categories/DBCAT000066). To ensure that the database is fully comprehensive, other literature works [45,17,50,13,23,16,49,54,31] and resources such as ViPR [43] were also scanned for any additional drug-viral associations. ViPR or NIAID Virus Pathogen Database and Analysis Resource (http://www.viprbrc.org/) is a repository of data and analysis tools for virology research [43] capturing various types of information derived from comparative genomics analysis and visualization tools.…”

Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2

“…They catalyze the proteolysis of polyproteins translated from the viral genome into non-structural proteins essential for packaging the nascent virion and viral repication. 4 Therefore, inhibiting the activity of these proteases would impede the replication of the virus. M pro processes the polyprotein 1ab at multiple cleavage sites.…”

“…5 Thus, M pro is viewed as a promising target for anti SARS-CoV-2 drug design; it has been the focus of several studies since the pandemic has emerged. 2,[4][5][6][7] An X-ray crystal structure of M pro reveals that it forms a homodimer with a 2fold crystallographic symmetry axis. 2,5 Each protomer, with a length of 306 residues, is made of three domains (I-III).…”

“…Domains II and I fold into a six-stranded β-barrel that harbors the active site. 2,4,5 Domain III forms a cluster of five antiparallel αhelices that regulates the dimerization of the protease. A flexible loop connects domain II to domain III.…”

“…These residues have been found to interact with the ligands co-crystallized with M pro in different studies. 2,4,5 Crystallographic data also suggested that Ser 1 of one protomer interacts with Phe 140 and Glu 166 of the other as the result of dimerization. 2,4 These interactions stabilize the P1 binding pocket, thereby, dimerization of the main protease is likely for its catalytic activity.…”