Rifampicin may be repurposed for COVID-19 treatment: Insights from an in-silico study

The ongoing Coronavirus disease 2019 (COVID-19) outbreak has led to global epidemics with high morbidity and mortality. However, there are no ecacious therapeutic options available for the treatment of COVID-19. Considering this medical emergency, drug repurposing is the need of the hour. Therefore, this study aimed to determine possible COVID-19 drug candidates using virtual screening of selected FDA approved drugs by targeting the main protease (Mpro) of novel coronavirus (SARS-CoV-2). In this study, rst, we have done an extensive literature search to nd out the marketed FDA approved drug compounds available for human therapeutic applications having anti-viral and anti-bacterial properties. The preliminary reports paved the way for further studies that revealed some of the drugs approved to treat other viral or bacterial infections may be a promising drug candidate and can be repurposed for the treatment of COVID-19. Using a computer-aided drug designing approach, novel candidates for drug repurposing were identied. Based on our preliminary screening supported by previous works of literature, we selected Rifampicin, Ciclesonide, Reserpine, Loperamide, Elvitegravir, Brivudine, Pentoxifylline, Eugenol, Isoniazid, Tinidazole, Diethylcarbamazine, and Vancomycin to target the SARS-CoV-2 main protease (Mpro). These compounds were examined based on docking studies using Autodock 4.2 Autodock 4.2., visualization of the docked results using Pymol version 1.7.4.5 Edu and prediction of two main toxicity aspects hepatotoxicity and cytotoxicity to check the safety of the hit compounds. Selected drugs were ranked for potential ecacy against COVID-19 based on their binding energy. The docking results were found in the order of Rifampicin (-11.16), Ciclesonide (-8.77), Reserpine (-7.76), Loperamide (-7.63), Elvitegravir (-6.58), Brivudine (-6.06), Pentoxifylline (-5.64), Eugenol (-4.78), Isoniazid (-4.71), Tinidazole (-4.51), Diethylcarbamazine (-4.66), Vancomycin (-4.11). ). In the current study, Rifampicin appeared as the most promising drug showing a very good binding energy. Based on our preliminary insights from in silico studies, we propose that Rifampicin may be repurposed for the treatment of COVID-19 however it is pertinent to mention here that our ndings need further validation by in vitro studies and clinical trials. In summary, the ndings of this study suggest Rifampicin might be a promising drug for targeting the COVID-19 protease.


Introduction
A newly identi ed coronavirus strain (or severe acute respiratory syndrome, SARS-CoV-2) was reported in Wuhan, China, in late December 2019 (Paraskevis et al. 2020). As of 8 April 2020, a total of 1,478,469 cases of COVID-19 were con rmed in the world including 86,748 deaths. The disease caused by noble coronavirus was named as the coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) released on 11 February 2020 (World Health Organization (WHO) 2020). According to the current situation, this pandemic is still ongoing, lacking e cacious therapeutic options. However, the steps taken to reduce the severity of infection remain limited to supportive strategies intended to avoid further complications of coronavirus infection (Zhang et al. 2020). Now researchers are racing against time to discover an effective drug candidate by targeting protease inhibitors to block the SARS-CoV-2 main protease (M pro ). The molecular structure of COVID-19 main protease (M pro ) as determined by Liu et al. (2020) represents a potential target for the inhibition of coronavirus replication . Some retrospective ndings of past outbreaks have helped to examine possible combinations like the protease inhibitor lopinavir/ritonavir, commonly used in the treatment of HIV patients (Lu. 2020). Taken together, these ndings indicate that protease inhibitors may block the main protease of the novel coronavirus and this could be an effective approach in checking the virus replication cycle ). The therapeutic strategies have been focused to identify the viral protease inhibitor that does not inhibit important human protease activity.
Drug repurposing (also known as drug repositioning or repro ling) is a technique to identify new applications for certi ed or investigational drugs outside the original medical indication. Thus, in this study, using in silico approach, several FDA approved drugs that exhibit the potential for drug repurposing, e.g., Brivudine, Ciclesonide, Diethylcarbamazine, Elvitegravir, Eugenol, Isoniazid, Loperamide, Lopinavir, Pentoxifylline, Reserpine, Rifampicin, Ritonavir, Tinidazole, Vancomycin (https://www.drugbank.ca/) have been virtually screened for prediction of the potential drug candidate and binding a nity against coronavirus main protease (M pro ). Therefore, great attention has been paid to the drug repurposing approach (Talevi and Bellera 2020). The initially conceived technique of using proven medications to treat other symptoms for one disorder has found success across medical elds. Such repurposing medication promises to provide patients with quicker access to drugs while reducing costs in the long and di cult drug development cycle (Parvathaneni et al. 2019). Therefore, the current study aimed to elucidate the Novel Drug Repurposing Strategy to investigate the candidate drugs for in vitro studies, in vivo studies and clinical trials and to attain mechanistic insights into the outcomes of such research studies. In this study, we employed in silico methods to investigate novel protease inhibitor for the suppression of the antiviral effect of coronavirus in order to target SARS-CoV-2 main protease (M pro ). which form a homodimer. Thereafter the chain A of PDB le was optimized for macromolecule preparation.

Materials & Methods
2.2 Literature search and drug selection: The list and structures of FDA approved drugs were obtained from the drug bank (https://www.drugbank.ca/). The 12 FDA-approved drug compounds (Table 1) used in the present study were Brivudine, Ciclesonide, Diethylcarbamazine, Elvitegravir, Eugenol, Isoniazid, Loperamide, Lopinavir, Pentoxifylline, Reserpine, Rifampicin, Ritonavir, Tinidazole, Vancomycin against viral protease that could block SARS-CoV-2 protease. Thereafter, the molecules which were optimized, saved in the PDB format for further processing.

Toxicity evaluation of FDA approved drugs involved in the study
The individual toxicities of FDA approved (marketed) drugs were predicted by using ProTox-II. The toxicity analysis was performed in order to predict the safety aspects of the FDA approved drug. The major toxicity endpoints were taken into consideration and the drugs which were not following the safety parameters of toxicity endpoints were not taken for further analysis in our priority list. As shown in Table  2, ProTox-II toxicity prediction software gave result mainly associated with three main toxicity aspects cytotoxicity, carcinogenicity and mutagenicity to check the safety aspects of the FDA approved market available drug compounds. According to the toxicological data, none of the selected FDA approved market available drug compound was shown potential cytotoxicity, carcinogenicity, and mutagenicity.  Among them, 4 compounds approved by the FDA have a docking score better than -7.0 kcal/mol. Thus among 12 compounds and the standard reference compounds, 4 hits were found to have good a nities in terms of docking scores. The results of our docking study revealed that among selected drugs, interestingly, two drugs Rifampicin and Ciclesonide showed the best a nity even better than the reference compounds Ritonavir and Lopinavir.

Discussion
The outbreak of COVID-19 has been declared a global pandemic by the WHO, and the development of effective treatment for COVID-19 is in an urgent need (World Health Organization 2020). COVID-19 main protease M pro is responsible for proteolytic processing, viral replication, and the establishment of the infection cycle. In a recent study by Liu et al. (2020), the crystal structure of COVID-19 main protease M pro has been reported (PDB ID: 6LU7) (Jin et al. 2020) and this may signi cantly facilitate the discovery of potential drug compounds to prevent COVID-19 infection by targeting its main protease M pro through existing drug libraries for drug repurposing. Lopinavir and ritonavir are well-established protease inhibiting drugs for HIV (Sheean et al. 2008). In several studies, both drugs were also proposed to treat SARS and the Middle East respiratory syndrome (MERS) (Chu et al. 2004). This combination has also been used in COVID-19 patients in order to control COVID-19 infection (Lim et al. 2020). Therefore, in this study, we have taken these drugs as a standard reference to compare the e cacy of the binding of our

Conclusion
Drug repurposing is an attractive option for life-threatening pandemics like COVID-19 for the rapid identi cation of potential therapeutics. This study aimed to examine several FDA approved drug compounds that could be used as repurposed drugs to inhibit COVID-19 infection. In this in silico study, Rifampicin was identi ed as the best hit among the selected drugs. Interestingly, the binding a nity of Rifampicin was found to be much better than the selected reference drugs as per docking results. Further, the major toxicity end pint prediction results were also good for rifampicin. Therefore the results of this study indicate that Rifampicin, a well-established medicine for the treatment of tuberculosis has a stronger binding a nity for COVID-19 main protease Mpro in comparison to the other drug compounds taken in this study. To sum up, our preliminary in silico ndings suggest that Rifampicin may be used as a repurposed drug for the treatment of COVID-19. However, it is pertinent to mention here that these ndings warrant further in vitro and clinical trials in order to precisely conclude our ndings.