key: cord-0848221-ck77orfk authors: Sayed, Ahmed M.; Khalaf, Ahmed M.; Abdelrahim, Mohamed E. A.; Elgendy, Marwa O. title: Repurposing of some anti‐infective drugs for COVID‐19 treatment: A surveillance study supported by an in silico investigation date: 2020-12-17 journal: Int J Clin Pract DOI: 10.1111/ijcp.13877 sha: c61afec735dd0a91eadcc19176b6ce21cbae0882 doc_id: 848221 cord_uid: ck77orfk OBJECTIVE: The repurposing of nitazoxanide, doxycycline and azithromycin may be effective to improve the symptoms in mild and moderate COVID‐19 subjects. This study aimed to detect and explain the efficacy of reusing of these drugs in treating COVID‐19. METHODS: The study was divided into two parts: clinical and computational parts. In the clinical part, 80 (30 females) subjects with reverse transcription‐polymerase chain reaction‐confirmed COVID‐19 with mild and moderate symptoms were enrolled in the study. Subjects were treated with azithromycin or doxycycline, and nitazoxanide was added to the treatment if the subject had diarrhoea. Subjects were divided into four groups: Group 1: subjects treated with azithromycin (20 subjects); Group 2: subjects treated with doxycycline (20 subjects); Group 3: subjects treated with a combination of nitazoxanide and doxycycline (20 subjects); and Group 4: subjects treated with a combination of nitazoxanide and azithromycin (20 subjects). In the computational part, we docked the three drugs against all currently available COVID‐19‐related protein targets (viral and non‐viral). Subsequently, top hits were subjected to molecular dynamic simulations (MDSs) (50 ns) and binding free energy calculations to further validate the docking experiments and to investigate the binding modes of the potential inhibitors. RESULTS: The symptomatic improvement of mild to moderate subjects was seen on the fifth day after starting treatment in Group 3 and Group 4 and on the seventh day in Group 2. However, for Group 1, the symptomatic improvement of mild to moderate subjects was not seen on the fifth day and required replacement by doxycycline to get the symptomatic improvement. None of the subjects needed intensive care admission and no deaths were reported. In silico, results were in good accordance with the clinical outcomes, where both nitazoxanide and doxycycline achieved the best docking scores against the viral ADP‐ribose phosphatase (ADPRP) and the human Adaptor‐Associated Kinase 1 (AAK1). MDSs revealed that both drugs were stable in their bindings indicating that they can be considered as lead molecules for targeting ADPRP and AAK1. CONCLUSION: The clinical and computational studies applied on three FDA‐approved antimicrobials together with their recent clinical findings revealed that both nitazoxanide and doxycycline have great therapeutic potential against COVID‐19. The future in vitro mechanistic investigation may confirm our primary computational outcomes, and in turn, these classes of compounds provide a promising starting point for further anti‐COVID‐19 therapeutics. IL-6. 7 Consequently, it has been proposed as a promising agent in controlling the cytokine storm associated with SARS-CoV-2. 8 Hence, nitazoxanide, doxycycline and azithromycin hypothetically can be considered as a potential therapeutics against SARS-CoV-2. Computation-based (ie, in silico) drug screening has emerged during this outbreak as a rapid and efficient tool in repurposing the already available therapy, so that the health systems around the globe can handle the rapidly spreading outbreak. Thus, we aimed in this study to highlight the efficacy of the three aforementioned antimicrobial drugs in the management of COVID-19 and proposing their possible mode of action via a comprehensive in silico investigation. The clinical and computational studies applied on three FDA-approved antimicrobials together with their recent clinical findings revealed that both nitazoxanide and doxycycline have great therapeutic potential against COVID-19. The future in vitro mechanistic investigation may confirm our primary computational outcomes, and in turn, these classes of compounds provide a promising starting point for further anti-COVID-19 therapeutics. • The repurposing of nitazoxanide, doxycycline and azithromycin may be effective to improve the symptoms in mild and moderate COVID-19 subjects. • This study aimed to detect and explain the efficacy of reusing of these drugs in treating COVID-19. The subjects were given a treatment of azithromycin or doxycycline and added nitazoxanide to the treatment if the subject had diarrhoea. Subjects were divided into four groups: Group 1: subjects treated with azithromycin (20 subjects); Group 2: subjects treated with doxycycline (20 subjects); Group 3: subjects treated with a combination of nitazoxanide and doxycycline (20 subjects); and Group 4: subjects treated with a combination of nitazoxanide and azithromycin (20 subjects). The dose of azithromycin was 500 mg once daily for 5 days; doxycycline 100 mg daily for 10 days; nitazoxanide was 600 mg twice daily for 5 days. The subjects were followed up daily for symptomatic improvement. In this study, 80 (30 females) subjects with RT-PCR-confirmed COVID-19 with mild and moderate symptoms from Beni-Suef University hospital clinic were included from June to July 2020. The oldest subject was 70 years and the youngest one was 18 years. Most subjects were The symptomatic improvement (especially if there was diarrhoea) of mild to moderate subjects was seen on the fifth day after starting treatment in Group 3 and Group 4 and was seen on seventh day after starting treatment in Group 2; however, for Group 1, the symptomatic improvement of mild to moderate subjects was not seen at fifth day after starting the treatment. So, the subjects in this group needed replacing azithromycin with doxycycline to get the symptomatic improvement. None of the subjects needed intensive care admission and no deaths were reported. Docking of the three proposed drugs against all available viral-based and human-based targets ( Figure 2 ) revealed that both nitazoxanide and doxycycline have the potential to modulate the viral ADP-ribose phosphatase (ADPRP) and the human Adaptor-Associated Kinase 1 (AAK1), respectively. ADPRP is a phosphatase that removes the terminal 1″-phosphate group of ADP-ribose-1″-phosphate (Appr 1″-p). Such a process can breakdown the cellular innate immunity, and hence, facilitate the completion of viral replication and release without a host immune response. 16 Recently, the activity of ADPRP has been linked to the cytokine storm syndrome that is commonly observed in COVID-19 severe cases. 17 Herein, nitazoxanide showed a high binding affinity towards ADPRP (Docking score = −10.1 kcal/mol) and comparable with that From the clinical results, we noticed that nitazoxanide, doxycycline and azithromycin in combination gave good results in treating COVID-19. The Egyptian protocol for treating COVID-19 includes hydroxychloroquine. However, the hydroxychloroquine and azithromycin combination has some safety problems, as drug-drug interactions and cardio-toxicity, specifically in old subjects. 22 The results of this study demonstrate that the treatment with azithromycin alone was associated with a non-significant symptomatic improvement of mild to moderate subjects on the fifth day after starting treatment and required replacement by doxycycline. Previously, subjects who received azithromycin alone were shown to have the strongest cumulative hazard. 23 The high evidence of effectiveness for azithromycin is because of its role as an antibacterial drug. Although there is no direct proven effect of azithromycin in COVID-19, some scientists have suggested that the antibacterial properties of azithromycin are still clinically useful as empirical treatment of COVID-19 infection. 24 Because of the risks of using azithromycin especially in combination with hydroxychloroquine, we suggest the use of doxycycline as an alternative to azithromycin. Doxycycline has anti-inflammatory effects with in vitro antiviral efficacy against many RNA viruses. The use of this drug in our study was associated with clinical improvement and decreased cytokine production in some infections with RNA viruses. 5 Doxycycline is safe to use in the treatment of acute respiratory distress syndrome. It could be a better option for COVID-19 treatment. 25 F I G U R E 7 Interacting amino acid residues with both nitazoxanide (A) and doxycycline (B) during the course of MDS Nitazoxanide is an orally broad-spectrum anti-parasitic and antiviral drug and unlike metronidazole, its metabolites are safer and free of mutagenic factors. 26 Nitazoxanide also potentiates the production of interferon alfa and interferon beta and it has been previously shown to exhibit an in vitro activity against MERS-CoV and other coronaviruses. 27 Moreover, when nitazoxanide was given in a dose of 600 mg twice daily for 5 days, it was noticed that it reduces the duration of symptoms in subjects with acute uncomplicated influenza with little adverse effects. 28 So, this dose regimen can be used in combination with azithromycin or doxycycline to decrease SARS-CoV-2 morbidity and mortality. The nitazoxanide when used in combination with azithromycin or doxycycline appeared to be a safe and more effective regimen that might replace hydroxychloroquine/azithromycin combination as standard care for COVID-19, especially if the preliminary data regarding the low efficacy of azithromycin alone have been detected. 29 In the computational study, we were able to virtually screen these drug molecules against several viral protein targets, thanks to the rapid characterisation of SARS-CoV-2 vital proteins. Additionally, previous reports on other viral diseases including SARS-CoV-1 revealed that other host-based target proteins can enhance viral virulence. Hence, these targets were also included in our primary screening step. As a result, two enzymes, ADPRP and AAK1, appeared to be the possible targets of both nitazoxanide and doxycycline. ADPRP is a key enzyme that enables the virus to bypass the host immunity, while AAK1 is a host-based kinase that controls the viral entry and processing inside the host cell. Consequently, targeting both of these enzymes can provide effective anti-COVID-19 Subsequent MDSs and binding free energy calculations tentatively illustrated the mode of interactions of nitazoxanide and doxycycline inside the active sites of both proteins. Surprisingly, each tested drug molecule achieved binding free energies higher than the reported co-crystallised inhibitors, and thus, they possess the interesting potential to be considered as drug candidates for further in vitro evaluation. Besides the observed therapeutic efficacy of these antimicrobial agents in treating COVID-19, they can alleviate the secondary bacterial infection associated with the disease, particularly in immunocompromised subjects, since 15% prevalence of secondary bacterial infection were found among hospitalised subjects. 30 The repurposing nitazoxanide, doxycycline and azithromycin are highly effective for the symptomatic improvement of mild and moderate COVID-19 subjects. Dual therapy by combining nitazoxanide with azithromycin or doxycycline is considered to be better than monotherapy. The use of azithromycin alone is not recommended, as it was not effective against symptoms compared with the other regimens used. The clinical and computational studies applied on three FDAapproved antimicrobials together with their recent clinical findings revealed that both nitazoxanide and doxycycline have great therapeutic potential against COVID-19. The future in vitro mechanistic investigation may confirm our primary computational outcomes, and in turn, these classes of compounds provide a promising starting point for further anti-COVID-19 therapeutics. No conflict to declare. 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