key: cord-0686584-gyqugmci
authors: Rabie, Amgad M.
title: Teriflunomide: A possible effective drug for the comprehensive treatment of COVID-19
date: 2021-09-11
journal: Current research in pharmacology and drug discovery
DOI: 10.1016/j.crphar.2021.100055
sha: 2605eef014d24ffdc36c886fdad57eb2f567b313
doc_id: 686584
cord_uid: gyqugmci

The coronavirus disease 2019 (COVID-19) pandemic has undoubtedly become a global crisis. Consequently, discovery and identification of new or known potential drug candidates to solve the health problems caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become an urgent necessity. This current research study sheds light on the possible direct repurposing of the antirheumatic drug teriflunomide to act as an effective and potent anti-SARS-CoV-2 agent. Herein, an interesting computational molecular docking study of teriflunomide, to investigate and evaluate its potential inhibitory activities on the novel coronaviral-2 RNA-dependent RNA polymerase (nCoV-RdRp) protein, was reported. The docking procedures were accurately carried out on nCoV-RdRp (with/without RNA) using the COVID-19 Docking Server, through adjusting it on the small molecule docking mode. Remdesivir and its active metabolite (GS-441524) were used as the active references for the comparison and evaluation purpose. Interestingly, the computational docking analysis of the best inhibitory binding mode of teriflunomide in the binding pocket of the active site of the SARS-CoV-2 RdRp revealed that teriflunomide may exhibit significantly stronger inhibitory binding interactions and better inhibitory binding affinities (teriflunomide has considerably lower binding energies of −9.70 and −7.80 kcal/mol with RdRp-RNA and RdRp alone, respectively) than both references. It was previously reported that teriflunomide strongly inhibits the viral replication and reproduction through two mechanisms of action, thus the results obtained in the present study surprisingly supports the double mode of antiviral action of this antirheumatic ligand. In conclusion, the current research paved the way to practically prove the hypothetical theory of the promising abilities of teriflunomide to successfully attack the SARS-CoV-2 particles and inhibit their replication in a triple mode of action through integrating the newly-discovered nCoV-RdRp-inhibiting properties with the previously-known two anticoronaviral mechanisms of action. Based on the previous interesting facts and results, the triple SARS-CoV-2/sextet COVID-19 attacker teriflunomide can further undergo in vitro/in vivo anti-COVID-19 assays together with preclinical/clinical studies and trials in an attempt to evaluate and prove its comprehensive pharmacological activities against the different SARS-CoV-2 strains to be effectively used in COVID-19 therapy in the very near future.

On the latest days of 2019, a novel type of the coronaviruses (2019-nCoV), known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), dramatically appeared in Wuhan (China) (Hui et al., 2020) . Transmission of this occult single-stranded positive nonsegmented RNA viral microbe is continuous resulting in the prevalence of the virus specific illness, coronavirus disease 2019 , with its main symptoms specifically present in the respiratory system of humans (reach to severe pneumonia and death in many cases) (Hui et al., 2020; . The SARS-CoV-2 has sheaths that enfold around the RNA genome (virion, which is the whole virus, is round/oval, usually polymorphic, with a diameter of approximately 50-200 nm) (Wu et al., 2020) . Except for the promising investigational drugs, cyanorona-20, CoViTris2020, ChloViD2020, and Taroxaz-104, there is no specific and effective potent drug therapy successfully recognized for COVID-19 to date at the middle of 2021 Rabie, 2021a; Rabie, 2021b; Rabie, 2021c; Rabie, 2021d; Rabie, 2021e; Rabie, 2021f) .

The old drugs known for their antiviral activities or any activities that hinder any stage(s) of the coronaviral life cycle are of special interests for medicinal chemists for the repurposing strategies against the COVID-19. Potent antirheumatic medicines, like hydroxychloroquine, leflunomide, and teriflunomide, are among the drugs that are under the microscope in this respect due to their potential antiviral activities (Ip et al., 2021; Rabie, 2021a; Mei-Jiao et al., 2019; Xiong et al., 2020) . To the best of my knowledge, there is not any reported comprehensive evidence-based and/or clinical study that investigated, discussed, and proved the possibility of repurposing J o u r n a l P r e -p r o o f leflunomide and/or its active metabolite teriflunomide against the resistant COVID-19 to date.

Teriflunomide (chemically, its structure is 2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide), a once-daily orally-administered potent immunosuppressive/immunomodulatory agent, was regulatory approved in most countries of the world (including the United States and the European Union) for the effective treatment of the moderate-to-severe multiple sclerosis (MS) and some other rheumatic conditions (Bar-Or et al., 2014; Breedveld and Dayer, 2000) . It was firstly identified as the major active metabolite of its parent antirheumatic drug leflunomide which is used mainly for the treatment of rheumatoid arthritis (Breedveld and Dayer, 2000) . Leflunomide is converted, by the action of the in vivo metabolic activation, into teriflunomide which is specifically responsible for the complete biological and therapeutic actions of the parent drug ( Fig. 1) (Breedveld and Dayer, 2000) . The only chemical difference between the two molecules is the opening of the isoxazole ring to free and form the two pharmacologically-important moieties cyano and hydroxyl groups, of the bioactive teriflunomide molecule, instead of being embedded as lessactive heteroatoms (nitrogen and oxygen atoms, respectively) in the heterocyclic isoxazole ring of the leflunomide molecule (Breedveld and Dayer, 2000; Rozman, 2002) . The molecular pharmacokinetic studies revealed that the teriflunomide molecule clinically exits in two different tautomeric structures, enol/keto forms, in the blood; the enol form, in turn, exists either in the Z-or E-configuration (Z-E interconversion), with the Z enolic form being the most predominant structure since it J o u r n a l P r e -p r o o f 

It is worth mentioning that there are two main concerns in the attack of COVID-19, the viral load (almost the first stage of the disease, which is mainly characterized by the speedy SARS-CoV-2 replication) and the immune response (the second stage of the disease, which is characterized by the severe immune-mediated damage) (Cantini et al., 2020) . If both pivotal stages are successfully managed and inhibited, the disease will be almost significantly controlled and cured. Additionally, the success in managing any consequent, secondary, and marginal health concerns during and after the coronaviral-2 attack should also be considered. In principle, teriflunomide, with its exceptional synergistic immunomodulatory/antiviral dual mode of action, can expectedly fulfill the managing strategy for COVID-19, no matter the infecting SARS-CoV-2 particles are mutated or not (this may be a very unique characteristic property of this drug in COVID-19 treatment) (Bar-Or et al., 2014; Xiong et al., 2020) . First, it can reduce and control the SARS-CoV-2 viral load, and, second, it can fight and overcome the massive cytokine immune outbreak. Recently, clinical findings proved that COVID-19 is very mild in patients treated with continuous therapeutic doses of teriflunomide and that continuing with teriflunomide therapy during SARS-CoV-2 infection and COVID-19 is very safe and even highly recommended for MS patients (Luetic et al., 2021; Capone et al., 2021; Ciardi et al., 2020) . The major objective of the present research work is to search for and find any additional anti-SARS-CoV-2 activities (anti-COVID-19 mechanisms of action) of teriflunomide to assess the possibility of its clinical use as an available choice to be incorporated in the optimal therapeutic strategies and protocols designed for the effective COVID-19 treatment.

J o u r n a l P r e -p r o o f RNA-dependent RNA polymerase (RdRp) is considered one of the most interesting and effective targets for designing new medicines against the renitent SARS-CoV-2 (Rabie, 2021a; Rabie, 2021c) . Structurally, RdRp is the nonstructural protein 12/7/8 (nsp12/7/8); nsp12 (chain A) is the polymerase which binds to its two major cofactors, nsp7 (chain C) and nsp8 (chains B and D) (Yin et al., 2020) . It is a very pivotal enzyme in the replication and transcription of the coronaviral genome, and as a result, the potent inhibition of the performance and activities of this enzyme will significantly hinder the replication of SARS-CoV-2 and, consequently, restrict the COVID-19 infection as a whole (Rabie, 2021a; Rabie, 2021c; Yin et al., 2020) . The close resemblance between the structures of teriflunomide and nucleoside analogs inspired me to propose the possible action of teriflunomide to act as a potential potent inhibitor of the novel coronaviral-2 RdRp (nCoV-RdRp or, simply, CoV-RdRp). To accurately evaluate this possibility, a complete computational molecular docking study was performed using the new and well-validated molecular docking web server COVID-19 Docking Server (COVID-19 Docking Server, 2021) . Remdesivir comparator protocol was used for the comparison and validation purposes using both remdesivir (a nucleotide analog) and its active metabolite (GS-441524; a nucleoside analog), since they are almost the only internationally-approved potent nCoV-RdRp inhibitors to date (Moirangthem and Surbala, 2021; Eastman et al., 2020; Yan and Muller, 2020) . The results were very promising, since they revealed the strong inhibitory binding affinities of teriflunomide with the active amino acid residues of the binding pocket(s) of the nCoV-RdRp main active site (either in its complicated state with RNA or in the free state). Surprisingly, teriflunomide considerably surpasses the J o u r n a l P r e -p r o o f native ligand remdesivir together with the active metabolite GS-441524 in the negative binding energies with the nCoV-RdRp.

Based on these results and all the previous literature data and knowledge (Cantini et al., 2020; Mei-Jiao et al., 2019; Xiong et al., 2020; Teschner and Burst, 2010; Bar-Or et al., 2014; Claussen and Korn, 2012; Moon et al., 2017; Breedveld and Dayer, 2000; Capone et al., 2021; Ciardi et al., 2020; Luetic et al., 2021) , my current comprehensive hypothesis can be confidently established, stating that teriflunomide can potentially act as a very effective drug candidate for the treatment of COVID-19 via two broad complementary efficient modes of action (i.e., a dual mode of action), each of which has three synergistic distinct mechanisms of action (Fig. 3) . The first triple pathway is the novel anticoronaviral (anti-SARS-CoV-2 replication) mode of action, which includes: 1. inhibiting the coronaviral replication through interfering with the nucleocapsid tegumentation of SARS-CoV-2, which results in disruption of the SARS-CoV-2 virion assembly; 2. interfering with the coronaviral replication in the infected cells through inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), which plays a critical key role in the de novo synthesis of pyrimidine and uridine monophosphate (rUMP), resulting in reduced pyrimidine de novo synthesis and depletion of the available pyrimidine pools, this in turn causes a large decrease in the nucleoside/nucleotide availability required for the RNA synthesis and SARS-CoV-2 proliferation (i.e., antiproliferative effect); and 3. blocking the coronaviral replication through acting as a potent direct nCoV-RdRp inhibitor (new potential effect). On the other hand, the second triple pathway is the potent immunomodulatory (immunoregulatory or anticytokine storm) mode of action, which J o u r n a l P r e -p r o o f includes: 1. reducing the cytokines generation, especially the interleukin 6 (IL-6), which is found to significantly contribute to the human acute respiratory distress syndrome (ARDS) in the SARS-CoV-2 infection; 2. inhibiting the immune cells activation (mainly, inhibiting the autoimmune lymphocytic activities, adhesion molecules expression, and immunoglobulin production) through disrupting the interactions with the antigen-presenting cells (by the integrin activation impairment and reduced protein aggregation); and 3. impairing the cellular (mainly, the activated autoimmune cell) reproduction and proliferation through the previously-mentioned strong action of blocking the pyrimidine de novo biosynthesis and, therefore, significantly depleting the intracellular pyrimidine pools. The first mode of action is concerned with the coronaviral microbe (SARS-CoV-2) particles, while the second one is especially concerned with the host (human) cells. Both triple modes of action are expected to synergistically act in a complementary/integrative and comprehensive strategic way in COVID-19 therapy.

In a word, an interesting computational molecular docking study of teriflunomide as a potential nCoV-RdRp inhibitor (anti-SARS-CoV-2 drug candidate) was reported in this current work, and thus this important research paved the way to logically and practically (i.e., clinically) establish and prove the theoretical hypothesis of the promising actions of teriflunomide to successfully treat the COVID-19 via attacking the SARS-CoV-2 and effectively inhibiting its replication in a triple antiviral mode of action (it acts as a potential triple attacker of the virus, and as a sextet attacker of the COVID-19 in general) through integrating the newly-discovered nCoV-RdRpinhibiting properties (investigated herein) with the previously-known two J o u r n a l P r e -p r o o f anticoronaviral mechanisms of action (together with the original three potent immunomodulatory mechanisms of action). 

To specifically assess the nCoV-RdRp-inhibiting properties among the overall potential anti-COVID-19 activities of the antirheumatic drug teriflunomide before its Server, 2021). For docking of just one small molecule each time, the "Docking" mode box as the computational type must be particularly chosen for each specific target (this is the used option in the current state). To obtain the most precise results, an average exhaustiveness option of "12" was selected. Teriflunomide was the tested ligand, while remdesivir and GS-441524 were used as the positive reference control ligands. The resulted binding complexes were clearly visualized in 3D models by JSmol. The data outputs of the COVID-19 Docking Server include both the binding free energy score values (in kcal/mol) and rescoring binding affinity random forest (RF) score values (expressed as pKd "= -log (Kd)") (Kd is the dissociation constant which is commonly used to quantify the strength with which a ligand binds to a specific protein. This important equilibrium constant measures the tendency for a specific protein-ligand complex to separate into its constituent components; it is used herein to describe the degree of tightness of proteins to their binding ligands "binders". That is, by interpreting complexes whose components are more likely to dissociate "high dissociation constants" as loosely bound "low binding affinities" and vice versa. In brief, higher pKd values reflect exponentially greater binding affinities). The detailed results of all the estimations for the top (best) model in each case are shown in Table 1 ( Fig. 4a,b and Fig. 5a- 

The perception of the SARS-CoV-2 protein-ligand interactions exemplifies a very critical key challenge in drug discoveries for the management and treatment of COVID-19. The primary and fundamental theoretical prediction of the nCoV-RdRpinhibiting properties of the newly-repurposed antirheumatic drug teriflunomide quite J o u r n a l P r e -p r o o f assists us to get an overview of the comprehensive anti-COVID-19 activities of this target compound. This computational expectancy also aids us to obtain a detailed conception about the major mode of anti-COVID-19 action of teriflunomide together with the drug degrees of effectiveness and potency. The validated docking procedures were sufficient for that, since they were performed in the nCoV-RdRp in each of its two states, the complex-with-RNA state and the free one. Table 1 . Score values of the two computationally-predicted anti-SARS-CoV-2 properties (against SARS-CoV-2 RdRp-RNA and SARS-CoV-2 RdRp, respectively) of the target teriflunomide and its two potent antiviral references (remdesivir and GS-441524), respectively, using the COVID-19 Docking Server methodology (the table shows the top docking model score value "ranked 1", i.e., the top binding mode score value or the least predicted binding free energy value, in kcal/mol, together with its corresponding highest binding affinity RF score value, expressed as pKd value, for each compound with each target RdRp site of the two ones). 

On close checking of the score values of nCoV-RdRp-RNA and nCoV-RdRp dockings using the COVID-19 Docking Server (shown in Table 1) , it is clearly observed that teriflunomide is specifically ranked first in its inhibitory binding affinities and potencies with binding free energies of -9.70 and -7.80 kcal/mol and with corresponding rescoring RF values of pKd of 6.99 and 5.66, respectively. The binding affinities of teriflunomide considerably exceed those of the two bioactive references remdesivir (it has binding free energies of -8.30 and -7.10 kcal/mol, and RF score values "expressed as pKd" of the inhibitory binding affinities of 5.38 and 5.27, respectively) and GS-441524 (it has binding free energies of -9.10 and -7.00 kcal/mol, and RF score values "expressed as pKd" of the inhibitory binding affinities of 6.47 and 4.86, respectively). Teriflunomide powerfully binds to the SARS-CoV-2 RdRp (with RNA) in their complex (i.e., teriflunomide molecule forms a very stable complex with the SARS-CoV-2 RdRp-RNA) with a relatively low binding free energy which is the lowest among all (i.e., significantly lower than the binding free energies of both remdesivir and GS-441524 in their complexes with the coronaviral-2 RdRp-RNA).

GS-441524 and its parent potent antiviral remdesivir come second and third, respectively, in their relative inhibitory binding, potency, and efficacy on nCoV-RdRp, therefore, the results clearly express the high superiority of the antirheumatic teriflunomide over both of them as a potent anti-COVID-19 candidate agent. For more explanation, Fig. 4a,b and Fig. 5a- (Rabie, 2021e) . This, in turn, results in more proper and sufficient (i.e., potent) inhibition of the replication activities mediated or controlled by the SARS-CoV-2 RdRp. Foreseeably, the highly-balanced considerable flexibility of teriflunomide molecule significantly increases its potential biological ability and activity to act as a very potent anti-COVID-19 agent.

Additionally, the possibility that teriflunomide molecule may undergo intracellular metabolism into less active forms by human cellular enzymes is extremely limited or even totally excluded, since, biologically and clinically, teriflunomide is almost the final active metabolite that could be biogenerated and obtained from the J o u r n a l P r e -p r o o f parent antirheumatic leflunomide. Thus, teriflunomide has an extra advantage, over remdesivir and most other investigated anti-COVID-19 drug candidates, of being nonconvertible to other inactive or less active forms in vivo. It is worth noticing that teriflunomide has significant chemical structural similarity with many underinvestigation anti-COVID-19 drug candidates, such as remdesivir, cyanorona-20 (Rabie, 2021a; Rabie, 2021b) , GS-441524, and favipiravir (Cai et al., 2020) , since it has the same key and principal anti-nCoV-RdRp structural elements and features (e.g., 

Comprehensively, in the light of the proved potent antiviral/immunomodulatory activities of teriflunomide, together with the promising computational docking results of the current detailed ligand-protein interaction study, we can conceptually establish a strong starting base for the promising abilities of teriflunomide to successfully strike the SARS-CoV-2 particles of different strains and combat their accompanying immunogenic cytokine storm/inflammatory condition in humans, thus providing an effective dual drug treatment for COVID-19. Teriflunomide significantly inhibits SARS-CoV-2 RdRp with encouraging low inhibitory binding energies, which reach about -9.70 kcal/mol, and strong inhibitory binding interactions (comparable to and J o u r n a l P r e -p r o o f even better than those of the only FDA/WHO-approved anti-COVID-19 drug remdesivir along with its potent active metabolite GS-441524). The current research study supports the hypothesis that teriflunomide may inhibit SARS-CoV-2 replication in a triple mode of action through integrating the newly-discovered mechanism of nCoV-RdRp-inhibiting action with the previously-known two mechanisms of anticoronaviral-2 action. Additionally, teriflunomide attacks the inflammatory COVID-19 immune storm also in a triple mode of immunomodulatory action.

Hopefully, the triple SARS-CoV-2 attacker (or the sextet COVID-19 attacker)

teriflunomide can be further subjected to in vitro/in vivo anti-COVID-19 assays together with preclinical/clinical studies and trials in an attempt to assess and successfully confirm its comprehensive pharmacological bioactivities against SARS-CoV-2 to be efficiently used as an available choice in the COVID-19 therapy in the near future. In a word, teriflunomide is computationally and hypothetically proposed to be a potential effective anti-COVID-19 drug (i.e., a potent candidate anticoronaviral-2 agent), since it can potentially take integrative and comprehensive roles in the treatment of COVID-19 via its dual mode of six synergistic mechanisms of action (which are perfectly complementary to each other in COVID-19 therapy).

I hereby declare that I totally have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this new paper.

J o u r n a l P r e -p r o o f 29 : Acknowledgments I gratefully thank and deeply acknowledge anyone who gave a hand to make this new discovery and work coming out to light.

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