key: cord-0851035-14alhxc3 authors: Low, Zheng Yao; Yip, Ashley Jia Wen; Lal, Sunil K. title: Repositioning Ivermectin for Covid-19 treatment: Molecular mechanisms of action against SARS-CoV-2 replication date: 2021-10-20 journal: Biochim Biophys Acta Mol Basis Dis DOI: 10.1016/j.bbadis.2021.166294 sha: e3e1d5be03b16423c901cf381e4b3195aaa56af0 doc_id: 851035 cord_uid: 14alhxc3 Ivermectin (IVM) is an FDA approved macrocyclic lactone compound traditionally used to treat parasitic infestations and has shown to have antiviral potential from previous in-vitro studies. Currently, IVM is commercially available as a veterinary drug but have also been applied in humans to treat onchocerciasis (river blindness - a parasitic worm infection) and strongyloidiasis (a roundworm/nematode infection). In light of the recent pandemic, the repurposing of IVM to combat SARS-CoV-2 has acquired significant attention. Recently, IVM has been proven effective in numerous in-silico and molecular biology experiments against the infection in mammalian cells and human cohort studies. One promising study had reported a marked reduction of 93% of released virion and 99.98% unreleased virion levels upon administration of IVM to Vero-hSLAM cells. IVM's mode of action centres around the inhibition of the cytoplasmic-nuclear shuttling of viral proteins by disrupting the Importin heterodimer complex (IMPα/β1) and downregulating STAT3, thereby effectively reducing the cytokine storm. Furthermore, the ability of IVM to block the active sites of viral 3CLpro and S protein, disrupts important machinery such as viral replication and attachment. This review compiles all the molecular evidence to date, in review of the antiviral characteristics exhibited by IVM. Thereafter, we discuss IVM's mechanism and highlight the clinical advantages that could potentially contribute towards disabling the viral replication of SARS-CoV-2. In summary, the collective review of recent efforts suggests that IVM has a prophylactic effect and would be a strong candidate for clinical trials to treat SARS-CoV-2. Several studies in the past have revealed the possible role of SARS-CoV-1 ORF6 interacting with the Karyopherin-α2 (KPNA2), retaining the IMPα/β1 of the Golgi membrane. Thereafter, inhibiting the STAT1 nuclear transport, antagonizing antiviral activity and downplaying the host's antiviral response (28, 29, 30) . Given the role of importin in many viruses, especially SARS-CoV-1, it is of great interest to explore the mechanism of action of IVM for its potential in viral inhibition. We briefly describe the antiviral action of IVM for each of the viruses listed above and postulate its role in the SARS-CoV-2 infection cycle. The Flavivirus genus spans over numerous types of viruses such as ZKV, WNV and many more. ZKV is an enveloped single-stranded positive-sense RNA virus, akin to the SARS-CoV-2 (31) . Differing from SARS-CoV-2, ZIKV is mosquito-borne via Aedes mosquitoes carrying the possibility of non-mosquito transmissions, such as pregnancy (mother-foetus) and sexual transmission (32) . ZKV increases the risk of neurological complications such as Guillain-Barré syndrome, neuropathy, and myelitis (33) . An in-vitro study conducted by Barrow and colleagues has discovered the antiviral effects of IVM on ZKV, particularly on the ZKV MEX_I_7 strain (32) . In this study, IVM showed the strongest inhibition of ZKV at concentrations of 10 μM and 16 μM in human liver cells (HuH-7) and human amnion epithelial cells (HAECs), respectively. Apart from this, another study has also shown IVM to act as an inhibitor of importin α/β that blocks non-structural protein 5 (NS5) interaction with IMPα/β transporter in ZKV, leading to 60% NS5 reduction in nucleus post-7 hours IVM treatment (34) . NS5 is an important protein for the methyltransferase and RNA-dependent RNA polymerase activities in ZKV, which is vital for viral replication (34) . the human population, the IAV needs to overcome MxA and nuclear viral ribonucleoprotein (vRNP) nuclear import restrictions (38). MxA antiviral proteins are produced by Myxovirus resistance gene1 (MX1) upon induction of the type-I (α/β) or type-III (λ) Interferons (IFN) during the early stages of IAV infection (38) . Interestingly, the determining factor for the strength of MxA inhibition relies upon the viral nucleoprotein (NP) (38). It was hypothesized that the MxA inhibits IAV in two different pathways. Firstly, it interferes with the transcription of viral RNA via retention of incoming vRNPs in the cytoplasm (39). Secondly, MxA inhibits the amplification of viral RNA (vRNA) via cytoplasmic sequestering of newly synthesized NP and PB2 (40) . IVM as a importin α/β complex inhibitor shows a complete suspension of the nuclear import for all vRNP complexes in both wild-type and antiviral MxA escape IAV mutants at 10 µM concentration. Therefore, it effectively inhibits the viral replication (41) . These findings provide a clear potential for IVM as an antiviral medication against IAV. Human immunodeficiency virus type 1 or HIV-1 is a single-stranded, positive-sense, enveloped RNA virus under the genus Lentivirus. Upon entry into the cell, the vRNA genome of HIV-1 is transcribed into dsDNA (cDNA) via reverse transcriptase (42) . This is followed by the import of dsDNA into the cell nucleus and integration into the cellular DNA by the viral encoded enzyme Integrase (IN), allowing for immune evasion and subsequent vRNA replication. HIV-1 depend heavily on IN for efficient viral production and replication (42) . IVM has been documented to show a reduction of IN nuclear accumulation in HIV-1 infected cells, with a half-maximal inhibitory concentration (IC 50 ) of 2 μM (43) . Another study showed that treatment of 25μM IVM in HIV-1 infected HeLa cells for 2 hours, significantly reduced viral replication with no observable cell toxicity induced (21) . These results the envelope, it consists of a ~29.9 kilobase (kb) RNA genome with 2/3 rd of the genome containing the main open reading frame 1a and 1b (ORF1ab) replicase gene from the 5'-end, encoding for the non-structural proteins (NSP 1-16) while the remaining 1/3 rd genome encodes for the structural proteins (spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N)) (46) . The NSPs play an important role for viral replication in infected host cells. NSP 1 and 3 are known to inhibit IFN signalling, interrupting the translation of RNA and innate immune responses (47) . NSP 3 and 5 promote cytokine expression and viral protein cleavage (47) . NSP-12 is an RNA-dependent RNA polymerase (RdRp) and has been shown to be inhibited by IVM in studies on SARS-CoV and MERS-CoV (48,49). This discovery discloses strong possibilities for SARS-CoV-2. RdRp, also known as RNA replicase, is a vital enzyme in the life cycle for RNA viruses since it primarily functions to catalyse the replication of RNA from an existing RNA template in the virus thereby initiating viral replication. The SARS-CoV accessory protein ORF6 has been shown to sequester IMPα/β1 on the rough endoplasmic reticulum which antagonizes the STAT1 transcription factor, resulting in an antiviral potential (50). The genomic similarity between SARS-CoV-2 and the previous SARS-CoV may reveal the role of the importin heterodimer complex (IMPα/β1) for viral protein (NSP12-RdRp) shuttling between the nucleus and cytoplasm upon infection. Currently, there is only one RdRp inhibitor approved by the FDA for Covid-19, namely Remdesivir (51) . However, a recent discovery from Monash University, Australia reported that IVM could inhibit SARS-CoV-2 within a 48 hours post-infection, drawing much attention worldwide (22) . IVM, a non-specific inhibitor of IMPα/β1-dependent nuclear import, now shows great potential in reducing SARS-CoV-2 viral replication via different modes. Apart from Transporting host proteins, such as STAT or NF-κB transcription factor families, in-and-out of the nucleus is a crucial function for normal nuclear activity in all eukaryotic cells. The signal for transport is mediated by the members of the IMP superfamily transporters, αand βtypes (54) . The import of host protein, such as STAT protein (>45kDa) into the nucleus Theoretically, the IVM binds to the IMPα, dissociating the IMPα from the heterodimer IMPα/β1 complex, thus preventing nuclear import of viral proteins via the NPC, halting the IMPα/β1 dependent nuclear import activities of viral SARS-CoV-2 proteins (NS12-RdRp) (56) . In the laboratory setting, numerous in-vitro studies on IVM have been carried out recently. An in-vitro infection study on a SARS-CoV-2 isolate, Australia/VIC01/2020 showed a 93% reduction in viral RNA at 24 hours and a 99.98% reduction (~5000-fold) at 48 hours upon administration of 5 μM IVM (22) . The same study also found no cytotoxicity from administration of IVM, making it a safe and efficacious candidate for further clinical studies (22 and viral clearance (58) . Meanwhile, an in-silico study revealed that IVM is capable of binding to the RdRp complex at the active residues (Ser759 and Asp760), further insinuating the potential of IVM for inhibiting SARS-CoV-2 viral replication (59). J o u r n a l P r e -p r o o f Journal Pre-proof IVM may be a good therapeutic tool to inhibit the cytokine storm and prevent ADRS in the following pathways. IVM blocks the IMPα/β1-dependent nuclear import of viral proteins (NS12-RdRp) as mentioned prior (56) . IVM also inhibits the activity of STAT3, subsequently reducing inflammatory IL-6 cytokine production. Besides that, IVM promotes the ubiquitination-mediated degradation of p21 activated kinase 1 (PAK1), a key protein that binds to STAT3 for IL-6 gene transcription, disrupting IL-6 production (60, 68). The Much like the previous SARS-CoV, SARS-CoV-2 also requires the binding of spike (S) protein to the host angiotensin-converting enzyme 2 (ACE2) receptors to infiltrate the host cells (69) . There are two regions in the receptor-binding motif on the S protein that forms an interface between S protein and ACE2. Key residues from the S protein, namely L455 and Q493, possess a high affinity for residues K31 and E35 on the ACE2 via hydrogen bonding, thus encouraging the binding of S protein to the ACE2 receptor (70). It was evident that the overexpression of ACE2 was highly associated to SARS-CoV-2 replication, from which the Moreover, there is an interaction between the alkyl group from IVM and aromatic rings of S protein residues (TYR449, TYR489, PHE456, LEU455, PHE490). The LEU455 and GLN493 possesses a high binding affinity with ACE2, showing that IVM binding to these residues will block the attachment of S protein to host ACE2, subsequently obstructing viral entry, and effectively reducing the viral load (52) . As described previously, the envelope of SARS-CoV-2 is a ~29.9 kilobases (kb) RNA genome exists containing the ORF-1a and 1b genes to encode NSPs and the structural protein (S, E, M, N) (46) . Upon infection, the SARS-CoV-2 binds to the ACE2 receptor via its S protein, subsequently hijacking the host ribosome and reprogrammes it to translate the viral RNA to large polypeptides (53) . These polypeptides must be auto-cleaved by papain-like proteases (PLpro) and 3-chymotrypsin like protease (3CLpro) to generate the NSP required for viral replication, such as the RdRp enzyme produced by the NSP12 (53) . 3CLpro or Mpro is characterized as a three-domain protein that is highly conserved amongst coronaviruses, in which mutation of 3CLpro sequences has shown to be fatal for many viruses. Thus, the risk of drug resistance from virus evolution is significantly reduced (76) . Shown here, the 3CLpro shares a high sequence identity of ≥95% between SARS-CoV and SARS-CoV-2, highlighting the potential of 3CLpro as a target for drug repurposing (77) . of 3CLpro (79) . The His41 acts as a proton acceptor while the Cys145 is primarily targeted by the carbonyl carbon of the substrates. As discussed by Dai et al., the inhibitory activity of drug candidates for 3CLpro confers within its ability to form covalent bonds with Cys145, disrupting dimerization (80, 81, 82) . From which, several key residues (H41, C145, Q192, T190, A191, D187, Q189, M165, S144, D187, M49, G143 and N142) that highly contributed to the binding affinity of 3CLpro inhibitors have been revealed, as H41 and C145 were the most conserved residues amongst coronaviruses (77) . In particular, perampanel, praziquantel, and nelfinavir have demonstrated better inhibitory activity for 3CLpro in SARS-CoV-2 due to its ability to bind with the abovementioned key residues (7,77). In further note, a Michael acceptor-based peptidomimetic inhibitor, N3 has shown great inhibitory activity against SARS-CoV-2 3CLpro over some competitive drug such as lopinavir, and ritonavir which generally requires a high concentration for a significant effect (83, 84, 85) . The N3 functions via covalent binding between the vinyl group of N3 and the aforementioned C145 of the catalytic dyad in 3CLpro. Moreover, N3 possesses several features (lactam ring, aliphatic isobutyl group and methyl group) which corresponds to its side chain (P1, P2 and P4), allowing it to fit tightly to the subsites of S1, S2 and S4 via multiple hydrogen bonds, to effectively hinder the activity of 3CLpro (79, 86) . Similar to the N3 inhibitor molecule, an in-silico study showed that IVM exhibits an inhibitory effect (>85%) against the 3CLpro in SARS-CoV-2 at 50 µM concentration, positioning it as another promising antiviral candidate (53) . It was later found that the carbonyl group in IVM also forms hydrogen bonds with the active site residues (Cys 145 and His 41 ) of 3CLpro monomer, destabilising the complex, leading to the loss of function and subsequently reducing its protease activity (53, 87, 88) . Besides that, IVM was also found to have interact with 3CLpro via ten polar residues (Arg188, Asp187, Asn142, His41, His164, Ser46, Glu166, Gly170, Gln189, and Thr169), and seven non-polar residues (Cys145, Leu27, Leu50, Leu167, Met49, Met165, and Pro168) of subunit 1 (75) . The interaction between the two polar residues (Gln306 and Ser1), and two non-polar residues (Phe305 and Val303) of subunit 2 were discovered as well. The stable hydrogen bond interactions were also illustrated by the docking simulation, in which IVM complexes with the subunit 2 of 3CLpro at Thr304, and glu166 residues, forming the greatest binding energy (T). Lastly, the inhibition of the 3CLpro complex has been proven a success in HIV-1 and SARS-CoV previously, making 3CLpro a viable target for IVM (89) . Numerous clinical studies are underway to study the efficacy of IVM. Ivermectin has illustrated great potency towards asymptomatic SARS-CoV-2-positive subjects in a randomised trial in Lebanon (90) . In that study, the IVM group (n=50) has an increased cycle-threshold (Ct) value from 15.13 to 30.14 whereas the control group (n=50) has Ct values from 14.20 to 18.96 at 72 hours (90) . A higher Ct-value denotes for an insignificant viral remnant or non-viable virus, from which the values of 30 and above are to be considered as negative (91) . Additionally, the subjects from the IVM group developed fewer symptoms compared to the non-IVM group from the reported incidence of fever (2% vs 22%), ageusia (6% vs 24%), anosmia (6% vs 32%), and myalgia (0% vs 18%) (90) . Other notable studies on the positive effects of IVM on COVID-19 patients are as follows. Despite numerous positive outcomes for IVM in SARS-CoV-2 treatment, there are several contradictions. For instance, a study has reviewed that the standard dosage of single-dose IVM (200ug/kg) showed no significant clinical and microbiological outcomes compared to the patients that did not receive any IVM (95) . Albeit there was no significant difference, a smaller proportion of the patients from the IVM group required intensive care as compared to the non-IVM group (69% vs 38%), postulating the need for a higher effective dose (95) . Another study pointed no differences in the viral load, outcomes of adverse events, and the laboratory parameters between the IVM treated group ( and safety and efficacies studies for the mass public (98). Another report showed that IVM alone has a low probability of success in treating COVID-19 as the approved dose or dose of 10x higher than the approved is unlikely to reach the concentration needed for 50% inhibition (IC50) in the lungs after single-dose oral administration, making it less ideal for COVID-19 treatment (99). The rapid emergence of SARS-CoV-2 has put drug repositioning in a critically important position. IVM, an FDA approved antiparasitic drug for onchocerciasis and strongyloidiasis, now sees great opportunity as an interim antiviral drug for SARS-CoV-2. 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