key: cord-1046791-50qjlarf
authors: Jans, David A.; Wagstaff, Kylie M.
title: The broad spectrum host-directed agent ivermectin as an antiviral for SARS-CoV-2?
date: 2020-10-21
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.10.042
sha: b9883fb5b42ee63bb273f72bcbf5d197d8288a36
doc_id: 1046791
cord_uid: 50qjlarf

FDA approved for parasitic indications, the small molecule ivermectin has been the focus of growing attention in the last 8 years due to its potential as an antiviral. We first identified ivermectin in a high throughput compound library screen as an agent potently able to inhibit recognition of the nuclear localizing Human Immunodeficiency Virus-1 (HIV-1) integrase protein by the host importin (IMP) α/β1 heterodimer, and recently demonstrated its ability to bind directly to IMPα to cause conformational changes that prevent its function in nuclear import of key viral as well as host proteins. Cell culture experiments have shown robust antiviral action towards a whole range of viruses, including HIV-1, dengue, Zika and West Nile Virus, Venezuelan equine encephalitis virus, Chikungunya, pseudorabies virus, adenovirus, and SARS-CoV-2 (COVID-19). Close to 70 clinical trials are currently in progress worldwide for SARS-CoV-2. Although few of these studies have been completed, the results that are available, as well as those from observational/retrospective studies, indicate clinical benefit. Here we discuss the case for ivermectin as a host-directed broad-spectrum antiviral agent, including for SARS-CoV-2.

The work identifying ivermectin, a macrocyclic lactone 22,23-dihydroavermectin B produced by the bacterium Streptomyces avermitilis, and subsequently demonstrating its activity as a novel therapeutic against "infections caused by roundworm parasites" was recognized, along with the seminal work on the antimalarial artemisin, with the award of the 2015 Nobel Prize for Physiology or Medicine [1, 2] . Soon after its discovery in 1975, ivermectin was approved as a treatment for parasitic infection indications in animals (1981) , and subsequently for human use to treat onchocerciasis (river blindness) 6 years later. It has since been approved for treatment of a range of human nematode (roundworm) infestations that cause river blindness, filariasis, ascariasis and strongyloidiasis, as well as pediculosis and scabies, caused by ectoparasites, and also for rosacea [1, 3] . Because of its importance in treating all of these indications, ivermectin is firmly on the WHO (World Health Organisation)'s List of Essential Medicines [6] , with millions of doses prescribed worldwide every year.

Starting in 2012, ivermectin's antiviral properties have been progressively documented towards a number of RNA viruses, including human immunodeficiency virus (HIV)-1, influenza, flaviruses such as dengue and Zika, and most notably, SARS-CoV-2 (COVID-19) [4, 5, [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] , as well as DNA viruses such as pseudorabies, polyoma and adenoviruses [18] [19] [20] . Ivermectin's antiviral activity is based on its ability to bind to and inhibit the transport function of the host importin α (IMPα) protein [11, 18, 20] ; IMPα is known to mediate nuclear import of various viral proteins and key host factors, although other actions of ivermectin have been proposed which may also contribute to its activity [eg. 12, [21] [22] . In light of the possibility that ivermectin has potential to be a key weapon to help control Transport into and out of the nucleus is an integral part of normal eukaryotic cell function, as well as in the case of viral infection, since viruses commonly hijack the system in order to antagonize the cellular antiviral response that is driven to a large extent by nuclear factors such as transcription factors [14, 27] . The key signal-dependent mediators of this transport are the members of the IMP superfamily of proteins, of which there are multiple α and β forms [14, 27] . Nuclear transport mediated by the IMPα/β1 heterodimer is the best characterized pathway by which host proteins enter the nucleus through nuclear envelope-embedded nuclear pores; host proteins transported into the nucleus include members of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and signal transducers and activators of transcription (STATs) inducible transcription factor families that play key roles in the response to infection. Specific viral proteins [see 27,28] also use this pathway (see Fig. 1 ). Briefly, IMPα within the IMPα/β1 heterodimer performs the adaptor role of signal-specific recognition of the nuclear import cargo, while IMPβ1 performs the main transport roles of binding to/translocation through the nuclear pores, and subsequent release of the nuclear import cargo within the nucleus upon interaction with another transport factor, the monomeric guanine nucleotide binding protein Ran (not shown in Fig. 1 ) [see 27]. Specific high affinity recognition by IMPα is critical to nuclear localization of various viral proteins such as dengue nonstructural protein (NS) 5, as shown in mutagenic studies [32]; significantly, dengue virus with impaired NS5 interaction with IMPα is severely attenuated, underlining the critical importance of the NS5-IMPα interaction for dengue infection. The importance of this interaction to dengue infection is the basis for the fact that multiple distinct small molecules that disrupt IMPα recognition of dengue NS5 are able to limit dengue infection [eg. 7, 8, 11, 31, 33] . Significantly in the case of ivermectin, this activity extends to a large number of different viruses (see below) [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] , including SARS-CoV-2 [18] .

We identified ivermectin in 2011 in a high throughput screen using bacterially expressed proteins and a 1200-compound library of small molecules for inhibitors of HIV-1 Integrase (IN) recognition by IMPα/β1 [34]; specific inhibitors targeting IMPα/β1 directly (such as ivermectin) and not IN (such as budesonide) were identified using a nested counterscreening strategy [34, 35] . Of several compounds now confirmed to be active against IMPα/β1 and possessing antiviral activity as a consequence [7, 14, 31, 36] , ivermectin is the best characterized, shown to have broad-spectrum activities (summarized in Tables 1-3) . It was initially shown to inhibit nuclear import not only of IN, but also of simian virus SV40 large tumour antigen (T-ag) and other IMPα/β1-(but not IMPβ1-) dependent cargoes, consistent with the idea that IMPα (not IN) is the direct target of ivermectin [34, 35] . Subsequent work has confirmed this, with ivermectin's ability to inhibit the nuclear accumulation of various different host, including NF-kB p65 [37, 38] and viral proteins demonstrated in transfected and infected cell systems (see Table 1 ) [14, 34] . Ivermectin's ability to inhibit binding of IMPα to the viral proteins NS5 and T-ag has also been confirmed in a cellular context using the biomolecular fluorescence complementation technique [11] (Table 1) .

Although targeting of IMPα by ivermectin was implicated by many years of research (see also below), direct binding was only recently formally demonstrated using a set of biophysical techniques, including thermostability, analytical ultracentrifugation, and circular dichroism (CD) [11] . Importantly, the CD/thermostability studies indicate that binding of ivermectin to IMPα induces structural changes which are likely the basis of ivermectin inhibition of IMPα binding to viral nuclear import cargoes. Strikingly, the structural change also appears to impair heterodimerisation of IMPα with IMPβ1 [11 -see however 20]; thus, ivermectin inhibits nuclear import not only by preventing signal recognition by IMPα, but also by ensuring that the IMPα/β1 complex essential to mediate subsequent transport through the nuclear pore is prevented from forming (see Fig. 1 ).

Consistent with the fact that many viruses are known to rely on IMPα/β1-dependent nuclear import of specific viral proteins for robust infection, including many viruses for which the lifecycle is entirely carried out in the cytoplasm [14, 27, 28] , a body of in vitro studies have confirmed ivermectin to be active in limiting infection by a range of different RNA viruses [10, 14] , including HIV-1 [7] , DENV (all 4 serotypes) and related flaviviruses [8, 11, 12] , influenza, and alphaviruses such as Venezuelan equine encephalitis virus (VEEV) and Chikungunya [9, 15, 16] (see Table 2 ); it is also active against DNA viruses [18] [19] [20] . Exciting recent studies indicate it is a potent inhibitor of SARS-CoV-2 [17] .

A striking aspect of this antiviral activity is that, where determined, the EC50 for viral inhibition as assessed by a range of different techniques is in the low uM range (see right column, Table 2 ), interestingly aligning perfectly with its activity in inhibiting recognition of viral nuclear import cargoes by IMPα (see Table 1 ). The clear implication is that the mechanism of inhibition of infectious virus production in the case of all of the viruses listed in Table 2 is largely through targeting IMPα to prevent its role in nuclear import, and of viral proteins in particular (see Fig. 1 ). Significantly, two other small molecules (GW5074 and gossypol) that appear to target IMPα in a very similar way to prevent its nuclear import function [31 and unpublished] have comparable antiviral properties [13, 31, 36] , consistent with the idea that the host protein IMPα is a key contributor to infection by a number of medically important viruses.

As in many other disciplines, one of the biggest challenges in antiviral research is to transition from laboratory experiments to preclinical/clinical studies, with the question of dosing in the case of ivermectin for viral infectious indications contentious [see 6, 39, 40] . It is important, firstly, to stress the obvious in this context: that the antiviral activities of ivermectin documented in Table 2 have been derived from laboratory experiments that largely involve high, generally non-physiological, multiplicities of infection, and cell monolayer cultures, often of cell lines such as Vero cells (African green monkey kidney cells impaired in interferon α/β production) that are not clinically relevant. Clearly, the results in Table 2 for low uM EC50 values reveal robust, dose-dependent antiviral activity in the particular model system used, but it would be naïve to believe that it is necessary to achieve uM concentrations of ivermectin in a patient for maximum clinical benefit.

In fact, a key consideration in clinical intervention using ivermectin is its host-directed (IMPαdirected) mechanism of action. Host-directed agents that impact cellular activities that are essential to healthy function must be tested with caution; although ivermectin has an established safety profile in humans [24, 26] , and is FDA-approved for a number of parasitic infections [1, 3, 5] , it targets a host function that is unquestionably important in the antiviral response, and titration of a large proportion of the IMPα repertoire of a cell/tissue/organ is likely to lead to toxicity. With this in mind, where a host-directed agent can be a "game-changer" in treating viral infection may well be in the initial stages of infection or potentially even prophylactically by keeping the viral load low so that the body's immune system has an opportunity to mount a full antiviral response [11, 17] .

Ivermectin's potential as an antiviral to treat infection can of course only be demonstrated in clinical studies. Preclinical studies in a lethal Pseudorabies (PRV) mouse challenge model showed that dosing (0.2 mg/Kg) 12 hours post-infection protected 50% of mice, which could be increased to 60% through administration of ivermectin at the time of infection [18] . Clinical data for a phase III trial against DENV infection similarly indicate antiviral activity, with daily dosing (0.4 mg/Kg) found to be safe, and virological efficacy demonstrated, although it was concluded that dosing regimen modification was required to achieve clinical benefit [41] . These studies underline ivermectin's real potential as an antiviral able to reduce viral load in a clinical context.

The current SARS-CoV-2 pandemic has eclipsed the porcine flu epidemic in terms of numbers of infections (currently approaching 40 million) and deaths (> 1.1 million, with > 220,000 of these in the US alone) worldwide. The search for antivirals through repurposing existing drugs has proved challenging (eg. see [42, 43] ), one important aspect of repurposing being the perceived need to achieve therapeutic levels in the lung. Published pharmacokinetic modelling based on both the levels of ivermectin achievable in human serum from standard parasitic treatment dosing and robust large animal experiments where lung levels of ivermectin can be measured, indicates that concentrations of ivermectin 10 times higher than the c. 2.5 µM EC50 indicated by in vitro experiments (Table 2) are likely achievable in the lung in the case of SARS-CoV-2 [39] ; modelling based on different assumptions predicts lower values, but highlights the long-term stability of ivermectin in the lung (for over 30 days) based on data from animals [40] . It should also be noted that liquid formulations for intravenous administration of long-acting ivermectin have been described, with aerosol administration also in development, to enable ivermectin administration to achieve even higher concentrations to tackle SARS-CoV-2, whilst the use of ivermectin in combination with other agents may enhance efficacy at lower doses.

Ivermectin as a treatment for SARS-CoV-2 in humans has already been approved in a number of states and countries, including the Republic of Peru [44] and Northeastern Beni region of Bolivia [45] . Importantly, close to 70 trials worldwide are currently testing the clinical benefit of ivermectin to treat or prevent SARS-CoV-2 (see Table 3 ); these include variations on dosing regimens, combination therapies (preliminary results for NCT04523831 in Table 3 , #45) [46, 47] , and prophylactic protocols. With respect to the latter, preliminary results from recently completed study NCT04422561 (Table 3 , #22, and footnote) examining asymptomatic family close contacts of confirmed COVID patients, reveal that two doses of ivermectin 72 h apart result in only 7.4% of 203 subjects reporting symptoms of SARS-CoV-2 infection, in stark contrast to control untreated subjects, of whom 58.4% reported symptoms, underlining ivermectin's potential as a prophylactic. Results from retrospective/observational trials [49] [50] [51] [52] are also consistent with clinical benefit. Mymensingh Medical College Hospital (Bangladesh) reported that none of 115 subjects receiving a single 12 mg dose of ivermectin developed pneumonia/cardiovascular complications, compared to 9.8% (pneumonia) and 1.5 % (ischemic stroke) in 133 control subjects [49] . Further, significantly fewer ivermectin group patients developed respiratory distress (2.6 versus 15.8 %), or required oxygen (9.6 versus 45.9 %), antibiotics (15.7 versus 60.2 %) or intensive care management (0.09 versus 8.3 %). Ivermectin-treated patients became SARS-CoV-2 negative significantly faster (median 4 compared to 15 days), had significantly shorter hospital stays (median 9 versus 15 days), and significantly lower mortality (0.9 vs 6.8 %). Although combinations of therapies were used, the results for a 196 patient propensity-matched cohort study (ICON -Ivermectin in COvid Nineteen -study) at Broward Health Medical Centre (Florida, USA) [50] indicate significantly reduced mortality (13.3%) in subjects receiving 0.2 mg/Kg ivermectin (optional second dose 8 days later), compared to 24.5% mortality in those not receiving ivermectin, with more significant differences for patients with severe pulmonary involvement (mortality rates of 38.8 versus 80.7%). Although these early results are consistent with efficacy, it is clear that only the results from large rigorous randomised clinical trials (Table 3) will definitively establish ivermectin's utility to treat or prevent SARS-CoV-2 infection. It is to be hoped that the results from these trials will emerge in the next few months to document ivermectin's credentials or otherwise as a viable therapeutic for COVID-19 infection, and potentially infection by many other viruses. Subsequently, IMPβ β β β1 mediates transport of the trimeric complex into the nucleus through the nuclear envelope (NE)-embedded nuclear pore (NPC, nuclear pore complex). Release within the nucleus enables the transcription factor to carry out normal function in transcriptional regulation, including in the antiviral response. IMPα α α α cannot mediate nuclear import, unless heterodimerised with IMPβ β β β1. B.

In viral infection, specific viral proteins (eg. NS5 in the case of DENV, ZIKV) able to interact with IMPα α α α utilize the IMPα/β α/β α/β α/β1 heterodimer to access the nucleus and antagonize the antiviral response [14, 27, 28] . This is critical for optimal virus production as shown by mutagenic and inhibitor studies.

The SARS-CoV-2 proteins that may access the nucleus via IMPα/β α/β α/β α/β1 in infected cells has not been nuclear import [11] , contributing to reduced virus production.

J o u r n a l P r e -p r o o f EC50 = 1.9/0.6 uM (luciferase, 2 hosts)/ 3 uM > 5000-fold reduction (pfu) [15] 3 uM > 1000-fold (pfu) [15] 3 uM > 200-fold (pfu) [15] 1 uM c. 20-fold (pfu) [9] est. EC50 = 2 uM (TCID/luciferase) [13] EC50 = c. 2.5 uM; 10 uM 20-fold reduction (qPCR) [20] 10 uM c. 8-fold reduction (qPCR) [20] Est. EC50 1.5 uM (pfu/CPE/qPCR) [19] Est. EC50 c. 0.8 uM 1000-fold [18] a In vitro b Inhibits helicase activity (FRET based assay) of DENV2, YFV and WNV NS3 (IC50 0.2-0.5 uM) [12] Abbreviations: HIV-1, human immunodeficiency virus; VLP, virus like particle; PSV, Pseudorabies virus; YFV (yellow fever virus); DENV, dengue virus; ZIKV, zika virus; WNV, West Nile virus; TCID; pfu, plaque forming unit (infectious virus assay); CPE, cytopathic effects; qPCR, quantitative polymerase chain reaction; CFI, cell fluorescence-based immunofluorescence assay 

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