In vitro Anti-influenza Activity of in silico Repurposed Candidate Drug Cycrimine

Introduction

Despite the ample availability of antiviral drugs and vaccines, influenza remains a serious worldwide public health threat causing up to 5 million cases of severe illness and about 290,000–650,000 deaths during seasonal outbreaks worldwide [1]. Seasonal influenza vaccination remains the primary method for the prevention of influenza A and B virus infections but efficacy varies from year to year [2]. Due to low effectiveness of seasonal vaccines, every year a significant part of the population is prone to influenza A infection regardless of vaccination status. In addition to vaccination as a first-line of defence against influenza, antiviral drugs play an important role as major prophylactic and therapeutic agents during epidemics and pandemics. Today's options for treatment and prophylaxis against seasonal influenza are limited to the neuraminidase inhibitors (NAIs) and recently developed cap-dependent endonuclease (CEN) inhibitor of the viral polymerase, baloxavir (marboxil), which is approved in Japan and the United States for the treatment of influenza A and B infections [3]. Two main NAIs, oseltamivir and zanamivir, are licensed globally while peramivir and laninamivir are licensed only in some countries [4]. Currently circulating strains have a low frequency of NAI resistance (<1%) [5], however, the therapeutic window for NAIs is very short and only patients that begin treatment within 24–48 h after the onset of ‘flu’ symptoms benefit from it [4]. CEN is part of the polymerase acidic (PA) protein within the RdRpol complex of influenza A and B viruses and baloxavir marboxil (Xofluza) was developed to inhibit its function and so prevent the transcription of viral mRNA [3]. However, the potential of the influenza virus to develop treatment-emergent resistance due to direct pressure from baloxavir marboxil has been documented and connected with I38T, I38M or I38F mutations in the PA gene [6]. The first approved FDA-licensed anti-influenza drug class was adamantanes, IAV matrix protein 2 (M2) ion-channel inhibitors, successfully used against influenza A virus infection for more than 30 years [7]. Because of the lack of activity against influenza B [8], adverse effects, quick emergence of resistance in the course of the treatment or even without selective drug pressure, adamantanes are no longer recommended [9]. The resistance to adamantanes is associated with several amino acid substitutions where M2-S31N mutation is the most common and present in more than 95% of the currently circulating influenza A viruses [10]. Expansion of resistant viruses with S31N mutation in M2 simultaneously with the S31 and D31 mutations in the same gene in viruses in Australia [11] points out the need for new anti-influenza M2 inhibitors that will target both wild-type (WT) and S31N mutant viruses.

We have previously proposed a simple theoretical criterion for fast virtual screening of molecular libraries for candidate anti-influenza M2 ion channel inhibitors for both WT and adamantane-resistant influenza A viruses [12]. After in silico screening of drug space using the EIIP/AQVN filter [13], and further filtering of drugs by ligand-based virtual screening and molecular docking, we proposed cycrimine as a candidate inhibitor of M2 [12]. In this study, the potential antiviral activity of cycrimine was validated in vitro. The experimental results show significant anti-influenza activity of cycrimine in cell culture.

Methods

Results

We examined in vitro antiviral activity of cycrimine, the top candidate selected by screening of DrugBank for possible anti-influenza agents [12]. Addition of cycrimine to cells infected with H1N1 and H3N2 pandemic influenza viruses resulted in significantly lower production of infectious virus in a dose-dependent manner.

Both 10 and 30 μM cycrimine treatment resulted in significant reductions in H1N1 viral titres at 12, 16 and 24 h post-infection (Figure 1A). Both 10 and 30 μM cycrimine treatment resulted in significant reductions in H3N2 viral titres at day 1 post-infection. 30 μM cycrimine treatment also resulted in significant reductions in H3N2 viral titres at 8, 12 and 16 h post-infection (Figure 1B).

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Figure 1

Dose-dependent effect of cycrimine on influenza replication

By luminescent signal quantification, which corresponds to the amount of present ATP, cell viability was determined. ATP is directly proportional to the number of metabolically active cells. The graph (Additional file 1) shows that 100 μM cycrimine was not cytotoxic, therefore 30 μM and 10 μM doses were not evaluated.

Discussion

As a result of increased clinical use of antiviral drugs, which led to the emergence of resistant viral strains, the existing prevention and treatment options for influenza A and B infections are insufficient and there is an urgent need for new antiviral drugs [14]. The findings of current study are important in at least two major aspects: firstly, we demonstrated that licensed drug cycrimine has an in vitro anti-influenza activity, and secondly, we have confirmed in the previous in silico drug repurposing study that identified cycrimine is the best M2 candidate inhibitor out of 2,627 approved drugs from DrugBank [12]. Our previous study selected five best candidate inhibitors of both WT and S31N mutants using in silico screening of Drugbank database. In that screening we used the AQVN/EIIP filter that determines the long-range interaction between the drug and the therapeutic target [12], followed by the ligand-based virtual screening and molecular docking. Among five drugs selected, according to binding affinities values and their ratios towards WT M2 and S31N mutant, cycrimine was predicted to be the best candidate [12]. Accordingly, we were able to demonstrate significant antiviral activity of this FDA approved drug against influenza A/CA/07/2009 (H1N1) and influenza A/New York/55/04 (H3N2) in a dose-dependent manner.

Cycrimine (trade name Pagitane) is a central anticholinergic drug used to reduce the levels of acetylcholine in the treatment of Parkinson's disease [15,16]. The anti-influenza drug amantadine, previously repurposed for treatment of Parkinson's disease, also causes anticholinergic-like side effects [17]. Amantadine has been approved for the treatment of motor complications in Parkinson's disease after clinical trials but the initial step for the investigation of amantadine for Parkinson's disease was based on a single doctor–patient interaction [18]. More importantly, as amantadine and cycrimine are in the same EIIP/AQVN domain, it could be expected that they share the same therapeutic targets [12]. Interestingly, other drugs against Parkinson's disease with adamantine scaffold such as biperiden, triperiden and trihexyphenidyl (also identified in our previous in silico studies as a candidate influenza inhibitor) showed inhibitory activity against the influenza A virus [19,20].

Previously, the EIIP/AQVN in silico approach was established as an efficient filter for virtual screening of molecular libraries for candidate inhibitors of HIV and Ebola virus infection [21,22]. By means of this approach, ibuprofen was selected as an inhibitor of the Ebola virus infection and later this activity was con-firmed in vitro [23,24]. In a quest for new preventive and therapeutic options to minimize drug resistance and threats of outbreaks of epidemic and pandemic viruses, the main obstacle is the fact that drug development is a quite costly and time-consuming process. Therefore, drug repurposing represents a promising therapeutic strategy for many viral diseases, including anti-influenza A and B. Various predictive computational approaches have been developed to identify drug repositioning opportunities against influenza viruses [25]. Approved drugs are highly appreciated, initial point for drug discovery. Analysis of drugs entering the market reveals that most of their drug progenitors (or lead structures) were known drugs or clinical candidates [26]. Currently, two clinical trials with repositioned drugs targeting influenza viruses are ongoing: the first trial (Phase IIb/III clinical trial) combines clarithromycin and naproxen along with oseltamivir in a triple-drug combination. The second trial is testing efficacy of an antiparasitic drug, nitazoxanide, against influenza viruses (Phase III) [27].

In conclusion, here we presented results suggesting cycrimine as a promising new therapeutic agent against the influenza A viruses. Additional studies are needed to test activity against other influenza A subtypes and to elucidate cycrimine antiviral effects in vivo.