Original Article in Vitro Anti-Influenza Activity of in Silico Repurposed Candidate Drug Cycrimine

Antiviral Therapy 2019; 24:589–593 (doi: 10.3851/IMP3348)

Original article In vitro anti-influenza activity of in silico repurposed candidate drug cycrimine

Stanislava Matejin1†, Natalya Bukreyeva2†, Draginja Radosevic3, Milan Sencanski3, Emily Mantlo2, Veljko Veljkovic4, Sanja Glisic3, Slobodan Paessler2,5*

1Center for Advanced Heart Failure (ACTAT), Houston, TX, USA 2Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA 3Laboratory for Bioinformatics and Computational Chemistry, Institute of Nuclear Sciences VINCA, University of Belgrade, Belgrade, Serbia 4Biomed Protection, Galveston, TX, USA 5Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA

*Corresponding author e-mail: [email protected] †These authors contributed equally to this work

Background: Due to the limitations of current antiviral Methods: In this study we examined the potential antiviral therapies because of drug resistance and the emergence activity of cycrimine in vitro. of new circulating viral strains, novel effective antivirals Results: The experimental results showed the anti-influenza are urgently needed. Results of the previous drug repur- activity of cycrimine against two different influenza A sub- posing by virtual screening of DrugBank revealed the types in cell culture. anticholinergic drug cycrimine as a possible inhibitor of Conclusions: The findings of this study suggest cycrimine the influenza virus infection. as a potential therapeutic agent for influenza.

Introduction

Despite the ample availability of antiviral drugs and globally while peramivir and laninamivir are licensed vaccines, influenza remains a serious worldwide only in some countries [4]. Currently circulating public health threat causing up to 5 million cases strains have a low frequency of NAI resistance (<1%) of severe illness and about 290,000–650,000 deaths [5], however, the therapeutic window for NAIs is very during seasonal outbreaks worldwide [1]. Seasonal short and only patients that begin treatment within influenza vaccination remains the primary method 24–48 h after the onset of ‘flu’ symptoms benefit from for the prevention of influenza A and B virus infec- it [4]. CEN is part of the polymerase acidic (PA) pro- tions but efficacy varies from year to year [2]. Due to tein within the RdRpol complex of influenza A and B low effectiveness of seasonal vaccines, every year a viruses and baloxavir marboxil (Xofluza) was devel- significant part of the population is prone to influenza oped to inhibit its function and so prevent the tran- A infection regardless of vaccination status. In addi- scription of viral mRNA [3]. However, the potential tion to vaccination as a first-line of defence against of the influenza virus to develop treatment-emergent influenza, antiviral drugs play an important role as resistance due to direct pressure from baloxavir mar- major prophylactic and therapeutic agents during epi- boxil has been documented and connected with I38T, demics and pandemics. Today’s options for treatment I38M or I38F mutations in the PA gene [6]. The first and prophylaxis against seasonal influenza are limited approved FDA-licensed anti-influenza drug class was to the neuraminidase inhibitors (NAIs) and recently adamantanes, IAV matrix protein 2 (M2) ion-chan- developed cap-dependent endonuclease (CEN) inhibi- nel inhibitors, successfully used against influenza A tor of the viral polymerase, baloxavir (marboxil), virus infection for more than 30 years [7]. Because which is approved in Japan and the United States for of the lack of activity against influenza B [8], adverse the treatment of influenza A and B infections [3]. Two effects, quick emergence of resistance in the course of main NAIs, oseltamivir and zanamivir, are licensed the treatment or even without selective drug pressure,

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adamantanes are no longer recommended [9]. The cells at approximately 85–95% confluency in 96-well resistance to adamantanes is associated with several plates in order to determine viral titres using a 50% tis-

amino acid substitutions where M2-S31N mutation sue culture infective dose (TCID50) assay. The growth is the most common and present in more than 95% curve for each virus was plotted based on individual of the currently circulating influenza A viruses [10]. titres for each sample collected at 8, 12, 16 and 24 h Expansion of resistant viruses with S31N mutation in post-infection. M2 simultaneously with the S31 and D31 mutations in the same gene in viruses in Australia [11] points Cytotoxicity determination out the need for new anti-influenza M2 inhibitors that The Luminescent Cell Viability Assay (Promega, will target both wild-type (WT) and S31N mutant Madison, WI, USA) was used to determine the cyto- viruses. toxicity of cycrimine. The number of viable cells in We have previously proposed a simple theoretical culture was based on quantification of ATP levels. criterion for fast virtual screening of molecular librar- In brief, MDCK cells were seeded in 96-well plates, ies for candidate anti-influenza M2 ion channel inhibi- grown for 24 h and then incubated with serially- tors for both WT and adamantane-resistant influenza diluted compound. Plates were harvested at the times A viruses [12]. After in silico screening of drug space indicated and treated according to manufacturer’s using the EIIP/AQVN filter [13], and further filtering of instructions. drugs by ligand-based virtual screening and molecular docking, we proposed cycrimine as a candidate inhib- Results itor of M2 [12]. In this study, the potential antiviral activity of cycrimine was validated in vitro. The experi- We examined in vitro antiviral activity of cycrimine, mental results show significant anti-influenza activity of the top candidate selected by screening of DrugBank cycrimine in cell culture. for possible anti-influenza agents [12]. Addition of cycrimine to cells infected with H1N1 and H3N2 pan- Methods demic influenza viruses resulted in significantly lower production of infectious virus in a dose-dependent In vitro efficacy testing of cycrimine against manner. influenza A H1N1 and H3N2 viruses Both 10 and 30 mM cycrimine treatment resulted in Cycrimine, which is supplied as a white solid, was significant reductions in H1N1 viral titres at 12, 16 and obtained from United States Biological (Salem, MA, 24 h post-infection (Figure 1A). Both 10 and 30 mM USA). 20 mM stock of cycrimine was prepared in cycrimine treatment resulted in significant reductions DMSO and stored at -20°C. Cycrimine then was in H3N2 viral titres at day 1 post-infection. 30 mM diluted in serum free media to reach the assay target cycrimine treatment also resulted in significant reduc- concentrations and the final DMSO concentration was tions in H3N2 viral titres at 8, 12 and 16 h post-infec- equal or below 0.5%. tion (Figure 1B). Influenza A/CA/07/2009 (H1N1) virus was premixed By luminescent signal quantification, which cor- with 10 or 30 µM of cycrimine and incubated at 37°C responds to the amount of present ATP, cell viability for 1 h. The same procedure was applied for influ- was determined. ATP is directly proportional to the enza A/New York/55/04 (H3N2) virus. The 12-well number of metabolically active cells. The graph (Addi- plates of Madin-Darby canine kidney (MDCK) cells tional file 1) shows that 100 μM cycrimine was not at approximately 85–95% confluency were washed cytotoxic, therefore 30 μM and 10 μM doses were not with serum free media twice and then infected with evaluated. influenza A (H1N1) or influenza A (H3N2) viruses/ cycrimine mixtures. 9 repeats of each treatment group Discussion were tested. After approximately 1 h of incubation at

37°C and 5% CO2, cells were washed with serum free As a result of increased clinical use of antiviral drugs, media once and 1x of each compound dose was added which led to the emergence of resistant viral strains, to the cells. Three wells were used as a negative con- the existing prevention and treatment options for trol and were mock-infected and nine wells served as a influenza A and B infections are insufficient and there virus control and were infected. All control wells were is an urgent need for new antiviral drugs [14]. The

untreated. Cells were incubated at 37°C and 5% CO2 findings of current study are important in at least two and samples were collected at 8, 12, 16 and 24 h post- major aspects: firstly, we demonstrated that licensed infection. Samples were stored at -80°C until the day of drug cycrimine has an in vitro anti-influenza activ- analysis. Each sample collected at various time points ity, and secondly, we have confirmed in the previ- was diluted at a 1:10 dilution and used to inoculate ous in silico drug repurposing study that identified

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Figure 1. Dose-dependent effect of cycrimine on influenza replication

A H1N1 B H3N2 108 108

107 107 d c

6 6 10 c 10 per ml per ml 105 105 50 50 c c 104 a 104 103 103 a c Titre, TCID Titre, Titre, TCID Titre, 102 b 102 d 101 101 8 12 16 20 24 8 12 16 20 24 Hours post-infection Hours post-infection

Untreated Cycrimine 10 µM Cycrimine 30 µM

(A) Influenza A/CA/07/2009 (H1N1) or(B) influenza A/New York/55/04 (H3N2) replication inMadin-Darby canine kidney (MDCK) cells after treatment with the indicated concentrations of cycrimine. Results are plotted as the means of nine observations, with error bars reflecting the standard deviations. Statistical significance a b c d is determined by a P-value of less than 0.05 on a one-tailed student’s t-test. P≤0.001. P≤0.0001. P≤0.01. P≤0.05. TCID50, 50% tissue culture infective dose.

cycrimine is the best M2 candidate inhibitor out of of motor complications in Parkinson’s disease after 2,627 approved drugs from DrugBank [12]. Our clinical trials but the initial step for the investigation previous study selected five best candidate inhibi- of amantadine for Parkinson’s disease was based on tors of both WT and S31N mutants using in silico a single doctor–patient interaction [18]. More impor- screening of Drugbank database. In that screening tantly, as amantadine and cycrimine are in the same we used the AQVN/EIIP filter that determines the EIIP/AQVN domain, it could be expected that they long-range interaction between the drug and the share the same therapeutic targets [12]. Interest- therapeutic target [12], followed by the ligand-based ingly, other drugs against Parkinson’s disease with virtual screening and molecular docking. Among five adamantine scaffold such as biperiden, triperiden drugs selected, according to binding affinities values and trihexyphenidyl (also identified in our previous and their ratios towards WT M2 and S31N mutant, in silico studies as a candidate influenza inhibitor) cycrimine was predicted to be the best candidate showed inhibitory activity against the influenza A [12]. Accordingly, we were able to demonstrate sig- virus [19,20]. nificant antiviral activity of this FDA approved drug Previously, the EIIP/AQVN in silico approach was against influenza A/CA/07/2009 (H1N1) and influ- established as an efficient filter for virtual screening enza A/New York/55/04 (H3N2) in a dose-dependent of molecular libraries for candidate inhibitors of HIV manner. and Ebola virus infection [21,22]. By means of this Cycrimine (trade name Pagitane) is a central approach, ibuprofen was selected as an inhibitor of the anticholinergic drug used to reduce the levels of ace- Ebola virus infection and later this activity was con- tylcholine in the treatment of Parkinson’s disease firmed in vitro [23,24]. In a quest for new preventive [15,16]. The anti-influenza drug amantadine, previ- and therapeutic options to minimize drug resistance ously repurposed for treatment of Parkinson’s dis- and threats of outbreaks of epidemic and pandemic ease, also causes anticholinergic-like side effects [17]. viruses, the main obstacle is the fact that drug devel- Amantadine has been approved for the treatment opment is a quite costly and time-consuming process.

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Accepted 14 February 2020; published online 28 February 2020

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