1521-0111/90/3/188–198$25.00 http://dx.doi.org/10.1124/mol.116.105346 MOLECULAR Mol Pharmacol 90:188–198, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Pharmacological Characterization of the Spectrum of Antiviral Activity and Genetic Barrier to Resistance of M2-S31N Channel Blockers

Chunlong Ma, Jiantao Zhang, and Jun Wang Department of Pharmacology and , College of , and BIO5 Institute, University of Arizona, Tucson, Arizona Received May 26, 2016; accepted June 30, 2016 Downloaded from

ABSTRACT Adamantanes (amantadine and rimantadine) are one of the two currently circulating influenza A viruses and their genetic barrier to classes of Food and Drug Administration–approved antiviral are unknown. In this report, we evaluated the used for the prevention and treatment of influenza A virus therapeutic potential of these two classes of M2-S31N inhibitors infections. They inhibit viral replication by blocking the wild-type (WJ379 and BC035) by profiling their antiviral against

(WT) M2 proton channel, thus preventing viral uncoating. How- multidrug-resistant influenza A viruses, in vitro drug resistance molpharm.aspetjournals.org ever, their use was discontinued due to widespread drug re- barrier, and synergistic effect with oseltamivir. We found that M2- sistance. Among a handful of drug-resistant mutants, M2-S31N is S31N inhibitors were active against several influenza A viruses the predominant mutation and persists in more than 95% of that are resistant to one or both classes of Food and Drug currently circulating influenza A strains. We recently designed two Administration–approved anti-influenza drugs. In addition, M2- classes of M2-S31N inhibitors, S31N-specific inhibitors and S31N inhibitors display a higher in vitro genetic barrier to drug S31N/WT dual inhibitors, which are represented by N-[(5- resistance than amantadine. The antiviral effect of WJ379 was cyclopropyl-1,2-oxazol-3-yl)methyl]adamantan-1-amine (WJ379) also synergistic with oseltamivir carboxylate. Overall, these results and N-[(5-bromothiophen-2-yl)methyl]adamantan-1-amine reaffirm that M2-S31N inhibitors are promising antiviral drug (BC035), respectively. However, their antiviral activities against candidates that warrant further development. at ASPET Journals on September 26, 2021

Introduction target to develop the next generation of antiviral drugs. The M2-S31N mutant is an ideal antiviral drug target because: 1) Influenza viruses are the causative agents that lead to M2-S31N persists in more than 95% of currently circulating annual seasonal influenza epidemics as well as sporadic, more influenza A viruses among humans, including oseltamivir- devastating influenza pandemics (Rappuoli and Dormitzer, sensitive and oseltamivir-resistant strains (Dong et al., 2015), 2012; Monto and Webster, 2013). On average, influenza virus thus targeting M2-S31N is likely to yield broad-spectrum infection leads to 36,000 deaths in the United States and antiviral drugs; and 2) M2 is a validated antiviral drug target, approximately 250,000–500,000 deaths globally each year and there is no such homolog proton channel encoded by the (Thompson et al., 2003, 2004). However, current countermea- human genome, thus M2 inhibitors are expected to have high sures against influenza infection are curtailed by the emer- selectivity. As a homotetrameric proton-selective channel, one gence of drug-resistant strains. Resistance to the only orally mutation in M2 actually results in four changes at the same bioavailable neuraminidase inhibitor, oseltamivir, has been time, which leads to a profound impact on the structure and continuously reported and was prevalent in the 2008–2009 function of this very constricted channel. As a result, only a influenza season (Cheng et al., 2009; Samson et al., 2013). The very limited number of M2 mutants confer both drug use of M2 channel blockers (amantadine and rimantadine) is resistance and transmissibility among humans, of which no longer recommended by the Centers for Disease Control M2-S31N is the predominant mutant. This small set of and Prevention due to prevalent drug resistance (Bright et al., transmissible mutants suggests that M2 is a highly conserved 2006; Fiore et al., 2008). Thus, novel antivirals are clearly drug target compared with other viral proteins, rendering it needed. Toward this goal, we chose M2-S31N as the drug an ideal drug target for the development of anti-influenza drugs (Wang et al., 2015). This work was supported by the National Institutes of Health National M2-S31N mutant was traditionally tagged as an “undrug- Institute of Allergy and Infectious Diseases [Grant AI119187], the PhRMA gable” target, and decades of traditional Foundation 2015 Research Starter Grant in Pharmacology and Toxicology, and the start-up funds from the University of Arizona and the BIO5 Institute. campaign failed to yield a single hit compound (Wanka et al., dx.doi.org/10.1124/mol.116.105346. 2013; Wang et al., 2015). Nevertheless, guided by information

ABBREVIATIONS: BC035, N-[(5-bromothiophen-2-yl)methyl]adamantan-1-amine; CPE, cytopathic effect; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MDCK, Madin-Darby canine kidney; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ST6Gal I, b-galactoside a-2,6-sialytransferase I; TEVC, two-electrode voltage clamp; WJ379, N-[(5-cyclopropyl-1,2-oxazol-3-yl)methyl]adamantan-1-amine; WT, wild-type.

188 Pharmacology of M2-S31N Inhibitors 189 gathered from previous structure-activity relationship studies Compounds. WJ379 and BC035 were synthesized in house as of amantadine (Wang et al., 2015) and breakthroughs in M2 previously described (Wang et al., 2013b; Wu et al., 2014). Amantadine structural biology and mechanistic studies (Hong and hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). DeGrado, 2012), steady progress has been made toward the Plaque Assay. Plaque assays were carried out as previously design of M2-S31N inhibitors (Wang et al., 2013a,b; Wu et al., described (Jing et al., 2008; Balannik et al., 2009), except MDCK cells expressing ST6Gal I were used instead of regular MDCK cells. In brief, 2014; Li et al., 2016). They are broadly classified as S31N- a confluent monolayer of ST6Gal I MDCK cells was incubated with specific inhibitors and S31N/wild-type (WT) dual inhibitors. ∼100-pfu virus samples in DMEM with 0.5% bovine serum albumin for S31N-specific inhibitors target the S31N channel only, not the 1 hour at 4°C, then 37°C for 1 hour. The inoculums were removed, and WT, and one representative example is N-[(5-cyclopropyl-1,2- the cells were washed with phosphate-buffered saline (PBS). The cells oxazol-3-yl)methyl]adamantan-1-amine (WJ379) (Wang et al., were then overlaid with DMEM containing 1.2% Avicel microcrystalline 2013b). S31N/WT dual inhibitors target both S31N and cellulose (FMC BioPolymer, Philadelphia, PA) and N-acetyl trypsin WT M2 channels, and one representative example is N-[(5- (2.0 mg/ml). To examine the effect of the compounds on plaque formation, bromothiophen-2-yl)methyl]adamantan-1-amine (BC035) (Wu the overlay media were supplemented with compounds at testing concen- et al., 2014). Their channel blockage and antiviral effects have trations. At day 2 after infection, the monolayers were fixed and stained been confirmed in electrophysiological two-electrode voltage with crystal violet dye solution (0.2% crystal violet, 20% methanol). Cytotoxicity Assay and Cytopathic Effect Assay. The cyto- clamp (TEVC) assays and antiviral plaque assays, respec-

of compounds was determined in MDCK cells. Cells were Downloaded from tively. Their modes of binding were revealed by both solution treated with different concentrations of WJ379 or BC035 and stained and solid-state NMR studies (Cady et al., 2010, 2011; Wang with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide et al., 2013b; Williams et al., 2013; Wu et al., 2014). (MTT) for 4 hours. A virus-induced cytopathic effect (CPE) assay In this study, we further evaluated the therapeutic potential was performed as previously described (Atkins et al., 2012; Beyleveld of both classes of M2 inhibitors, S31N-specific inhibitors and et al., 2013). In brief, MDCK cells were grown in DMEM supplemented S31N/WT dual inhibitors. Specifically, from the standpoint of with 10% FBS in 96-well plates. When 90% confluence was reached, developing therapeutics, we are interested in testing whether the cells were washed once with 100 ml of PBS buffer and infected with molpharm.aspetjournals.org potent antiviral effect of these compounds on the model virus, virus at multiplicity of infection (MOI) of 0.002. Then DMEM (without A/WSN/33 (H1N1), can be extended to the currently circulating FBS) with indicated concentrations of compounds was added. After incubating at 37°C for 72 hours, 20 mlof5mg/ml MTT reagent was influenza A strains among humans. Furthermore, with these tool added to each well, and the plates were incubated at 37°C for 4 hours. compoundsinhand,wewouldliketoaddresstheissueofdrug Then the cells were washed once with PBS, and 100 ml of 0.1 N HCl in resistance. For example, will the S31N mutant virus become isopropanol was added. The absorbance was read at 560 nm with a resistant to the newly developed S31N inhibitors under drug plate reader. The assays were repeated in quadruplicate. selection pressure? If yes, which mutations will be selected? How Serial Passage Experiments and Resistance Mutation many passages does it take for the S31N mutant virus to evolve Identification. MDCK cells were infected with A/WSN/33 (H1N1) drug resistance? In addition, as we already learned the lessons of virus or A/WSN/33 N31S (H1N1) at an MOI of 0.001 for 1 hour. Then at ASPET Journals on September 26, 2021 antibiotics and antivirals, resistance is unfortunately inevitable. It the inoculum was removed, and MDCK cells were incubated with is not a question of yes or no but rather a question of when (Clavel compounds at concentrations shown in Table 2 or in the absence of – and Hance, 2004; Hayden and de Jong, 2011; McKimm-Breschkin, compounds to evaluate cell culture adapted mutations. In the first 2013; Walsh and Wencewicz, 2014; Blair et al., 2015). With this passage, the applied compound concentrations were slightly below their corresponding EC values. In the following passages, the in mind, what one can do is to slow down the resistance evolution 50 compound concentrations were gradually increased, as shown in rather than eradicate it. Toward this goal, we would like to test Table 2. In each passage, the viruses were harvested when significant the combination therapy potential of M2-S31N inhibitors with cytopathic effect was observed, which usually takes 2–3 days after oseltamivir, as this offers a means to delay resistance evolution virus infection. The titers of harvested viruses were determined by against both oseltamivir and M2-S31N inhibitors. plaque assay. At selected passages, as shown in Table 2, influenza M segments were subjected to sequencing. Influenza viral RNA was isolated using QIAamp viral RNA Mini Kit (Qiagen, Hilden, Ger- Materials and Methods many). The M segment cDNA was generated with reverse-transcription polymerase chain reaction (PCR) using M-specific primers Cell Lines, Viruses, and Viral Infection. Madin-Darby canine (59-AGCAAAAGCAGGTAGATATTGAAAG-39 and 59-TAGTTTTTTACTC- 9 kidney (MDCK) cells were grown at 37°C in 5% CO2 atmosphere in CAGCTCTATGTTG-3 ). Reverse-transcription PCR products were Dulbecco’s modified Eagle’smedium(DMEM;highglucose,withL-glu- analyzed with agarose gel electrophoresis and purified with Wizard tamine) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml SV Gel and PCR Clean-up System (Promega, Madison, WI). Purified penicillin, and 100 mg/ml streptomycin. MDCK cells overexpressing PCR products were sequenced by Genewiz, Inc. (South Plainfield, NJ). b-galactoside a-2,6-sialytransferase I (ST6Gal I) were obtained from Dr. Electrophysiological TEVC Assay. The corresponding drug- Yoshihiro Kawaoka at the University of Wisconsin (Madison, WI) resistant mutations identified from serial drug-passage experiments were through material transfer agreement and were maintained in the introduced to M2 sequences in pGEM3 vector with the QuikChange Site- presence of 7.5 mg/ml puromycin, except when they were used for viral Directed Mutagenesis Kit (Agilent, Santa Clara, CA) as described infection. Influenza A virus strains A/California/07/2009 (H1N1) and previously (Khurana et al., 2009). mRNA preparation and microinjection A/Texas/04/2009 (H1N1) were obtained from Dr. James Noah at the and electrophysiological TEVC recordings were carried out as previously Southern Research Institute (Birmingham, AL), and influenza A virus described (Jing et al., 2008). The inhibition kinetics time constant was strains A/Denmark/524/2009 (H1N1) and A/Denmark/528/2009 (H1N1) determined by fitting the period of recording trace when the compounds were obtained from Dr. Elena Govorkova at St. Jude Children’sResearch are present to a one-phase exponential decay equation [f(t) 5 A*exp(2t/G) 1 Hospital (Memphis, TN). Virus stocks were amplified in MDCK cells in C] in Clampfit 10.3 (Molecular Devices, Sunnyvale, CA). the presence of 2 mg/ml N-acetyl trypsin. Two days post infection, the Combinational Therapy. Combination evaluations were per- culture media were harvested, and cell debris was removed by centrifu- formed in quintuplicate using the CPE assay described earlier except gation at 3000 rpm for 30 minutes. Virus titers were determined by that neutral red was used for staining instead of MTT. In brief, MDCK plaque assay using MDCK cells expressing ST6Gal I. cells were seeded in 96-well plates for 24 hours. Seven serial 190 Ma et al. half-logarithmic dilutions of WJ379 and oseltamivir carboxylate were TABLE 1 prepared and added to MDCK cells infected with influenza A virus Channel blockage and antiviral activity of M2-S31N inhibitors A/WSN/33 (H1N1) (MOI 0.001) in the 96-well plate. After incubation Structure at 37°C in a humidified incubator with 5% CO2 for 72 hours, the medium was removed, and cells were stained with 100 ml of neutral red in DMEM (40 mg/l) for 4 hours. After washing once with 150 mlof PBS, the neutral red was extracted from cells with 150 ml of neutral red destain solution [ethanol/water/glacial acetic acid: 50/49/1 (v/v/v)] to form a homogenous solution (Repetto et al., 2008). The absorbance at 540 nm was measured using a Multiskan FC Microplate Photom- eter (Fisher Scientific, Houston, TX). Drug-drug interaction was M2-S31N channel 85%/65%b 76%/N.T.c evaluated using the MacSynergy II software program, which auto- inhibitiona matically calculates volumes of synergy or antagonism for each three- Plaque assay 0.49 2.2 d EC50 (mM) dimensional plot of data (Prichard and Shipman, 1990; Tarbet et al., m e – – – m 2% m  CPE EC50 ( M) 0.36 6.20 2012). The value ranges 0 25, 25 50, 50 100, or over 100 m ( m Cellular toxicity 125 123c mm  %) are considered as insignificant, minor but significant, f CC50 (mM) moderate, or strong synergy or antagonism, respectively. Synergy SIg 255 56c

plots were made at the 95% confidence limit. Downloaded from N.T., not tested; SI, selectivity index. M2 Sequence Analysis. All M2 protein full-length sequences of aM2-S31N channel inhibition was measured using two-electrode voltage clamp influenza virus A isolated from humans were obtained from the assay as described (Wang et al., 2013b; Wu et al., 2014). The data are expressed as Influenza Research Database (http://www.fludb.org, accessed on January percentage inhibition at 100/30 mM compound concentration. bReported in Wang et al. (2013b). 18, 2016). A total of 18,393 M2 sequences were aligned by MEGA6 (www. cReported in Wu et al. (2014). d megasoftware.net) using the ClustalW method. The frequencies of the EC50 in plaque assays was determined with the A/WSN/33 (H1N1) virus. e EC50 in CPE assay was determined with the A/WSN/33 (H1N1) virus using MTT. appearance of substitutions of L26I and I32T were identified. f CC50 was assayed using MTT after incubating MDCK cells with increasing concentrations of compounds for 72 hours. molpharm.aspetjournals.org g SI was calculated by dividing CC50 values over plaque assay EC50 values. Results WJ379 and BC035 Are Potent M2-S31N Channel oseltamivir-sensitive), A/Texas/04/2009 (H1N1) (amantadine- Blockers and Antivirals against S31N Containing the resistant, oseltamivir-resistant), A/Denmark/524/2009 (H1N1) A/WSN/33 (H1N1) Virus. We previously reported on two (amantadine-resistant, oseltamivir-sensitive), and A/Denmark/ classes of S31N inhibitors, S31N-specific inhibitors (Wang 528/2009 (H1N1) (amantadine-resistant, oseltamivir-resistant). et al., 2013b; Li et al., 2016) and S31N/WT dual inhibitors As these human clinical isolates did not produce countable (Wang et al., 2013a; Wu et al., 2014), which are represented by plaques using regular MDCK cells (data not shown), an

WJ379 and BC035, respectively. To select noncytotoxic drug engineered MDCK cell line overexpressing the human ST6Gal at ASPET Journals on September 26, 2021 concentrations for the following serial viral passage experi- I was used for this purpose as it expresses N-acetyl sialic acid ments, the cytotoxicity and selectivity indexes of these two linked to galactose by an a-2,6-linkage on its cell surface compounds were determined. The CC50 values of WJ379 and (Hatakeyama et al., 2005). This cell line mimics human BC035 were 125 and 123 mM, respectively (Table 1). Their epithelial cells and is the only cell line that is suitable to antiviral activities were further quantified in both plaque assays perform plaque assays for human influenza viruses and cytopathic effect assays using the influenza A virus stain (Matrosovich et al., 2003; Hatakeyama et al., 2005). With this A/WSN/33 (H1N1) (Table 1). Both the plaque assay and CPE cell line and optimized conditions, we were able to get uniform assay were standard antiviral assays used to determine the and reproducible plaques with all four strains. Representa- efficacy of antiviral drugs. The difference is that the plaque assay tive images of plaque assays with A/California/07/2009 quantifies the change of viral titer upon drug treatment, whereas (H1N1) and A/Texas/04/2009 (H1N1) are shown in Fig. 1. the CPE assay quantifies the change of cell survival upon viral The antiviral EC50 values of compound WJ379 were in the infection and drug treatment (Atkins et al., 2012; Beyleveld submicromolar range, whereas for compound BC035, the EC50 et al., 2013). In general, there is a positive correlation between values were in the low micromolar range (Fig. 1, D and E). Of these two assays. In the plaque assays, the EC50 values of WJ379 note, oseltamivir carboxylate only reduced plaque size, but not and BC035 against the A/WSN/33 (H1N1) virus were 0.49 and plaque number, whereas M2-S31N inhibitors completely 2.2 mM, respectively, which were consistent with those of WJ379 inhibited plaque formation. and BC035 in the CPE assays (0.36 and 6.20 mM, respectively) M2-S31N Inhibitors Have a Higher In Vitro Genetic (Table 1). The selective indexes of WJ379 and BC035 were Barrier to Drug Resistance than Amantadine. To eval- 255 and 56, respectively. On the basis of these results, the uate the in vitro genetic barrier to drug resistance of these two highest drug concentrations used for the following serial viral S31N inhibitors, serial viral passage experiments in MDCK passage experiments were set to 5 and 16 mMforWJ379and cells were performed with the influenza A/WSN/33 (H1N1) BC035, respectively, which conferred minimal cytotoxicity. virus. The drug concentrations of BC035 and WJ379 applied M2-S31N Inhibitors Are Potent Antivirals against at passage 1 were slightly below their EC50 values and were Human Clinical Isolates of Multidrug-Resistant In- gradually increased in subsequent passages (Table 2). In the fluenza A Viruses. To determine whether the potent antivi- first two passages, the progeny viruses maintained full ral activity of WJ379 and BC035 on A/WSN/33 (H1N1) can be sensitivity to their respective compounds, as indicated by extended to other S31N-containing influenza A viruses, WJ379 the EC50 values (Table 2). At passage 3, the EC50 values of and BC035 were tested in plaque assays against human WJ379 and BC035 were moderately increased by 4.2- and 2.8- clinical isolates of multidrug-resistant influenza A viruses, fold, respectively. Significant resistance was observed at including A/California/07/2009 (H1N1) (amantadine-resistant, passage 4 in which the EC50 values increased more than Pharmacology of M2-S31N Inhibitors 191 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Fig. 1. Antiviral efficacy of compounds WJ379 and BC035 on clinically isolated influenza A viruses in plaque assay. The assay was carried out with MDCK cells expressing the ST6Gal I gene. (A) Plaque assays of WJ379 on A/California/07/2009 (H1N1). (B) Plaque assays of WJ379 on A/Texas/04/2009 (H1N1). (C) Plaque assays of BC035 on A/California/07/2009 (H1N1). (D) Dose-response curves of WJ379 on various influenza A strains. (E) Dose- response curves of BC035 on various influenza A strains.

10-fold in both cases. To test the persistence of the resulting was applied at 0.2 mM in passage 1 (Table 2), which was close drug-resistant mutants, the viruses were continuously pas- to its EC50 value. This condition was chosen to make a side-by- saged for two more rounds in the presence of compounds, and side comparison with the passage experiments of WJ379 and then five rounds in the absence of compounds. We found that BC035. In contrast to the emergence of complete resistance to the viruses at passage 11 were resistant to both WJ379 and WJ379 and BC035 by the A/WSN/33 (H1N1) virus at passage BC035. 4, complete amantadine resistance against the WSN N31S To rule out the possibility that the observed resistance was (H1N1) virus was observed as early as passage 1 (30% plaque due to cell culture–adapted mutations, the A/WSN/33 (H1N1) remaining even when treated with 30 mM amantadine). These virus was also passed in the absence of compound, and the results indicate that M2-S31N inhibitors, such as WJ379 and drug sensitivities of the resulting viruses against WJ379 and BC035, have a higher genetic barrier to resistance than BC035 were tested in plaque assays. At passage 6, the EC50 amantadine. values of WJ379 and BC035 were 0.65 and 2.9 mM, respec- Sequencing M2 Genes Reveals Mutations that Confer tively, which were similar to their corresponding EC50 values Resistance to M2-S31N Inhibitors. To identify how M2- at passage 0, indicating that the elevated EC50 values in serial S31N evolved to become resistant to WJ379 and BC035, the passage experiments in the presence of WJ379 and BC035 M2 genes from viruses at passages 0, 3, 4, 5, 6, and 9 were were not due to the virus adaption in cell culture. reverse-transcribed and sequenced. The sequencing traces are As a comparison, we carried out a serial viral pas- shown in Fig. 2, and the resulting M2 mutations at each sage experiment with the A/WSN/33 N31S (H1N1) virus passage are shown in Table 2. For WJ379, no M2 mutation against amantadine. The A/WSN/33 N31S (H1N1) virus is was identified up to passage 3. At passage 4, a substantial amantadine-sensitive. The EC50 value of amantadine against population of L26I mutation emerged, and its percentage the A/WSN/33 N31S virus (H1N1) was 0.26 mM. Amantadine continued to rise throughout passages 5 and 6 and completely 192 Ma et al.

TABLE 2 Resistance development under selection pressure

EC50 values were measured after each passage.

Passage No. WJ379 EC50 Mutation BC035 EC50 Mutation Ama EC50a

mM mM mM mM mM mM 0 0.49 N31 2.2 N31 0.26 1 0.30 0.33 N.T. 2.0 1.2 N.T. 0.20 Resb 2 0.60 0.38 N.T. 4.0 2.6 N.T. Not continued after passage 1 due to significant resistance 3 1.20 1.6 No mutation 8.0 7.2 No mutation 4 2.50 35.9 L26I 16.0 25.3 N31D/I32T 5 5.0 28.0 L26I 16.0 27.3 N31D/I32T 6 5.0 15.5 N31S/L26I 16.0 N.T. N31D/I32T 7 0 N.T. N.T. 0 N.T. N.T. 80.30 N.T. 0 20.0 N.T. 90.30 L26I (100%) 0 22.7 I32T (100%) 10 0 N.T. N.T. 0 N.T. N.T. 11 0 .30 N.T. 0 .20 N.T.

Ama, amantadine; Res, rimantadine; N.T., not tested. Downloaded from aA/WSN/33 M2 N31S virus was used. bComplete resistant viruses were selected as shown by the plaque-reduction assays. No complete plaque inhibition was observed even when amantadine was added at 30 mM (30% of plaque remained compared with no drug control). overtook L26 at passage 9. In addition, another mutation, inhibition at a 2-minute time point after compound treat- N31S, was also identified at passage 6. However, this mutant ment. Figure 3 shows the percentage of current inhibition reverted back to N31 (WT) at passage 9 when the drug of the M2 mutants by 100 mM WJ379 or 100 mM BC035. molpharm.aspetjournals.org selection pressure was removed. Nevertheless, the virus For the M2 mutants selected by WJ379, M2-N31S was nearly containing the single mutant L26I at passage 9 was still completely resistant to WJ379, as its conductance was resistant to WJ379 (Table 2). As M2-S31N could revert back to inhibited less than 10% by 100 mM WJ379 (Dong et al., M2-N31S (WT) to become resistant to S31N-specific inhibi- 2015). Another single mutant, M2-S31N/L26I, remained tors, such as WJ379, it is expected that S31N/WT dual sensitive to WJ379, although it was less sensitive than the inhibitors might have a higher genetic barrier to drug re- original M2-S31N. The percentage of current inhibitions for sistance, as they inhibit both the S31N and the N31S (WT) M2 M2-S31N/L26I and M2-S31N were 64% and 82%, respec- channels. To test this hypothesis, we repeated the serial tively, in the presence of 100 mM WJ379. Dose-response passage experiments with BC035, one of the S31N/WT dual experiments revealed that the IC50 of WJ379 against the M2- at ASPET Journals on September 26, 2021 inhibitors. Similar to the results of WJ379, no mutation was S31N/L26I mutant was 72.9 mM, in contrast to 27.9 mM identified up to passage 3. At passage 4, a small but obvious against M2-S31N (Fig. 4A). Similar to the results of the population of N31D mutants emerged, and its percentage single mutant M2-N31S, the double mutant M2-N31S/L26I continued to rise throughout passages 5 and 6. Similarly, the was nearly completely resistant to WJ379, showing 11% N31D mutation also completely reverted back to N31 at inhibition by 100 mM WJ379 (Fig. 3A). For the mutants passage 9 when the drug selection pressure was removed, selected by BC035, 100 mM BC035 had minimal inhibition whereas another mutation, I32T, which emerged at passage 5, against the M2-N31D single mutant and the M2-N31D/I32T continued to increase throughout passages 6–9 and became double mutant (Fig. 3B). However, the M2-S31N/I32T single the predominant mutation at passage 9 by complete replace- mutant remained sensitive to BC035 (Fig. 3B). The IC50 of ment of I32. BC035 against M2-S31N/I32T was 50.5 mM, only a 2.5-fold M2 Mutants Selected from Serial Passage Experi- increase compared with the IC50 value of BC035 against ments Have Reduced Drug Sensitivity to M2-S31N M2-S31N (Fig. 4B). Inhibitors in TEVC Assays. In the aforementioned serial Correlation between Electrophysiological TEVC IC50 passage experiments, we identified two amino acid substitu- Values with Antiviral EC50 Values Reveals the Impor- tions, L26I and N31S, for WJ379 and two amino acid tance of Drug Inhibition Kinetics. In general, there is a substitutions, N31D and I32T, for BC035. As we cannot positive correlation between an M2 channel blocker’s electro- distinguish whether these two substitutions occur in the same physiological IC50 value and its antiviral EC50 value. A more M2 protein or at two different M2 proteins, we therefore potent channel blocker normally has higher antiviral activity generated all possible M2 constructs and tested their drug (Wang et al., 2013b; Li et al., 2016). However, when correlat- sensitivity in TEVC assays. Specifically, in the case of WJ379, ing the IC50 and EC50 values of WJ379 and BC035 against M2 we recorded the channel blockage of WJ379 against M2-S31N/ mutants, we found different results (Table 3). The single L26I (single mutation), M2-N31S (single mutation), and M2- mutant (S31N/L26I) selected by WJ379 only conferred a 2.7- N31S/L26I (double mutations) in TEVC assays. In the case of fold increase in TEVC IC50 values (calculated based on BC035, we recorded its channel blockage against M2-S31N/ 2-minute time points); however, it led to more than a 60-fold I32T (single mutation), M2-N31D (single mutation), and M2- increase in antiviral EC50 value. This discrepancy led us to N31D/I32T (double mutations). The TEVC assay is one of the further investigate the kinetics of drug binding in both S31N gold-standard assays to study the proton conductance prop- and S31N/L26I channels. It is of note that the percentage of erty of M2 channels as well as their drug inhibition (Pinto channel blockage was recorded 2 minutes after compound et al., 1992; Wang et al., 1993; Pinto and Lamb, 2006). treatment at pH 5.5. An examination of the current-inhibition Channel blockage is expressed as percentage of current traces revealed that, for S31N channel inhibition, equilibrium Pharmacology of M2-S31N Inhibitors 193 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 26, 2021

Fig. 2. M2 gene sequencing traces. M2 genes at passages 0, 3, 4, 5, 6, and 9 were sequenced by Sanger sequencing. The regions (residues 18–37) that cover detected mutations are shown. Mutations are indicated by arrows. Sequencings from WJ379 serial passage experiments are shown in (A–F): WJ379, passage 0 (A); WJ379, passage 3 (B); WJ379, passage 4 (C); WJ379, passage 5 (D); WJ379, passage 6 (E); WJ379, passage 9 (F). Sequencings from BC035 serial passage experiments are shown in (G–L): BC035, passage 0 (G); BC035, passage 3 (H); BC035, passage 4 (I); BC035, passage 5 (J); BC035, passage 6 (K); BC035, passage 9 (L). 194 Ma et al.

respectively, and the representative TEVC trace of WJ379 in inhibiting M2-S31N at 10 mMisshowninFig.6D.In summary, for the M2-WT channel inhibition by amantadine and the M2-S31N channel inhibition by WJ379, the IC50 value in TEVC assays at 2-minute time points underesti- mates their true IC50 values. After adjusting the binding kinetics, there appears to be a good correlation between the IC50 and EC50 fold increase from passage 0 to passage 11 (Table 3): the IC50 increased 18.2-fold (calculated based on 10-minute time points), and the EC50 increased more than 60-fold. The percentage of current inhibition was typically Fig. 3. Percentage of M2 current inhibition by 100 mM WJ379 or 100 mM recorded at 2-minute time points because oocyte cells used BC035 in TEVC assays at 2-minute time points. (A) Percentage of current for the current recording cannot survive under pH 5.5 for an m conductance inhibition of M2 channels by 100 M WJ379. (B) Percentage extended period of time. To get data at 5 and 10 minutes, of current conductance inhibition of M2 channels by 100 mM BC035. repetitive recordings had to be performed with multiple oocytes. was not reached at the 2-minute time point (Fig. 5A) when In summary, although M2-S31N/L26I and M2-S31N/I32T Downloaded from tested at 100 mM, which means the true IC50 is actually lower remained sensitive to WJ379 and BC035, respectively, the fast than the recorded IC50 if enough time is given to achieve binding kinetics rendered these two drugs much less effective. equilibrium. In contrast, for M2-S31N/L26I inhibition by The M2-S31N Inhibitor WJ379 Shows a Synergistic WJ379 and M2-S31N/I32T inhibition by BC035, the TEVC Antiviral Effect with Oseltamivir. One useful strategy to IC50 values at 2-minute time points were true IC50 values, as reduce the pace of drug resistance development is combination equilibriums were reached before the 2-minute time point therapy, which has established efficacy in the treatment of molpharm.aspetjournals.org (Fig. 5, A and B). Fitting the current-conductance trace of human immunodeficiency virus infection (Gulick et al., 1997, WJ379 in inhibiting M2-S31N/L26I using a one-phase expo- 1998; De Clercq, 2007). Unlike M2-S31N inhibitors, oseltami- nential decay equation gave a time constant of 8.5 seconds, vir is a neuraminidase inhibitor, which interrupts the viral which is much faster than the 45.3-second time constant in the replication cycle by preventing virus release from infected cell case of M2-S31N inhibition by WJ379. Similar results were surfaces. Therefore, oseltamivir is expected to show a syner- observed for BC035: the time constants for BC035 in inhibit- gistic effect with M2-S31N inhibitors in combination therapy. ing M2-S31N and M2-S31N/I32T were 29.5 and 5.5 seconds, To test this hypothesis, we combined WJ379 and oseltamivir respectively. Another example of slow drug inhibition kinetics carboxylate using the two-drug combination protocol (Hayden, is amantadine in inhibiting the WT-M2 channel (Fig. 5C). 2013; Dunning et al., 2014). The efficacy of oseltamivir at ASPET Journals on September 26, 2021 To get the true IC50 values of amantadine in inhibiting carboxylate alone was first determined by CPE assay. The M2-WT and WJ379 in inhibiting M2-S31N due to their slow EC50 of oseltamivir carboxylate against A/WSN/33 (H1N1) in binding kinetics, we chose to record the percentage of current MDCK cell culture was 18.4 nM in the CPE assay. In the inhibition at 5- and 10-minute time points instead of the combination therapy, a matrix was created to combine 10– regular 2-minute time point. As shown in Fig. 6A, when the 10,000 nM WJ379 with 1–1000 nM oseltamivir carboxylate. WT channel was treated with amantadine, the IC50 values Compounds were added to confluent MDCK cells that were were 12.5, 4.7, and 2.5 mM when the percentage of current infected with the A/WSN/33 (H1N1) virus at MOI 0.01. Cell inhibition data were collected at 2-, 5-, and 10-minute time viability was evaluated at 72 hours after infection by neutral points, respectively. A representative TEVC trace of amanta- red staining. The synergistic effect was calculated with dine in inhibiting M2-WT at 10 mM was shown in Fig. 6B, MacSynergy II software (Prichard and Shipman, 1990; which clearly showed different percentage of inhibition at 2-, Prichard et al., 1993; Smee et al., 2010). As shown in Fig. 7, 5-, and 10-minute time points. Similarly, in the case of M2- a region of significant synergy (29.0–42.4 mM2%) was observed S31N inhibition by WJ379 (Fig. 6C), the IC50 values were 27.1, for WJ379 and oseltamivir carboxylate at concentrations of 8.7, and 4.0 mM when recorded at 2, 5, and 10 minutes, 320–1000 and 32–100 nM, respectively. The calculated net

Fig. 4. Dose-response curves of WJ379 and BC035 on M2 channels in TEVC assay with 2-minute time points. (A) WJ379 dose-response curve. (B) BC035 dose-response curve. Each data point represents at least three oocytes. Pharmacology of M2-S31N Inhibitors 195

TABLE 3

Correlations of antiviral EC50 and TEVC IC50 values.

Passage 0 Passage 11 Fold Increase WJ379 TEVC IC50 (mM) 27.1 (2 minutes) (S31N) 72.9 (S31N/L26I) 2.7 4.0 (10 minutes) (S31N) 72.9 (S31N/L26I) 18.2 Antiviral EC50 (mM) 0.49 (S31N) .30 (S31N/L26I) .60 BC035 TEVC IC50 (mM) 20.5 (S31N) 50.5 (S31N/I32T) 2.5 Antiviral EC50 (mM) 2.2 (S31N) .20 (S31N/I32T) .9

effect across the entire surface was a volume of synergy of 166, bioavailable drug on the market. With the continuous pre- indicating a strong synergy. scription of oseltamivir, it will only be a matter of time before predominant flu strains become resistant to it. The alarming fact is that oseltamivir-resistant strains have been continu-

Discussion ously reported, and certain strains appear to adapt to the Downloaded from fitness of transmission among humans, which could lead to Despite the existence of influenza vaccines and antivirals, the next influenza pandemic (Hurt et al., 2009; Kelso and influenza virus infection is still one of the leading causes of Hurt, 2012; Hurt, 2014). In addressing this unmet medical death in the United States (Thompson et al., 2003, 2004). need, we revisited the M2 proton channel and aim to develop Among the two classes of Food and Drug Administration– the next generation of antivirals by targeting the M2-S31N approved anti-influenza drugs, adamantanes are no longer

mutant. M2-S31N is the predominant mutant among cur- molpharm.aspetjournals.org recommended, which leaves oseltamivir as the only orally rently circulating influenza A viruses, including those that are resistant to oseltamivir (Dong et al., 2015). Thus, targeting M2-S31N is expected to yield broad-spectrum antivirals that are active against both oseltamivir-sensitive and oseltamivir-resistant strains (Wang, 2015). To test this hypothesis, we chose two M2-S31N inhibitors, WJ379 and BC035, which represent S31N-specific inhibitors and S31N/ WT dual inhibitors, respectively, and tested their antiviral

efficacy in plaque assays. It was found that WJ379 had at ASPET Journals on September 26, 2021 potent antiviral activity against all four human clinical isolates with submicromolar efficacy, including two 2009 pan- demic strains that are resistant to both amantadine and rimantadine (A/Texas/04/2009 and A/Denmark/528/2009). BC035 similarly inhibited both oseltamivir-sensitive and oseltamivir-resistant influenza A strains with low micromo-

lar EC50 values. These results highlight the great therapeu- tic potential of the first-in-class M2-S31N inhibitors: they can serve as the second line of defense should oseltamivir fail to confine the next influenza outbreak caused by influenza A strains. With these tool compounds in hand, we began to investigate the potential issue of drug resistance. This is critical, as addressing the efficacy is only the first step in developing antiviral drugs. Ideal antiviral drugs should also bear a high genetic barrier to drug resistance such that they can be used for a longer period of time. The genetic barrier is defined in this study as the ease of resistance generation, although it is also defined in other reports as the number of mutations needed for resistance (Vingerhoets et al., 2005; Fofana et al., 2013). The first-generation M2 channel blockers amantadine and rimantadine were rendered ineffective due to the emerg- ing M2-S31N mutant. Related to the second generation of M2 Fig. 5. Representative TEVC recording traces showing different inhibition channel blockers, which are the M2-S31N inhibitors, the kinetics. (A) WJ379 in inhibiting M2-S31N and M2-S31N/L26I channels. concern is whether the M2-S31N channel will similarly WJ379 inhibited the M2-S31N channel much slower than the M2-S31N/ L26I channel. (B) BC035 in inhibiting M2-S31N and M2-S31N/I32T develop resistance to S31N inhibitors. To address this, two channels. BC035 inhibited the M2-S31N channel much slower than the S31N inhibitors, WJ379 and BC035, were subjected to serial M2-S31N/I32T channel. The inhibition kinetics time constant was deter- passage experiments in an attempt to select resistant viruses. mined by fitting the period of recording trace when the compounds are present to the one-phase exponential decay equation [f(t) = A*exp(2t/G)+C] Not surprisingly, resistant strains were selected for both in Clampfit 10.3. (C) Amantadine (Ama) in inhibiting the M2-WT channel. compounds at passage 4. In comparison with the genetic 196 Ma et al. Downloaded from

Fig. 6. Dose-response curves in TEVC assays with 2-, 5-, and 10-minute time points. (A) Amantadine in inhibiting WT M2 (S31). (B) TEVC trace of amantadine in inhibiting M2-WT at 10 mM for 10 minutes. (C) WJ379 in inhibiting M2-S31N mutant. (D) TEVC trace of WJ379 in inhibiting M2-S31N at

10 mM for 10 minutes. Each data point represents at least three oocytes. molpharm.aspetjournals.org barriers to drug resistance for the two classes of Food and BC035, we observed similar phenomena. As BC035 is a potent Drug Administration–approved anti-influenza drugs, M2- inhibitor of both the S31N and the S31 (WT) channels, M2- S31N inhibitors outperformed amantadine and are only S31N could not revert back to S31 to become resistant to it; slightly less effective than oseltamivir. In general, complete instead, it changed to N31D. N31D is also a dominant positive resistance to oseltamivir was generated around passage mutation, and the M2-N31D/I32T was completely resistant to 5 (Triana-Baltzer et al., 2011; Renzette et al., 2014). Sequenc- BC035. The single mutation M2-S31N/I32T remained sensi- ing the resulting resistant viruses revealed interesting find- tive to BC035, and this mutation only caused a 2-fold EC50 at ASPET Journals on September 26, 2021 ings. For the S31N-specfic inhibitor WJ379, M2-S31N simply increase. Interestingly, both of these highly resistant mu- reverted back to N31S to become resistant to WJ379. This tants, N31S and N31D, appeared to be less fit than the N31 mutation is a dominant positive mutation, as the M2-N31S/ mutant in cell culture; both N31S and N31D reverted back to L26I double mutant was also completely resistant to WJ379. N31 after releasing drug selection pressure. The single mutant M2-S31N/L26I alone was only partially Fast drug-binding kinetics were observed for both WJ379 in resistant to WJ379. For the M2-S31N/WT dual inhibitor inhibiting M2-S31N/L26I and BC035 in inhibiting M2-S31N/ I32T. It is possible that M2-S31N/L26I and M2-S31N/I32T might either adapt different structures or have different dynamics than M2-S31N, which allows drugs to easily escape from the channel cavity. For example, M2-S31N/L26I and M2- S31N/I32T might have an enlarged N-termini channel en- trance such that drug can easily escape from the channel. Alternatively, M2-S31N/L26I and M2-S31N/I32T might be more dynamic than M2-S31N; thus, drugs have a higher probability to escape the channels when they adapt the

Openout-Closedin conformation (Khurana et al., 2009). Although resistant mutants (L26I and I32T) were selected from serial passage experiments in cell culture under the drug selection pressure of WJ379 and BC035, respectively, both mutants appear to be rare among human influenza A viruses. We retrieved and analyzed all of the human influenza A virus M2 sequences from the Influenza Research Database (http:// Fig. 7. Three-dimensional plot of the combination therapy of WJ379 and www.fludb.org, accessed on January 18th, 2016). L26I oc- oseltamivir carboxylate on influenza A A/WSN/33 virus. A series of curred 70 times, and I32T only occurred twice out of 18,393 M2 combinations of WJ379 (0, 10, 32, 100, 320, 1000, 3200, and 10,000 nM) sequences. These results indicate that viruses carrying either and oseltamivir carboxylate (0, 1, 3.2, 10, 32, 100, 320, and 1000 nM) were added to MDCK cells that were infected with influenza virus A/WSN/33 in of these two mutants might have a reduced fitness of trans- a 96-well plate. The plate was subsequently incubated for 72 hours. Cell mission among humans, although more stringent experiments viability was determined by neutral red staining. The synergy or need to be followed to test their fitness and transmissibility. antagonism volumes of drug-drug interaction were generated by the MacSynergy II software program. The synergy for the various combina- As resistance development is, unfortunately, inevitable, one tions was significant at the 95% confidence limit. standard approach to delay resistance evolution under drug Pharmacology of M2-S31N Inhibitors 197 selection pressure is combination therapy. Combination therapy Bantia S, Kellogg D, Parker CD, and Babu YS (2010) Combination of peramivir and rimantadine demonstrate synergistic antiviral effects in sub-lethal influenza A has a proven track record of reducing resistance evolution and (H3N2) virus mouse model. Antiviral Res 88:276–280. side effects, and it is highly recommended for people with Beyleveld G, White KM, Ayllon J, and Shaw ML (2013) New-generation screening assays for the detection of anti-influenza compounds targeting viral and host compromised immune systems who might need extended anti- functions. Antiviral Res 100:120–132. viral treatment to clear the viruses (Ison, 2013). As an example, Blair JMA, Webber MA, Baylay AJ, Ogbolu DO, and Piddock LJV (2015) Molecular mechanisms of antibiotic resistance. 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