Scoring indicators observed daily for 5 days.
Copper complexes have previously been developed to target His37 in influenza M2 and are effective blockers of both the wild type (WT) and the amantadine-resistant M2S31N. Here, we report that the complexes were much less toxic to zebrafish than CuCl2. In addition, we characterized albumin binding, mutagenicity, and virus resistance formation of these metal complexes, and employed steered molecular dynamics simulations to explore whether the complexes would fit in M2. We also examined their anti-viral efficacy in a multi-generation cell culture assay to extend the previous work with an initial-infection assay, discovering that this is complicated by cell culture medium components. The number of copper ions binding to bovine serum albumin (BSA) correlates well with the number of surface histidines and BSA binding affinity is low compared to M2. No mutagenicity of the complexes was observed when compared to sodium azide. After 10 passages of virus in MDCK culture, the EC50 was unchanged for each of the complexes, i.e. resistance did not develop. The simulations revealed that the compounds fit well in the M2 channel, much like amantadine.
- medicinal metals
- proton transport
- plaque assay
The influenza A M2 protein is a homotetrameric channel  that is particularly selective for protons  and is essential for uncoating of the virus . The proton selectivity is due to the cluster of His37 imidazole side chains in the channel [4, 5]. This channel has been a primary antiviral target. Amantadine (AMT) and rimantadine (RMT) were highly successful as M2 blockers [6, 7, 8], but they became ineffective in 2005 when a mutation from serine to asparagine at residue 31 (S31N) in M2 occurred [9, 10].
Attempts have been made to develop variants of AMT, RMT and others that could block the V27A, L26F, or S31N mutations [11, 12, 13, 14]. We explored a different approach that could, in theory, target all functional forms of M2 .
Drawing from the observation that divalent cations, particularly copper, block M2 current  binding in the His37-Trp41 side chain quadruplex , divalent copper complexes of AMT were synthesized and found to be effective influenza A inhibitors with reduced cytotoxicity compared to CuCl2 . Because Cu2+ binds strongly to imidazole, it was suggested that the Cu2+ complexes also block M2 through His37-imidazole binding. In addition, the His37 cluster is highly conserved in nature , making it a prime target in the M2 channel.
The copper ligands developed were based on AMT and the lesser-known, equally effective M2 WT blocker, cyclooctylamine (CO) [19, 20], and extended via the amine with the functional groups iminodiacetate or iminodiacetamide. Six Cu2+ complexes (Figure 1) were synthesized and characterized using NMR, IR, MS, UV-Vis, and ICP-MS. The complexes demonstrated H37-specific block of M2 current in two electrode voltage-clamp (TEVC) studies with low μM potencies. The copper-free ligands did not show proton current block, demonstrating that the copper was key to the current-blocking process .
Because of the reduced toxicity to cultured cells found previously, we were interested to learn whether the six metal complexes were toxic to simple organisms. Zebrafish embryos were chosen because they have immune and nervous systems similar in many ways to more advanced organisms, because they are in an early, vulnerable stage of development, and because the compounds are readily administered at infection-relevant concentrations in their bathwater. We also explored and report additional properties of these copper complexes, including their efficacies in the cytopathic effect antiviral assay, their binding to albumin, mutagenicity testing in a bacterial assay, virus resistance development when passaged with cell culture in the presence of the compounds, and molecular dynamics simulations to explore how well the compounds fit in the M2 channel.
2. Materials and methods
2.1 Cytopathic effect assay
Confluent MDCK cells were transferred into 60 wells of a 96-well plate in DMEM (Gibco Thermo Scientific Waltham, MA, 4.5 g/L D-Glucose) with 5% Fetal Bovine Serum (FBS, Hyclone, Logan, UT). The cells were washed with a diluted solution of 50% SEM/50% serumless DMEM. SEM (simple electrolyte medium) consists of 4.33 g NaCl, 0.244 g KCl, 0.103 g CaCl2
The crystal violet staining technique described previously  was used to determine the fraction of cells that survived the exposure to the virus. After 48 h, the test medium was removed, and the cells were washed three times with 150 μl PBS. The cells were stained for 10 min with 50 μl crystal violet solution (0.03% crystal violet (w/v) in 20% methanol). The cells were then washed three times with 150 μl distilled water before adding 100 μl lysis buffer. After 20 min, the optical density (OD) of each well was measured at 590 or 620 nm and averaged over the set of six wells for each concentration.
Because viral dosing was sufficient to eliminate essentially all cells in treatment-free controls, their average OD was subtracted as baseline from the average of the treated well ODs. The result was divided by the average of the uninfected control well ODs to obtain a normalized vitality. Because the vitality can be affected by both reduction of virus cytopathic effect and increase of treatment toxicity as concentration is increased, we fitted the normalized concentration-dependent vitality, V(C), with a joint probability function:
Here, EC50 is the 50% effective dose of treatment that prevents viral cytopathic effect, CC50 is the 50% cytotoxic dose of the treatment, and n1 and n2 are their respective Hill coefficients. If the selectivity index, CC50/EC50, and the Hill coefficients are sufficiently high, this function rises to unity at doses that are sufficient to prevent viral replication but below toxic levels. Non-linear least squares fitting weighted with standard errors of means was done with the Marquardt algorithm in KaleidaGraph4 (Synergy Software, Reading, PA). In practice, it was necessary to fix the Hill coefficients to evaluate the effective doses, then manually adjust the Hill coefficients to improve the fit (due to low numbers of data points). Hence, the reported standard errors of the parameters obtained from the error matrix may be underestimated.
2.2 Protein binding assay
Each copper complex was dissolved in 25 ml of water to obtain a 1 mM and 800 μM solution. All water used in the protein binding assay was collected from a Millipore first-generation beige Milli-Q system. These solutions were sonicated until the crystals were fully dissolved. Four 1:2 serial dilutions were performed from the 800 μM solution to obtain 400, 200, 100, and 50 μM solutions, and a 1:5 dilution was performed from the 50 μM solution to obtain a 10 μM solution. 13.3 mg of BSA was then dissolved in 10 ml of each solution. The solutions were mixed thoroughly and allowed to stand at room temperature for approximately 20 min.
Spin filtration was performed using a swinging bucket rotor at 4000 rpm for 6 min. The spin filters used were Amicon Ultra-15 centrifugal filters. The filtrates from each spin were collected to test for copper content in ICP-MS. Solutions for ICP-MS were prepared from both the original solutions and the filtrates. For each solution, 1 ml of solution was added to 1 ml of 4% HNO3 and 8 ml of 2% HNO3 to obtain a 1:10 dilution of each solution in 2% HNO3. Nitric acid used for ICP-MS analysis was OmniTrace trace-metal grade obtained from EMD Millipore Corporation. We used BSA to model copper binding histidine in solution and calculate relative dissociation constants (
2.3 Zebrafish toxicity test
Following an approved BYU IACUC protocol, two AB wild-type male and female zebrafish were placed in an embryo media filled tank. The fish remained in a light and temperature-controlled facility until the following morning. Later that day, the fish were transferred into original tank. Embryos were moved into embryo media filled petri dishes (60 embryos/dish) and housed in an incubator for 2 days. Media was changed daily.
Fish embryos were dechorionated at 48 hpf. In a multi-well plate, 10 embryos were selected and 5 were added to each of two wells for each concentration with fresh embryo media. Drug solution (0–200 μM) was then added to test toxicity and observed over 5 days. Drug solutions were changed daily. After 5 days, the fish were scored using a morbidity scale (Table 1) indicating response, spine shape, edema, equilibrium, and death. The average for each complex was normalized using the maximal morbidity score of 50/well. The fish were then euthanized.
|Zebrafish scoring indicators|
|Equilibrium||Upright position||Lying on side||NA||NA|
|Response||Quick escape||Sluggish escape||No escape||NA|
|Spine shape||Straight||Slightly curved||Strongly curved||NA|
|Edema||None||1 place and minor||2 places or major||2 places and major|
2.4 Ames testing
The Modified Ames ISO kit (Environmental Bio-Detection Products Inc., Mississauga, ON) was used with
The complexes were compared against the mutagenicity of a positive control (NaN3) and vehicle (water). The complexes were serially diluted 1:2 to compare the complexes’ mutagenic ability at each of six concentrations.
TA100 was hydrated and incubated with histidine overnight at 37°C. Following the kit’s instructions, in 96-well plates’ exposure solution, diluted bacteria mix, and serial two-fold dilutions of complexes were combined with reversion media containing Bromocresol Purple, which serves as a pH indicator to identify infected wells. The 96 well plates were incubated for 6 days at 37°C without agitation. When a sample is mutagenic, it will revert the bacteria to WT, causing the media to turn slightly acidic and show a yellow color.
The number of reverted wells with complex was compared to the average number of reverted wells in the negative control. Significance was calculated using a one-tailed t-test.
The 2KQT M2 structure was used and oriented in a DMPC lipid bilayer with a center-of-mass harmonic constraint. The copper complexes were oriented such that the copper was near (~2.0 Å) at least one of the four imidazole nitrogens. Water molecules within 2.2 Å of the complexes were deleted. The protein-bilayer system was solvated with a tetragonal 60 Å × 60 Å × 90 Å water box as shown in Figure 2. The system was minimized for 1000 steps of steepest descent and heated to 300 K. The M2 channel was equilibrated for 1 ns. The complexes were pulled using a constant force for 10 ps during the production runs. Frames were saved every 50 steps, which is every 50 fs, of production for a total of 200 frames. Standard CHARMM version 37b1 parameters were used. Copper dihedral parameters were created using a 20 kcal/mol/rad2 energy penalty, which kept a conservatively rigid structure throughout the channel (Table A1).
The distance between imidazole nitrogens and copper on the complexes was calculated using CHARMM’s CORREL subroutine for each frame. The time for each complex was recorded when the copper reached 30 Å away from the imidazole nitrogens. This distance was chosen to represent the complex leaving the mouth of the channel.
2.5.1 Decisions affecting pulling force
The pulling force for each of the complexes was determined by normalizing the pulling forces to a 2.34 nN pulling force on AMT. The 2.34 nN force allowed comparisons to be made between compounds as they left the channel on the 10 ps timescale.
These steered molecular dynamics (SMD) simulations were analyzed by computing the mean and standard deviation of five independent. The five independent simulations were assigned random starting velocities and then analyzed to explore the time needed to pass the 30 Å threshold relative to the starting point from the copper atom on the complex.
The analysis examined whether the pulling forces, copper ligation mechanism, or scaffold (CO, AMT, or neither), significantly affected the exit times relative to free Cu2+.
2.6 Miniplaque assays, resistance testing, and sequencing
MDCK cells were seeded into a six-well plate and grown in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich, St. Louis, MO) augmented by 5% with fetal bovine serum (FBS, Hyclone, Logan, UT) until confluent. After 48 h, the growth media was removed and replaced with DMEM. At this point the virus (A/CA/07/09) was introduced into the medium (200 pfu/ml) and allowed to adsorb for 1 h. The medium was then removed and replaced with fresh DMEM containing a specified concentration of complex and 5 ml of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Thermo-Fischer Scientific, Waltham, MA, 1 mg/ml) was added to activate the virus. The plate was incubated at 33°C for 3 days. Then the medium was removed and centrifuged at 2000 rpm in order to remove cell debris. This virus-containing medium was then separated into 1-ml aliquots and frozen in Eppendorf tubes at −80°C. This process was repeated for each successive passage.
The concentration of virus was determined through an immunofluorescence assay (as previously described by ), which gave a multiplicity of infection (MOI) of 0.6. MDCK cells were seeded onto glass coverslips in vials containing 1 ml DMEM and trypsin in order to obtain 90% confluency after 24 h. The cells were allowed to grow overnight at 37°C, after which the growth medium was removed and replaced with DMEM. The sample of virus was then diluted by factors of 10, and the various dilutions of virus were stirred into the vials with coverslips. They grew at 33°C for 18 h. After this incubation period, the medium was removed, the cells were fixed with cold acetone (−80°C), and the coverslips were washed and stained with a fluorescein isothiocyanate labeled anti-IAV monoclonal antibody (Millipore Sigma, Burlington, MA, Cat. #5017). Excess antibody was washed off using a solution of 0.05% Tween20 in phosphate buffered saline and then again with distilled water. They were then viewed microscopically and individual infected cells (miniplaques) were counted.
This same process was followed in determining the new EC50 against the specific complex of each resistant strain. Except, 100 pfu of virus was used in each vial. Several different concentrations of the complex with which it was passaged were introduced into the vials, with concentrations ranging from 2 to 70 μM. The cells were infected with the virus in a solution of SEM rather than DMEM. The EC50 was calculated in KaleidaGraph using the Levenberg-Marquardt algorithm. The fitting parameters (sigmoidal function) were used to calculate the EC50 and the standard error of the mean.
To sequence the genome, the viral sample was concentrated 10-fold using a spin filter (VWR North America, Radnor, PA, Cat. #82031-352). After that, viral RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD). The isolated RNA was stored at −20°C. RNA was then reverse-transcribed using Invitrogen’s Superscript III One-Step RT-PCR Platinum Taq HiFi kit (Thermo-Fischer Scientific, Waltham, MA).
The resulting isolated DNA was stored at −20°C. The DNA was then amplified with PCR using the Phusion High-Fidelity PCR kit (New England Biolabs, Ipswich, MA). The solution was purified using Qiagen’s QIAquick PCR Purification Kit (Qiagen, Germantown, MD). It was sequenced using custom forward (TGTAAAACGACGGCCAGTACGAAAAGCAGGTAG) and reverse (CAGGAAACAGCTATGACCAGTAGAAACAAGGTAGT) primers for the segment of the new DNA that codes for the M2 protein.
3. Results and discussion
3.1 Cytopathic effect assay
3.2 Protein binding assay
To illustrate the potential of the metal complexes to bind to proteins, binding to BSA was measured in which a protein solution was mixed with various concentrations of a CuCl2 or copper complex solution. The copper content of the original sample was measured and compared to that of the filtrate. Taking the volume proportions into account, the “free copper concentration” in the filtrate relative to the “total copper concentration” in the original sample was fitted to a model assuming that each protein molecule had n equivalent copper or copper complex binding sites. Table 2 shows the best fit Kd values, assuming that each albumin monomer has n equivalent binding sites. The two parameters interacted and were therefore poorly constrained in the optimization of the deviations squared, but Table 2 indicates that the number of binding sites is well above 10, consistent with the count of 13 surface histidines in monomeric albumin (Figure A1). Complexes
BSA has 13 surface histidines (Figure A1), however, all of the fits optimized n at >13 copper binding sites. This difference could suggest non-specific binding to other sites on BSA. The high Kd’s for the complexes relative to CuCl2 indicate that the complexes remain intact during binding to BSA. The binding the copper complexes to BSA is very weak compared to that of the M2S31N (AMT resistant) channel, where block was ~80% for
3.3 Zebrafish toxicity test
Toxicity was evaluated for zebrafish exposed to various concentrations of CuCl2 or copper complex (
Compared to CuCl2, the copper complexes show less toxicity, suggesting that the ligands are coordinating to the copper and helping to reduce its toxicity through day 2 of high dosage. All of the copper complexes produce some toxicity in the zebra fish for all experimental concentrations, but compound
3.4 Ames testing
The mutagenicity of the copper complexes was tested using the Ames test. Table 3 shows the percent reversion out of 48 wells of three complexes. They were tested for mutagenicity against
3.5 Resistance testing and sequence
Because the putative target for the metal complexes, the His37 quadruplex, is highly conserved in nature and functionally critical for vRNP uncoating, we explored the propensity for virus resistance formation with passaging in MDCK cell cultures. Because the incubation had to be done in DMEM, which is known to inhibit complex efficacy, we used higher concentrations of complexes for the incubations such that the efficacy of block was projected to be ~50%, thus creating a concentration where mutation could occur. Ten passages (~5 weeks) of incubated virus in DMEM dosed with increasing metal complex concentrations (ranging from 50 to 100 μM) was chosen as a rigorous test. Resistance would be identifiable by an increase in miniplaque EC50 after passaging relative to the original value. As shown in Table 4, the new EC50 (column 3) is comparable to the original EC50 (column 2). Because none of the copper complexes significantly increased the EC50 after 5 weeks of incubation, we conclude that resistance is slow to develop. This contrasts with rapid resistance development when passaging with AMT .
|Complex||Original A/CA/09 (μM)||10 passages with complex (μM)|
|1||6.9 ± 1.2||3.7 ± 0.5|
|2||4.9 ± 0.8||2.1 ± 1.1|
|3||0.7 ± 0.1||1.1 ± 0.4|
|4||11.6 ± 1.1||3.9 ± 6.8|
|5||8.2 ± 2.0||1.3 ± 0.2|
|6||4.4 ± 0.6||2.9 ± 0.3|
The vRNA M segment was extracted from the passaged virus exposed to
3.6 MD simulations
Constant force steered molecular dynamics (MD) simulations were carried out to explore the steric limitations on metal complex exit from the M2 transmembrane domain AMT binding site. A 2.34 nN force was used to pull the complexes pass the 30 Å threshold and beyond the Val27 cluster within 10 ps. The 2.34 nN force gave a sufficient spread in leaving times to allow assessment of the ease of unbinding relative to AMT. For these simulations, the force was applied to the center-of-mass of the complex. Example trajectories for AMT (green) and
Table 5 shows the average time to leave from five independent simulations (identical starting configurations, but randomly assigned atomic velocities) for each complex to pass the 30 Å threshold. All metal complexes took longer to leave the channel than AMT. AMT exited the channel in 2.77 ps. Complex
|Complex||Average time to leave (ps)|
|1||4.60 ± 1.14|
|2||6.85 ± 1.01|
|3||4.67 ± 1.07|
|4||7.05 ± 0.53|
|5||3.75 ± 0.89|
|6||4.00 ± 1.06|
|AMT||2.77 ± 0.26|
The copper complexes are relatively non-toxic in zebrafish embryos compared to CuCl2 over a 5-day period. Also, they are efficacious in a 3-day assay (but with limitations due to serum protein binding and amino acid interference), are non-mutagenic compared to sodium azide, are slower to leave the M2 binding site compared to AMT, and, also compared to AMT, are not prone to resistance development.
Further testing of these copper complexes should include isothermal titration calorimetry (ITC) experiments with influenza A M2 channel to obtain binding energies, two-electrode voltage clamp (TEVC) experiments to obtain rate constants of binding to M2, and testing in an animal model that more accurately represents the effects of the copper complexes on humans.
Busath DD. Influenza A M2: Channel or Transporter? Advances in Planar Lipid Bilayers and Liposomes. Vol. 10. Burlington: Academic Press; 2009. pp. 161-201
Chizhmakov IV, Geraghty FM, Ogden DC, Hayhurst A, Antoniou M, Hay AJ. Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells. The Journal of Physiology. 1996; 494(Pt 2):329-336
Helenius A. Unpacking the incoming influenza virus. Cell. 1992; 69(4):577-578
Venkataraman P, Lamb RA, Pinto LH. Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus. The Journal of Biological Chemistry. 2005; 280(22):21463-21472
Wang C, Lamb RA, Pinto LH. Activation of the M2 ion channel of influenza virus: A role for the transmembrane domain histidine residue. Biophysical Journal. 1995; 69(4):1363-1371
Davies WL, Grunert RR, Haff RF, McGahen JW, Neumayer EM, Paulshock M, et al. Antiviral activity of 1-adamantanamine (amantadine). Science. 1964; 144(3620):862-863
Krylov VF, Alekseeva AA, Liarskaia T, Poliakova TG, Kupriashina LM. Therapeutic effectiveness of bonafton and rimantadine in influenza. Voprosy Virusologii. 1976;(2):186-191
Wang C, Takeuchi K, Pinto LH, Lamb RA. Ion channel activity of influenza A virus M2 protein: Characterization of the amantadine block. Journal of Virology. 1993; 67(9):5585-5594
Hata M, Tsuzuki M, Goto Y, Kumagai N, Harada M, Hashimoto M, et al. High frequency of amantadine-resistant influenza A (H3N2) viruses in the 2005-2006 season and rapid detection of amantadine-resistant influenza A (H3N2) viruses by MAMA-PCR. Japanese Journal of Infectious Diseases. 2007; 60(4):202-204
Krumbholz A, Schmidtke M, Bergmann S, Motzke S, Bauer K, Stech J, et al. High prevalence of amantadine resistance among circulating European porcine influenza A viruses. The Journal of General Virology. 2009; 90(Pt 4):900-908
Balannik V, Wang J, Ohigashi Y, Jing X, Magavern E, Lamb RA, et al. Design and pharmacological characterization of inhibitors of amantadine-resistant mutants of the M2 ion channel of influenza A virus. Biochemistry. 2009; 48(50):11872-11882
Wang J, Ma C, Wang J, Jo H, Canturk B, Fiorin G, et al. Discovery of novel dual inhibitors of the wild-type and the most prevalent drug-resistant mutant, S31N, of the M2 proton channel from influenza A virus. Journal of Medicinal Chemistry. 2013; 56(7):2804-2812
Wu Y, Canturk B, Jo H, Ma C, Gianti E, Klein ML, et al. Flipping in the pore: Discovery of dual inhibitors that bind in different orientations to the wild-type versus the amantadine-resistant S31N mutant of the influenza A virus M2 proton channel. Journal of the American Chemical Society. 2014; 136(52):17987-17995
Zhao X, Jie Y, Rosenberg MR, Wan J, Zeng S, Cui W, et al. Design and synthesis of pinanamine derivatives as anti-influenza A M2 ion channel inhibitors. Antiviral Research. 2012; 96(2):91-99
Gordon NA, McGuire KL, Wallentine SK, Mohl GA, Lynch JD, Harrison RG, et al. Divalent copper complexes as influenza A M2 inhibitors. Antiviral Research. 2017; 147:100-106
Gandhi CS, Shuck K, Lear JD, Dieckmann GR, DeGrado WF, Lamb RA, et al. Cu(II) inhibition of the proton translocation machinery of the influenza A virus M2 protein. The Journal of Biological Chemistry. 1999; 274(9):5474-5482
Su Y, Hu F, Hong M. Paramagnetic Cu(II) for probing membrane protein structure and function: Inhibition mechanism of the influenza M2 proton channel. Journal of the American Chemical Society. 2012; 134(20):8693-8702
Durrant MG, Eggett DL, Busath DD. Investigation of a recent rise of dual amantadine-resistance mutations in the influenza A M2 sequence. BMC Genetics. 2015; 16(Suppl. 2):S3
Pinto CA, Haff RF. Antival activity of cyclooctylamine hydrochloride in influenza virus-infected ferrets. Antimicrobial Agents and Chemotherapy. 1968; 8:201-206
Lin TI, Heider H, Schroeder C. Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein. The Journal of General Virology. 1997; 78(Pt 4):767-774
Schmidtke M, Schnittler U, Jahn B, Dahse HM, Stelzner A. A rapid assay for evaluation of antiviral activity against coxsackievirus B3, influenza virus A, and herpes simplex virus type 1. Journal of Virological Methods. 2001; 95:133-143
Kolocouris A, et al. Aminoadamantanes with Persistent in Vitro Efficacy against H1N1 (2009) Influenza A. Journal of Medicinal Chemistry. 2014; 57(11):4629-4639