Influenza A virus (IAV) is a significant and ongoing cause of human morbidity and mortality worldwide. While prophylactic vaccines can confer immunity against circulating virus strains, their effectiveness and extent of immunization coverage vary across populations from year to year. This variability contributed in part to the recent and notable 2017-2018 seasonal outbreak which is estimated by the Centers for Disease Control and Prevention to have caused approximately 710,000 hospitalizations and up to 79,400 deaths in the United States alone (CDC, 2018; Rolfes et al., 2019) . Of particular concern are less common but historically regular pandemic outbreaks in populations with limited or no immunity that can result in hundreds of thousands to millions of deaths worldwide.
Moreover, there still remains a significant technical gap to expedite a rapid vaccine production response, particularly in the face of an increasingly global spread of newly-emergent virus strains (Renaud et al., 2011; Hurt, 2014) . Thus, additional countermeasures against IAV in the form of prophylactic and therapeutic antivirals continue to be needed.
The M2 protein is required for IAV replication and spread and is an established antiviral target.
M2 is a proton-gated proton channel that belongs to a growing viroporin family of virus proteins. M2 ion channel activity is required for IAV replication, and adamantane-based M2 inhibitors including amantadine (1) and rimantadine (2; Figure 1 ) were historically used as influenza antivirals. However, adamantane-resistant virus strains are now prevalent to the point that these drugs are no longer recommended for therapeutic use (Bright et al., 2005; Deyde et al., 2007; Fiore et al., 2011; Hayden and De Jong, 2011) . More than 95% of circulating adamantane-resistant viruses harbor a serine to asparagine mutation at amino acid position 31 in M2 (Ser31Asn; Bright et al., 2005) , which distorts adamantane interactions within the pore without significantly affecting M2 ion channel activity or the fitness of viral replication Grambas et al., 1992; Pinto et al., 1992; Stouffer et al., 2008) . Therefore, a major goal of current studies is to identify new inhibitors of adamantane-resistant M2.
Recent advances in molecular and structural biology, electrophysiology, and virology have produced crucial information about the basis of M2-based drug resistance. This in turn has shed light on novel drug development strategies and transitioning of those ideas to practical tools. Here we review the current state of IAV M2 viroporin research and ongoing efforts to identify and develop new antivirals against its drug-resistant forms.
Viroporins are small (often ~60-120 amino acids), virally-encoded transmembrane proteins that regulate ion conduction across lipid membranes and are a frequent feature of both RNA and DNA viruses (Nieva et al., 2012; Nieto-Torres, 2015; Ouyang & Chou, 2014) . Viroporins are frequently described as exhibiting a "channel-pore dualism," where some sequences are broadly permeable to ions and small molecules, while others are tightly-regulated channels that conduct specific ions. Although the functions of most viroporins are not well understood, they are generally involved in conducting ions to facilitate viral entry, assembly, and/or release from host cells. Table 1 lists viroporins of representative viruses.
The M2 protein of IAV is arguably the best understood viroporin. It consists of a 97 amino acid, type I transmembrane domain-containing protein which forms a tetrameric ion channel that is both proton-gated and proton-selective (Hong and DeGrado, 2012; Nieto-Torres et al., 2015) . M2 is a multifunctional, modular protein where each segment performs one or more distinct function(s) (Wang et al., 2011c) . Following viral entry, M2 expressed on the virion membrane transports protons from host cell endosomes to acidify the virion interior ( Figure 2 ). This low pH environment facilitates conformational changes in viral hemagglutinin (HA) and dissociation of viral ribonucleoprotein complex from M1 proteins to promote virion membrane fusion and release of viral RNA into the host cell. M2
proton gating function is, however, not required per se to convert HA into the membrane fusion competent state (Bui et al., 1996; Gutman et al., 1993; Martin and Heleniust, 1991) . During viral egress, M2 also equilibrates the pH of trans-Golgi lumina and the cytoplasm, which may delay trafficking of virion particles and/or prevent HA from inappropriately adopting a low pH conformation (Alvarado-Facundo et al., 2015; Ciampor et al., 1992; Li et al., 2014; Pinto and Lamb, 2006; Sugrue et al., 1990) .
Compared to other influenza proteins, the sequence of M2 is highly conserved but has been shown to co-evolve with HA (Grambas et al., 1992; Rossman and Lamb, 2011) . As such, M2's conductance rate closely correlates with HA's fusogenic propensity, where increased proton conductance by M2 correlates with more acid labile HA sequences (Grambas and Hay, 1992; Scholtissek et al., 1998) . M2 has also been found to localize at the virus budding site on the host cell surface, where the amphipathic helix of M2 alters membrane curvature leading to membrane scission and release of new viral progeny (Chen et al., 2008; Rossman and Lamb, 2011; Rossman et al., 2010) . Although protons are the preferred substrate, M2 can also act as a Na + and K + antiporter (Leiding et al., 2010) . In particular, the ability of M2
to pass bulkier K + ions becomes physiologically relevant in the endocytic pathway, where high Na + concentration in early endosome clusters is replaced by K + to allow for the subsequent release of the viral ribonucleoprotein bundle (Stauffer et al., 2014) .
The existence of M2 was initially reported by (Lamb et al., 1985 Lai, 1981) Pinto et al. (1992) 
Xenopus laevis oocytes injected with M2 RNA and measured by the two-electrode voltage clamp (TEVC) method of electrophysiology, which has become the most common assay by far to measure M2 conductance and its inhibition by small molecules. However, several groups have used a variety of electrophysiological techniques including whole-cell patch-clamp of mammalian cells to probe the ion conduction properties of M2 (Chizhmakov et al., 1996; Holsinger et al., 1995 Holsinger et al., , 1994 Jalily et al., 2016; Shimbo et al., 1996; Tu et al., 1996; Wang et al., 1995 Wang et al., , 1993 For example, Chizhmakov et al. (1996) expressed M2 in mouse erythroleukemia cells and also observed selective conduction of protons.
Subsequent mutagenesis studies have further defined the specific M2 amino acid residues that are required for proton conduction and regulation ( Figure 3 ). Notably, a fragment of M2 encompassing the transmembrane domain and spanning as little as amino acids 21-51 was observed by TEVC to be sufficient to produce amantadine-sensitive, proton-dependent proton currents ). To a first approximation, transmembrane mutations that are predicted to increase the pore radius (i.e., mutation to residues with smaller side chains) result in increased proton conduction, presumably either through the enhanced formation of water wires or transfer by His37 through conformational changes, while mutations to residues with bulkier side chains that reduce the pore radius also reduce conductance. For example, the introduction of Ala at Val27, which faces the extracellular and intraluminal surfaces and is thought to form the most constricted part of the channel, increases the pore entrance size and obliterates the N-terminal gating mechanism, thereby allowing for easier pore hydration and enhanced conduction Holsinger et al., 1994; Pielak and Chou, 2010) . In contrast, introduction of bulky and/or hydrophobic residues such as Phe or Trp at Val27 results in non-or low-conducting M2 proteins. Similarly, mutations that reduce pore size at other locations including Ala30Trp, Ala30Pro, and Gly34Glu also slow the rate of proton conduction and frequently result in loss-of-function .
Notably, a highly-conserved sequence of His37-XXX-Trp41 within the C-terminal end of the M2 transmembrane domain is regarded as the functional core of proton conductance (Pinto and Lamb, 2006; Tang et al., 2002; Venkatraman et al., 2005) . Mutation of His37 to Gly or Gln results in enhanced conductance but also loss of proton selectivity and/or lack of pH dependence Wang et al., 1995) . Moreover, mutation of Trp41 to Ala, Cys or Phe results in larger inward currents but also outward currents, indicating that Trp41 regulates unidirectional conductance Tang et al., 2002; . Another key residue, Ser31, is likely to face the pore interior, and its mutation to hydrophobic residues such as Ala negatively affects pore hydration, resulting in diminished conduction. At the other end of the His-Trp quartet, mutating Asp44 to hydrophobic residues such as Ala affects proton exit at the C-terminal end of the channel by increasing the energy barrier . These and other mutations might also impact M2 function by indirectly affecting the inherent conduction moiety defined by the His37 tetrad or the gating mechanism defined by Trp41 (Gu et al., 2013; .
Recently reported structures of M2 have been instrumental toward understanding how adamantanes inhibit this ion channel and how drug resistance overcomes them, in addition to generally informing new M2 drug discovery and ion channel biology. Experimentally-determined protein structures derived from X-ray crystallography, solid-state and solution nuclear magnetic resonance (NMR), and protein-ligand complex structures originating from computational studies have also formed the basis of structure-based drug design. As of this writing, there were more than 35
structures of wild-type (WT) and drug-resistant A/M2 proteins available in the Protein Data Bank (PDB).
Most of these were solved by X-ray techniques although some were elucidated using NMR.
While the mechanisms of proton shuttling that enable conduction remain incompletely understood, it is likely that experimental conditions such as pH, peptide length, lipid/detergent composition and thickness, and binding of small molecules affect the fundamental properties of M2 (Acharya et al., 2010; S. Cady et al., 2011; Du et al., 2012; Kolokouris et al., 2019; Kovacs et al., 2000; Thomaston et al., 2013; Zhou and Cross, 2013) . Among the available M2 structures in PDB, 2RLF and 3C9J were among the first structures solved using solution NMR and X-ray crystallography in micelles, respectively (Schnell and Chou, 2008; Stouffer et al., 2008) . Hu et al. (2010) and Sharma et al. (2010) subsequently applied solid state NMR and molecular dynamics to explore the activation and conduction mechanisms of M2 at low pH (using PDB entries 2KQT, and 2L0J; Cady et al., 2010; Sharma et al., 2010) .
Although both studies support a "proton-relay" model of conduction, they differ with regard to the sidechain conformation of His37. The study by Hu et al. (2010) proposes a tightly-packed His37 tetrad which undergoes numerous reorientations and side chain rotations to relay protons. In contrast, the model by Sharma et al. (2010) suggests three distinct states for the His-Trp quartet: the ''histidine-locked state'', the ''activated state'', and the ''conducting state'', with transition between these states allowing for proton conductance. In this model, the "histidine locked state" exists when a proton is shared between two adjacent histidine residues (HisH + -His) and the channel is locked in the non-conducting state. They further described the activation cascade at low extracellular pH, when a single hydronium ion approaches the non-protonated histidine in the HisH + -His dimer, breaks this ionic bond, and transitions the channel to the "activated state", resulting in the formation of stable cation-p interactions between
His37 and Trp41. The Trp41 tetrad forms a gate at the C-terminal end, and the perturbation of this cation-p interaction leads to conformational changes which in turn transition the quartet to the "conduction state". Here, protons are then donated to water molecules and relayed to the intracellular side ( Figure 4 ). The transition between these three steps requires small changes of the χ 2 angles of the His37 and Trp41 side chains. This contrasts with the model by Hu et al. (2010) , where considerable reorientation of His37 and Trp41 residues are required to allow proton passage. Further experimental and theoretical data supporting both models are described in detail elsewhere (Hong and DeGrado, 2012; Thomaston et al., 2019; Watkins et al., 2019; Zhou and Cross, 2013) .
Ion channel activity of WT M2 is effectively antagonized by two FDA-approved adamantane-class drugs, amantadine (1) and rimantadine (2) (Figure 1 ), in addition to numerous other adamantane and non-adamantane derivatives (Tables 2-3 ; Tataridis et al., 2007; Wang et al., 2013a; Wang et al., 2013b; Wu et al., 2014) . During virus entry, amantadine's inhibition of M2 results in incomplete dissociation of M1 from ribonucleoprotein complexes, which in turn fail to enter the nucleus to initiate further replication. As M2 can also equilibrate pH across the trans-Golgi network and the cytoplasm, amantadine-based M2 inhibition may also disrupt viral egress (Lamb, R.A. and Krug, 2001; Takeda et al., 2003) .
Amantadine first obtained FDA approval in 1966 under the brand name "Symmetrel" for systemic use in humans for prophylaxis of Asian influenza (H2N2) (Davies et al., 1964; Schwab et al., 1969; Wingfield et al., 1969) . However, during the first decade following its licensure, Symmetrel was not widely prescribed due in part to side effects such as agitation, confusion, and hallucinations (Keyser et al., 2000) . Additionally, the molecular mechanism describing a direct link between amantadine and virus inhibition was not described until more than 25 years later (Duff and Ashley, 1992; Pinto et al., 1992) .
Rimantadine, sold under the brand name Flumadine, is a methylated derivative of amantadine which was licensed by FDA for the same indications in 1994.
Two pharmacologically relevant binding mechanisms were initially proposed for adamantanes ( Figure 5 ). In the first reported crystallographic structure of M2-bound amantadine at pH 5.3 (PDB: Stouffer et al. (2008) suggested that amantadine binds in a pocket located inside the intraluminal cavity and surrounded by the nonpolar side chains of Val27, Ala30, the C β group of Ser31, and the C α group of Gly34 ( Figure 5A ). In this pore binding model, the authors proposed that amantadine, by entering the lumen of the channel, "plugs" the M2 pore and thus prevents the transport of protons. The model further suggests that Ser31Asn confers adamantane resistance by decreasing the pore size and occluding stable access of amantadine to the pore (Stouffer et al., 2008) .
In a separate series of studies, an alternative but initially controversial binding mechanism for adamantanes, located external to the M2 pore, was also proposed (Pielak et al., 2009; Schnell and Chou, 2008 ; Figure 5B ). In these studies, rimantadine was found to bind to Asp44 on the C-terminal, lipidfacing side of the helices. The solution NMR structure (PDB: 2RLF), solved at pH 7.5, showed an allosteric mechanism of inhibition where four drug molecules were proposed to stabilize the inactivated state of M2, thereby preventing proton-gated M2 opening and proton transfer. In this binding mechanism, perturbation of the extra-luminal drug binding pocket as a result of Ser31Asn mutation rendered rimantadine incapable of effectively binding to the allosteric site (Pielak et al., 2009 ).
To reconcile these findings, a series of both experimental and computational studies were rapidly reported (Jing et al., 2008; Balannik et al., 2009; Cady et al., 2010; Carpenter et al., 2008; Chuang et al., 2009; Stouffer et al., 2008) . Jing et al. (2008) demonstrated that the intraluminal binding site is more physiologically relevant through conducting whole-cell electrophysiology and antiviral assays using mutant and chimeric M2 forms. By mutating the critical Asp44 residue to alanine, Jing and coworkers continued to observe inhibition by amantadine in both electrophysiological and antiviral plaque assays, suggesting that the allosteric-binding site is not pharmacologically essential (Jing et al., 2008) .
Additionally, in a chimeric ion channel based on the M2 of influenza B, which is normally amantadineinsensitive due to a larger and more hydrophilic transmembrane pore (Mould et al., 2003) , substitution with the M2 transmembrane domain from influenza A restored amantadine sensitivity (Jing et al., 2008) .
Using solid-state NMR, Cady et al. (2010) also reported that the channel pore was the preferred binding site (PDB: 2KQT), where amantadine preferentially localized to a hydrophobic cage formed by Ala30, Ser31, and Gly34. Hydrophobic interactions between Val27 side chains and the adamantane cage were also observed, and the ammonium group of amantadine showed hydrogen bonding to water molecules and pore-facing residues that stabilized its interactions in the occluded pore. The authors also reported that interactions with the allosteric binding site were observed only at high amantadine concentrations and were thus less probable as the primary mode of M2 inhibition. In further support of the porebinding model, mutation of Asp44, proposed as the key residue of the allosteric binding mechanism, to six different amino acids (Asp44Ala, Asp44Lys, Asp44Asn, Asp44Phe, Asp44Gly, and Asp44Thr) did not affect inhibition by amantadine . Notably, in further testing their initial hypothesis of an allosteric binding site of amantadine to M2, next generated an M2 chimera where the C-terminus was derived from the amantadine-insensitive M2 of influenza B virus (PDB: 2LJB).
Contrary to their previously proposed allosteric binding mechanism, using solution NMR, they described the rimantadine binding pocket to be preserved within the pore of the chimeric M2 protein. In a series of surface plasmon resonance experiments, the pore-binding site was also found to be more energetically favorable than the allosteric binding site, although the latter did remain sensitive to amantadine (Rosenberg and Casarotto, 2010) .
Another computational study using small molecular probes and solvent mapping techniques found the pore-binding site to be more energetically favored, although the allosteric site was also observed under some conditions (Chuang et al., 2009) . Gu et al. (2011) also applied molecular dynamics approaches to study the antagonistic effects of known M2 inhibitors and observed that, while interactions with the pore-binding site were more thermodynamically stable, ligands bound slowly and poorly dissociated due to a high energy barrier of binding. In contrast, the allosteric binding was readily accessible, the energy barrier for binding was minimal, and drug binding was less stable and more readily dissociated. Moreover, the initial NMR structure (PDB: 2RLF) did not show evidence of an intraluminal binding site but rather binding at the allosteric peripheral site in the lipid interface.
However, this can also be interpreted by noting that the NMR structure was narrower as compared to the counterpart X-ray structure (PDB: 3C9J) due to a smaller tilt angle which resulted in transmembrane helices being overly parallel. This finding further alerted the field to the importance of the protein crystallization environment and emphasised the risk of crystal packing distortions across all published crystal structures .
Later, through synthesizing organosilane probes and measuring intermolecular NOESY spectra in DPC micelles, Wang et al. (2011a) also mapped the drug binding site at the N-terminal lumen of A/M2 near Val27. Recent high-resolution X-ray crystal structures of M2 in complex with adamantanes and other M2 channel blockers published by DeGrado lab also unambiguously recognized the intra-luminal binding site (Thomaston et al., 2018; . Taken together, these follow-up studies support that amantadine inhibits IAV M2 by plugging the pore ( Figure 5A ).
More recently, the binding affinities of the R and S enantiomers of rimantadine to M2 have also been questioned. Surprisingly, by comparing the isotropic chemical shift changes of rimantadine's two enantiomers through ssNMR experiments and molecular dynamics simulations, Wright et al. (2016) proposed that the 2-R enantiomer may bind to full-length M2 differentially and with a higher affinity relative to the 2-S enantiomer. However, a subsequent series of in vitro and cellular assays, electrophysiology experiments, and molecular dynamics simulations indicated that both enantiomers exhibited similar channel blockade in TEVC experiments, comparable antiviral activity in plaque assays, and similar free energies of binding in isothermal titration calorimetry and computational simulations (Drakopoulos et al., 2017) . The latter observations are also consistent with experimental data showing similar efficacy of these two enantiomers in mice (Aldrich et al., 1971) .
In addition to Ser31Asn (and further consistent with the pore-binding model described above), the mutations Leu26Phe, Val27Ala, Ala30Thr and Gly34Glu also confer adamantane resistance (Bright et al., 2006; Cady et al., 2009a Cady et al., , 2009b . Large-scale sequence analyses of circulating IAV strains indicate that Leu26Phe, Val27Ala and Ser31Asn are the three major adamantane-resistance mutations found in transmissible viruses (Furuse et al., 2009a (Furuse et al., , 2009b . Among these, Ser31Asn is the prevalent mutation in more than 95% of adamantane-resistant strains (Dong et al., 2015; Nelson et al., 2009) , although a recent study reported a potential rise in the frequency of virus strains containing the Val27Ala and Ser31Asn double mutations (Durrant et al., 2014) . A recent crystallography study reported that elimination of hydrophobic contacts that are essential for adamantane drug binding is the underlying cause behind adamantane resistance to Val27Ala mutant (Thomaston et al., 2020) . In contrast to the Val27Ala single mutation that results in complete resistance to amantadine, M2 sequences containing Ser31Asn can still be inhibited by amantadine in vitro, albeit with approximately 200-fold higher concentrations in TEVC experiments and plaque reduction assays (PRAs) and substantially higher than what is therapeutically-achievable (Wang et al., 2013a) . Notably, the Ser31Asn mutation has been isolated in curated virus strains that were in circulation before use of adamantanes, and so the prevalence of the Ser31Asn mutation in the wild may not be exclusively the result of drug selection pressure. This contrasts with mutations such as Val27Ala which appear more likely than Ser31Asn to have evolved as a result of drug selection pressure (Furuse et al., 2009a (Furuse et al., , 2009b .
The effects of the Ser31Asn mutation on M2 pore size and proton conduction have been elucidated in exquisite detail. For example, the solution NMR structure (PDB: 2RLF; Schnell and Chou, 2008) presents Ser31 between two alpha-helices, and its mutation to the larger asparagine expands the pore by creating a kink in each monomer which would be expected to result in higher conductance. In contrast, the crystal structure (PDB: 3C9J; Stouffer et al., 2008) shows Ser31 facing the pore, where mutation to asparagine decreases the pore size and would be expected to reduce conductance.
However, Holsinger et al. (1994) did not observe a difference in the rate of proton conduction due to the Ser31Asn mutation as measured by single electrode electrophysiology recordings. This result was further confirmed by Balannik et al. (2010) in two-electrode voltage clamp assay in which S31N mutant had indistinguishable specific conductance as M2 WT. Regardless, being located at a pore lining residue, the Ser31Asn mutation is likely to alter the diameter as well as the polarity and dynamics of the channel pore, resulting in changes between the observed interactions of the Ser31Asn channel and amantadine when compared to WT M2 (Gleed et al., 2015) . Interestingly, in the solved X-ray crystal structure for M2
containing the Ser31Asn mutation (PDB: 5C02; (Thomaston and DeGrado, 2016; Thomaston et al., 2018;  2019), in the absence of a drug molecule in the pore and in the Inward open state, Asn31 was found to face the pore and was stabilized by H-bonds formed with the carbonyl groups of neighboring Asn31
( Figure 3A ). This arrangement sterically constricts the adamantane binding site in the pore. In contrast, in the Inward closed state, Asn31 was tucked away from the centre of the pore and stabilized by forming Hbonds with carbonyls at the monomer-monomer interface, thereby twisting the helices and narrowing the pore near the binding site ( Figure 3B) . Alternatively, using a solution NMR structure demonstrated that the Asn31 carboxamide could be stabilized in a pore-facing conformation through a bidentate interaction with the M2-S31N inhibitor M2WJ332 (compound 4; Table 2 ; Figure 6B ).
These observations further emphasize that the orientation of Asn31 in the pore is dependent on the conformational state of the protein and/or the presence of interacting drug molecule in the lumen of the pore.
An extensive combination of molecular dynamics simulations (Khurana et al., 2011; Wang et al., 2011a ), X-ray crystallography (Acharya et al., 2010; Stouffer et al., 2008) , and NMR spectroscopy Wang et al., 2009; Wang et al., 2011a; Wang et al., 2013a) along with iterative cycles of medicinal chemistry, electrophysiological testing, and antiviral assaying have led to the discovery of several classes of compounds that inhibit at least one of the three major drug-resistant mutations (Leu26Phe, Val27Ala, or Ser31Asn) with efficacies comparable or better than amantadine against WT M2. While several other compounds are reported throughout the literature to inhibit M2 Ser31Asncontaining viruses, the mechanism of action of many of them is not confirmed to be mediated through M2 as many of these compounds were not tested in M2-specific assays. The most promising compounds have therefore been validated by both antiviral and M2 conductance assays, the vast majority of which have been assessed by TEVC. These "second-generation" inhibitors of drug-resistant M2 can be broadly categorized in two main groups: adamantanes (Table 2 ) and a series of chemically diverse non-adamantane molecules (Table 3) . While numerous compounds are reported by many groups, in addition to those cited in Tables 2 and 3, we have highlighted particular compounds with electrophysiology- based IC 50 s and PRA-based EC 50 s at low or sub-micromolar concentrations, which approximate the activities of amantadine and rimantadine in these assays with wild-type M2. Clearly, the efficacy of a given inhibitor as measured by TEVC and PRA does not necessarily translate to efficacy in vivo. However, a subset of these inhibitors has recently advanced to preclinical and animal efficacy studies, where promising results are reported. Additional compounds that inhibit WT M2, in addition to early but weaker inhibitors of M2 Val27Ala and Ser31Asn, have been reviewed elsewhere (Duque et al., 2011; Wang et al., 2015) .
Among the many molecules synthesized and tested by the DeGrado group, compounds 3 and 4 Leu26Ile, which conferred drug resistance. This mutation was also co-observed with an occasional Asn31Ser mutation which reverted back to Asn31 upon drug withdrawal. In contrast, passaging with the dual inhibitor compound 8 resulted in Asn31Asp and Ile32Thr mutations which also reverted after drug withdrawal (Ma et al., 2016) . Both sets of mutations were confirmed to be resistant to their respective selecting drugs by TEVC. to Ser31Asn and Leu26Ile resulted in substantially reduced M2 proton conductance and viral replication.
These observations may suggest that the viable evolutionary space of M2 in response to selection with these compounds may be limited.
In further M2 resistance studies, Compound 16 was also reported as a potent inhibitor of both M2
Ser31Asn by TEVC (47.9% inhibition at 100 µM, although its proposed slow-binding kinetics may underestimate its efficacy) and in vitro virus replication (EC 50 as low as 0. Interestingly, Leu46Pro did not significantly change proton conductance or amantadine sensitivity on its own in WT M2. However, in the presence of Ser31Asn, the Leu46Pro mutation rendered compounds 14-16 ineffective in TEVC experiments. Through molecular modelling and molecular dynamics simulations, the authors proposed that Leu46Pro, despite being located outside of the canonical M2 binding sites of adamantanes, could affect the size of the M2 pore where compounds 14-16 were proposed to interact (Musharrafieh et al., 2019) . Taken together, these results suggest that when compared to amantadine and WT M2, the genetic barrier for drug resistance is likely to be higher for adamantane-derived compounds that target M2 Ser31Asn, at least in vitro.
The preclinical and in vivo potential of M2 Ser31Asn-inhibiting adamantane derivatives are now beginning to be elucidated. For example, Compounds 5, 8, 10, and 16 have been reported to inhibited virus strains with resistance to the licensed neuramindase inhibitor oseltamivir (Hu et al., 2017a; Ma et al., 2016; Wang et al., 2018) . In oseltamivir-sensitive viruses, 5 and 16 also synergized with oseltamivir, raising the possibility of combination therapies where lower doses of drugs can be used to minimize the risk of toxicities without sacrificing antiviral efficacy (Ma et al., 2016; Wang et al., 2018) . Compound 16 also exhibited favorable in vitro pharmacokinetic properties such as half-life of at least 145 minutes in mouse and human liver microsome stability assays. It also exhibited good cellular permeability in Caco-2 cells (~28 x 10 -6 cm/s) and did not inhibit a panel of five CYP enzymes. . Additional M2
Ser31Asn adamantane-derived inhibitors have shown similar supportive preclinical results, indicating that these properties are fairly common for this chemical class . Furthermore, injection of mice with the dual-inhibitor compound 9 at up to 100 mg/kg/day resulted in no changes in body weight. Importantly, at up to 100 mg/kg/day, compound 9 also rescued mice from lethal infection of viruses containing either WT M2 or M2 Val27Ala, thereby demonstrating in vivo efficacy (Hu et al., 2017a) . While initial results are encouraging, in vivo results for most adamantane derivatives remain limited and await further study.
Several non-adamantane chemical scaffolds have also been successfully explored for their ability to block adamantane-resistant M2. Examples of non-adamantanes that inhibit drug-resistant M2 are shown in Table 3 .
A polycyclic pyrrolidine (17) In a separate endeavor, derivatives of spiranamine (21) were also explored Wang et al., 2011a) . For example, a silaspirane derivative (22) exhibits activity against both WT and Val27Ala with 95% and 68% inhibition at 100 µM in TEVC experiments. (Wang et al., 2011b) .
Another notably explored chemical class is exemplified by hexamethylene amiloride (HMA, 23) , an amiloride-based compound which is reported to block viroporins and/or replication of IAV, hepatitis C virus, HIV-1, Dengue virus, and severe acute respiratory syndrome virus (SARS-CoV; Gazina and Petrou, 2012; Pervushin et al., 2009; Premkumar et al., 2004) . However, HMA has several off-target effects and is cytotoxic at low micromolar concentrations (Jalily et al., 2016; Premkumar et al., 2004) . Recently, a series of HMA derivatives were designed through iterative medicinal chemistry and electrophysiological testing approaches (Jalily et al., 2016) . Among these compounds, 24 exhibited activity against WT M2 in single electrode voltage clamp (SEVC) experiments, which was comparable to amantadine in these studies (IC 50 0.2 μM vs 0.6 μM), but was not obviously toxic to cells. While 24 was found to be active against WT M2 only, an elongated derivative (25) exhibited dual-inhibitory effects against WT and Ser31Asn M2 in SEVC (respective IC 50 s = 0.6 µM and 4.4 µM) and PRA (respective EC 50 s = 40 µM and 18 µM). Additionally compound 26, although inhibiting WT M2 with lower efficacy (20% inhibition at 100 µM) than 25 in SEVC, was more effective at inhibiting viruses encoding WT and Ser31Asn M2 in PRA (EC 50 s = 6.9 and 1.5 µM, respectively). Preliminary data indicate that 23 exhibits stability in human microsome assays (t 1/2 = 70 minutes) and mouse plasma following injection in vivo (t 1/2 = 60 minutes).
Moreover, injection of compound 23 at 2 mg/kg did not affect the monitored ECG or hemodynamic parameters in mice, and 200 µM of cumulative dosing of 23 over a course of 3 hours did not cause obvious cardiac toxicity (P. Jalily, I. Tietjen, M. Pourrier, and D. Fedida, unpublished data) .
The inhibitory effect of Cu 2+ on M2 conductance was initially studied by Gandhi et al. (1999) ,
where they showed that the inhibition is bi-directional across the membrane and dependent on the presence of His37. The WT protein also exhibited high specificity for Cu 2 + and was only partially inhibited by high concentrations (1 mM) of bivalent nickel, zinc, or platinum (Gandhi et al., 1999) . More recently, Gordon et al. (2017) reported the discovery of divalent copper complexes such as compound 27 as nonselective M2 inhibitors. The authors proposed that the mechanism of action of these copper complexes is independent of mutation at Leu26, Val27, or Ser31 but is instead dependent on the conduction moiety, His37, as no inhibition was observed with 27 against His37Ala mutant. Compound 27 was able to inhibit 80% of M2 proton currents at 100 µM as measured by TEVC and had an EC 50 of 0.7 μM against A/CA/07/2009 encoding M2 Ser31Asn in PRA.
Finally, Jang et al. (2018) reported that the monovalent ionophore salinomycin (28) was able to counteract the proton conductance function of both influenza A and B in vitro. However, it indirectly inhibits M2 channel function by preventing cellular endosome acidification, similar to the mechanism of chloroquine. Salinomycin inhibited proton conduction by both M2 WT and Ser31Asn incorporated into virus-like particles (54% and 72% vs. WT and N31 at 100 uM), in addition to proton conduction by the highly divergent M2 channel of influenza B (~60%). Salinomycin also synergized with oseltamivir in vitro.
However, while salinomycin on its own exhibited limited antiviral activity in vivo; it did rescue mice treated with a sub-optimal concentration of oseltamivir and lethally-infected with oseltamivir sensitive or resistant viruses. (Jang et al., 2018) .
The two-electrode voltage clamp assay remains the most effective assay for M2 ion channel studies, as it is less technically demanding and more likely to be replicated across independent laboratory groups when compared to SEVC. In general, there is a liner correlation between the percentage channel blockage from TEVC and the antiviral activity (Li et al., 2017) . However, we also note that the efficacies of certain M2 inhibitors as measured by TEVC and PRA can be discordant. However, kinetic measurements are labor intense and are often reserved as a secondary assay to characterize lead candidates that have already shown potent antiviral activity such as compound 16.
Nevertheless, it is understandable that the lack of a straight linear correlation between the results from the in vitro TEVC or SEVC assay and the cellular CPE or PRA assay might also be additionally due to other factors such as a compound's membrane permeability, virus strain differences, or other off-target effects. However, this is not a specific issue related to M2 channel blockers per se, as other drug candidates commonly face this challenge as well. In some cases, discordance also extends to M2 proteins and viruses containing different mutations. For example, compound 9 inhibits M2 Val27Ala with lower IC 50 s than M2 WT (0.3 and 18 µM , respectively), while the EC 50 s in PRA are the opposite (i.e.
1.8 and 0.3 µM, respectively). Similar results are also seen for compounds 7, 10, and 20 and could reflect, for example, differences in virus strains used and/or subtle changes in the binding kinetics of these compounds against different M2 forms. Thus, as with other drug development efforts, interpretation of specific results for individual compounds must be performed carefully and on a caseby-case basis.
The advent and improvement of electrophysiological techniques have substantially advanced our ability to directly measure the membrane currents from single cells expressing M2 or other viroporins of interest. The optimization of methods including TEVC and SEVC has also allowed us to study the fundamental biophysical properties of M2 variants and how small molecules engage and inhibit these channels. However, the inherent limitations of traditional electrophysiology such as low throughput, technical demands, and inaccessibility of the technique to many research groups have largely restricted screening and discovery of novel M2 inhibitors to a few specialized laboratories .
Toward addressing these limitations, recently developed systems with the capacity or potential of automated electrophysiology screening could substantially reduce the time, complexity and cost associated with manual traditional patch clamp experiments, particularly in the context of SEVC.
Notably, the companies Sophion (QPatch), Nanion (Patchliner) and Fluxion (IonFlux) have developed automated systems where screening of tens or hundreds of compounds could, in principle, be performed by SEVC in one day (Priest et al., 2017) .
To enable high-throughput screening of M2 inhibitors with ready availability to more research groups, S. cerevisiae yeast strains have also been developed which are induced to express WT or adamantane-resistant M2 proteins in the presence of galactose. In these assays, M2 expression inhibits yeast growth, as measured by culture turbidity, which in turn is restored by administration of M2 inhibitors. As shown by Balgi and Roberge (2009) , amantadine restored the growth of yeast expressing WT M2 from 27% of control to 95% at 1 µM. This assay was further optimized for high throughput screening: for example, the authors subsequently screened ~250,000 compounds and identified 21 new and potent inhibitors of WT M2 (Balgi et al., 2013) . These reports demonstrate that growth restoration assays are a sensitive, economical and technically simple technique for high throughput screening for inhibitors of M2 and presumably other viroporins. A similar expression system using E. coli has also been used to assess the properties of random M2 mutations (Santner et al., 2018b) , although results from these assays do not consistently agree with results obtained by TEVC (Musharrafieh et al., 2019; Santner et al., 2018a) .
In a separate approach, M2 was incorporated into virus-like particles, and proton conductance was measured using a potentiometric fluorescent dye readout (Sulli et al., 2013) . Using this technology, the authors successfully screened 107,572 compounds in 384-well format and discovered 19 new M2
WT-specific inhibitors with IC 50 values ranging from 140 nM to 13 µM (Sulli et al., 2013) , although no inhibitor was identified to target the drug resistant mutants of M2.
When designing drugs against viral targets, an inherent challenge is the higher degree of structural flexibility exhibited by viral proteins in comparison to many prokaryotic and eukaryotic targets (Das et al., 2010; Fischer and Hsu, 2011 ). Yet another challenge imposed against rational drug design is the dependence of such highly flexible proteins on their surrounding environment, which in turn affects their function. In contrast to thermostable proteins that encompass tight hydrophobic cores, viral proteins are frequently loosely packed, resulting not only in a smaller difference in energy levels between folded and unfolded states, but also a reduced contribution of random mutations or interactions with chemical inhibitors to overall stability (Paciaroni et al., 2002) . This equips viruses with enormous adaptive mechanisms to buffer the deleterious effects of random mutations; even more in the case of RNA viruses which intrinsically mutate faster than DNA viruses (Tokuriki et al., 2009 ). These challenges also apply to M2, where until recently the lack of high-resolution structures had impeded the advancement of rational drug design. Other obstacles that have prevented the successful design of a universal M2 inhibitor include the constricted drug binding site, technical difficulties in effective measurement of drug inhibition, and absence of reliable protocols to assess protein drug interactions.
To overcome these challenges, computation now plays a critical role in the field of rational drug design, as computers now have sufficient processing power to run virtual screening of large chemical libraries against multiple targets. While these strategies can identify multiple antiviral leads including putative viroporin inhibitors (Radosevic et al., 2019; Tietjen et al., 2015) , the likelihood of false-positive hits necessitates their validation by experimental techniques. Another role of computation is now the extended ability to run complex molecular dynamics simulations in shorter times and for larger complexes to predict and visualize the interaction of viral protein domains and putative inhibitors, complexes of proteins, and simulation of complexes of viral proteins and cellular factors.
Taken together, several emerging technologies are now available to design and assess the efficacy and activity of novel M2 inhibitors. However, it is important to note that both TEVC and PRA remain "gold standards" for these studies and should be applied as secondary assays to rule out false positives obtained from higher-throughput methods.
The next influenza virus pandemic, albeit unpredictable, is not a matter of "if" but "when." New therapeutics, especially those that are distinct from current antivirals and with the ability to inhibit existing drug-resistant viral strains, may be essential toward mitigating the next inevitable outbreak. The M2 viroporin, a proton-dependent proton channel required for virus entry and egress, has historically been an effective antiviral target for drugs like amantadine and rimantadine. However, mutations in M2, particularly lining the pore where adamantanes interact, are now widespread and have rendered these therapies ineffective. Over the past decade, new inhibitors of drug-resistant M2 consisting of both adamantane derivatives and new chemical scaffolds have been found that inhibit M2 ion conduction, as demonstrated using both electrophysiological techniques in vitro influenza replication assays. The most promising leads also inhibit multiple adamantine-sensitive and resistant forms of M2 (e.g., compounds 7-10, 12, 17-18, 22, 25), inhibit viruses with major resistance to other IAV antivirals like oseltamivir (5, 8, 10, 16) and/or synergize with oseltamivir (5, 16, 28), exhibit high genetic barriers to resistance following long-term in vitro passaging (5, 8, 14-16) , have supportive preclinical parameters such as good stability and low toxicity (9, 16, 23) , and rescue mice from lethal infection (9). Going forward, further discovery of new chemical scaffolds and/or optimization of existing leads by medicinal chemistry may be enabled by emerging technologies to screen for and monitor M2 conductance. In addition, demonstration of clinical efficacy of new chemical leads remains to be achieved. The coming decade will likely show whether new M2 inhibitors can significantly add to the evolving armamentarium of antivirals to combat current and emerging seasonal and pandemic influenza strains.
Funding was provided by the Canadian Institutes for Health Research (CIHR) [grant number PJT-153057] (to IT). The influenza antiviral work in the Wang laboratory was supported by NIH grants AI119187 and AI144887. The authors declare no conflicts of interest. Waters are shown as spheres (Thomaston et al., 2017) . 