TY - JOUR
AU1 - Bezrukov, Sergey M.
AU2 - Nestorovich, Ekaterina M.
AB - Emergent rational drug design techniques explore individual properties of target biomolecules, small and macromolecule drug candidates, and the physical forces governing their interactions. In this minireview, we focus on the single-molecule biophysical studies of channel-forming bacterial toxins that suggest new approaches for their inhibition. We discuss several examples of blockage of bacterial pore-forming and AB-type toxins by the tailor-made compounds. In the concluding remarks, the most effective rationally designed pore-blocking antitoxins are compared with the small-molecule inhibitors of ion-selective channels of neurophysiology. rational drug design, structure-based drug discovery, channel-blocking antitoxins, single molecule/protein interaction, multivalent interactions, physical forces of efficient blockage INTRODUCTION A widespread mechanism of virulence of multiple organisms, such as bacteria, plants, mushrooms, sea anemones, earthworms and even mammals, is the secretion of proteins that form pores in the target cell membranes. Alouf (2001) had estimated that from the 325 bacterial toxins identified at that point in time, at least 115 (35%) attacked human and/or animal cells by damaging the cytoplasmic phospholipid bilayer membranes. The majority of these toxins act by forming transmembrane pores which compromise the plasma membrane barrier function by allowing for the flow of ions down their electrochemical gradients and for the consequent reduction in the membrane potential (Bernheimer 1996). To reflect the specific mechanism of their action, these membrane-damaging toxins can be referred to as the membrane-perforating toxins. Another big group of bacterial exotoxins, the so-called AB-type toxins act in the cytosol, enzymatically modifying their specific intracellular substrates (Geny and Popoff 2006). Two forms of the AB-type toxins are distinguished. The classical AB-type toxins are secreted as single-chain proteins containing at least two functionally different domains, the receptor binding B domain and the active/enzymatic A domain. The binary AB-type toxins are formed by two (or three, in the case of anthrax toxin; Collier 2009) non-linked proteins, a binding B subunit and an active A subunit (Barth et al. 2004). Apart from the cell binding, the B domains/subunits are also believed to form channels in endosomal membrane that mediate intracellular transport of the toxin's A domains/subunits (Blaustein et al. 1989; Schmid et al. 1994; Knapp et al. 2002). Therefore, membrane-perforating and AB-type bacterial toxins, although both utilizing pore- or channel-forming proteins, have very distinct mechanisms of cell intoxication. Nevertheless, for the practical purpose of this article we will use the terms ‘pore’ and ‘channel’ interchangeably. Regardless of the action mechanism, the water-filled channels formed by bacterial toxins in the host plasma or organelle membranes represent an interesting and well-defined target to explore. The idea of toxin inhibition by channel blockage is suggested by nature that created different small-molecule and peptide-based neurotoxins specifically targeting ion channels of excitable cells. Ion channels are universally recognized targets of many pharmaceutical agents. Thus, approximately 13% of marketed drugs modify activity of ligand- or voltage-gated ion channels (Overington, Al-Lazikani and Hopkins 2006) producing approximately US$12 billion in worldwide sales (Wickenden, Priest and Erdemli 2012). Several viroporins, primarily the M2 channel from the viral envelope of influenza A virus, have also been investigated as feasible targets for the development of pore-blocking drug molecules (Scott and Griffin 2015). Relatively recently, similar approaches were developed to design antidotes to the poisonous action of certain bacterial toxins by inhibiting pore-forming toxins with small molecule or multivalent pore blockers (reviewed in Bezrukov and Nestorovich 2015). The progress in the field is supported by theoretical studies that include both analytical theories of channel transport based on the concept of a potential of mean force acting on the molecule in the channel (reviewed in Berezhkovskii and Bezrukov 2014) and examinations of the particular physical interactions that contribute to this potential. The examples discussed below highlight the importance of attractive Coulomb forces between the cationic blockers and predominantly anionic local environment of the target pores as well as different kinds of short-range interactions. In this minireview written for the thematic issue ‘Bacterial toxins – action and use’ in connection with the 17th European Workshop on Bacterial Proteins Toxins, we focus on the existing efforts to design inhibitors that directly block channel-forming bacterial toxins. In the concluding part, we compare the most active antitoxins with the leading small molecule blockers discovered previously for inhibition of ion-selective channels of excitable cells. INHIBITING MEMBRANE-PERFORATING BACTERIAL TOXINS The idea of disabling the membrane-perforating bacterial toxins via direct physical obstruction of the ‘virulent’ pores that these toxins form in the host membranes is very straightforward and, therefore, easy to appreciate. However, the number of such studies remains surprisingly limited. At the same time, several pore-forming bacterial toxins, such as α-hemolysin of Staphylococcus aureus and aerolysin of Aeromonas hydrophila, were extensively studied in model lipid bilayer membranes in the light of their potential nanobiotechnological applications reviewed in Majd et al. (2010) and Kasianowicz et al. (2015). These applications are mostly based on the molecular sensing capabilities of the pore-forming toxins, where single protein channels are used as detectors in the stochastic resistive-pulse technique (Bezrukov, Vodyanoy and Parsegian 1994; Gu et al. 1999). Because the principles of the molecular sensing nanopore technology can be applied in rational design of the effective pore blockers in a quite straightforward way, we anticipate that a number of new original developments would soon break this backlog. Below we briefly review the main studies where the idea of obstructing a toxin pore by blocking was discussed and tested. Staphylococcal membrane perforating toxins The exotoxins secreted by S. aureus (Van de Velde 1894; Denys and Van de Velde 1895; Bernheimer and Schwartz 1963; Chesbro et al. 1965; Donahue and Baldwin 1966; Arbuthnott, Freer and Bernheimer 1967) have recently prompted special attention (reviewed in Prevost et al. 2015) because of the widespread of a multidrug-resistant strain of the bacterium, the so-called methicillin-resistant S. aureus, or MRSA (Otto 2010, 2012) responsible for several difficult-to-treat infections in humans. Amongst multiple toxins, S. aureus secretes several that form pores in host membranes, which include α-hemolysin (αHL), γ-hemolysin and leukocidins. After being secreted as a water-soluble monomeric polypeptide, αHL binds to a host cell membrane forming a heptameric water-filled pore (reviewed in Berube and Wardenburg 2013). More than 30 years ago, Krasilnikov, Ternovsky and Tashmukhamedov (1981) successfully reconstituted αHL into a model lipid bilayer membrane and reported formation of large, slightly anion selective pores of approximately 1 nS conductance (1 M KCl, room temperature). Fifteen years later, a 1.9-Å resolution crystal structure of the αHL channel has shown a hollow mushroom-like 100 Å × 100 Å (length by diameter) heptamer consisting of the stem, cap and rim domains and two constriction zones with radii of 0.9 and 0.6–0.7 nm (Song et al. 1996). The αHL molecular sensing properties, stability and structural robustness have determined its wide usage for the stochastic resistive-pulse sensing of different types of molecules, ranging from small molecules to macromolecules and polymers (recently reviewed in Gurnev and Nestorovich 2014). Though these applications are not based on the αHL toxicity, they undoubtedly generate a large body of knowledge on the pore/molecule binding reaction kinetics, which can be helpful in intelligent drug design. For example, the ability of αHL to integrate β-cyclodextrin (βCD) molecules has been explored (Gu et al. 1999). It was shown that a single heptameric αHL pore equipped with a non-covalently bound 7-fold symmetrical βCD adapter is able to mediate αHL's ion current blocking by a number of organic analytes, such as adamantanamine hydrochloride, adamantine carboxylic acid, promethazine and imipramine. More recently, βCD's ability to obstruct the αHL lumen was utilized in a targeted design of αHL multivalent inhibitors (Table 1) that have the same structural symmetry as the target pore (Karginov et al. 2006b; Ragle and Bubeck Wardenburg 2009; Yannakopoulou et al. 2011). One of the newly designed seven positively charged hepta-6-substituted β-cyclodextrin derivatives, IB201, effectively blocked ion current through the αHL pores in the model bilayer membranes and protected rabbit red blood cells from αHL-induced hemolysis. In a murine model of S. aureus infection, IB201 prevented the αHL-mediated alveolar epithelial cell lysis and mortality associated with pneumonia. The single-pore in vitro experiments have provided important details of the mechanism of αHL inhibition by IB201. Within the time period of the experiment (several hours), αHL interaction with IB201 was irreversible. Application of the IB201 blocker to the cis side of the membrane, which corresponds to the cap side of the heptamer, prompted the αHL channel to switch to a blocked state with the residual conductance ranging between 1% and 15% of the open pore conductance. The αHL/IB201 binding reaction was shown to depend on the transmembrane voltage; in particular, the binding became stronger when trans-negative voltages were applied indicating that the cationic IB201 was likely pulled into the αHL lumen by the electric field. However, because only a very small number of the tested 7+βCD inhibitors showed the effect similar to IB201, it is too early to speculate on the particular molecular interactions involved in the αHL pore inhibition. Table 1. α-, β-, and γ-CD blockers of αHL (Yannakopoulou et al. 2011).       Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  1  α    >5000  >100  2  β    ∼50  3.3 ± 2.3  3  γ    >5000  >100        Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  1  α    >5000  >100  2  β    ∼50  3.3 ± 2.3  3  γ    >5000  >100  View Large Table 1. α-, β-, and γ-CD blockers of αHL (Yannakopoulou et al. 2011).       Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  1  α    >5000  >100  2  β    ∼50  3.3 ± 2.3  3  γ    >5000  >100        Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  1  α    >5000  >100  2  β    ∼50  3.3 ± 2.3  3  γ    >5000  >100  View Large In contrast to the homogeneous αHL oligomers, a family of bicomponent Staphylococcal cytolysins (reviewed in Alonzo and Torres 2014), γ-hemolysin (Hlg), leukocidin (Luk) and Panton-Valentine leukocidin (PVL) are formed via interaction of two distinct class F and class S polypeptides (Kaneko and Kamio 2004; Yamashita et al. 2011). In the model bilayer membranes, the bi-component octameric leukocidin, Luk, was reported to form large, approximately 2.5 nS in 1 M KCl, water-filled pores (Miles et al. 2001; Miles, Movileanu and Bayley 2002; Miles, Jayasinghe and Bayley 2006) making Luk an attractive candidate for the single-pore molecular sensing applications. In a recent study, a library of inhibitors potentially compatible with the leukocidin pore lumen was created and tested (Laventie et al. 2013). Among the compounds of different hydrodynamic radii, shapes, symmetries and charges, p-sulfonato-calix[n]arenes (SCns) SC6 and SC8 had an explicit antitoxin effect when tested on RBCs and liposomes. SC8 was shown to reduce the inflammation induced by 600 ng of PVL in rabbit eyes in a rabbit non-infectious model. Remarkably, despite the authors’ choice of the antitoxin candidates based on their potential ability to block the upper ring of the leukocidin pores, the mechanism of action seems to be somewhat different. Most probably, the antitoxins prevented the binding of class S proteins to membranes, thus inhibiting pore formation. Epsilon toxin of Clostridium perfringens The activated epsilon toxin (ETX) (recently reviewed in Stiles et al. 2013; Wioland et al. 2013; Alves et al. 2014), the major virulence factor produced by B and D types of C. perfringens, represents one of the most potent bacterial toxins after botulinum and tetanus neurotoxins. After being secreted as a low active prototoxin, ETX is activated by the proteolytic removal of 11 or 13 N-terminal and 29 C-terminal amino acid residues. ETX then assembles into heptameric prepores, later transformed into β-barrel heptameric transmembrane pores. In the model bilayers, ETX forms wide, slightly anion-selective pores with a single-channel conductance ranging between 440 and 640 pS in 1 M KCl (Petit et al. 2001; Knapp et al. 2009). Polyethylene glycol (PEG) partitioning experiments (Nestorovich, Karginov and Bezrukov 2010) suggest that the channel is asymmetrical with the trans opening of the ETX pore being wider (approximately 1.0 nm) compared to its cis opening (approximately 0.4 nm). The charge distribution along the ETX pore lumen was investigated by measuring ETX ion selectivity in the oppositely directed gradients of KCl aqueous solutions. Because the selectivity was ‘salted-out’ more easily from the wide trans opening of the ETX pore, the authors suggested that the positively charged amino acid residues responsible for the anionic selectivity of the ETX pore are not located in the narrowest part of the channel, but rather shifted toward the trans opening of the pore. However, even with the reported insights into the ETX pore structural features, effective small-molecule pore blockers are yet to be designed. The only study searching for ETX pore inhibitors allowed to identify three moderately active ETX inhibitors: N-cycloalkylbenzamide, furo[2,3-b]quinoline and 6H- anthra[1,9-cd]isoxazol from library of 151 616 compounds (Lewis, Weaver and McClain 2010). Because these three compounds did not inhibit ETX cell binding or oligomerization, the authors suggested that they might have acted by blocking the ETX pore lumen. INHIBITING BINARY BACTERIAL TOXINS The existing efforts to design the pore-forming bacterial toxin blockers primarily focus on targeting the binary bacterial toxins, including anthrax toxin of Bacillus anthracis and a family of related clostridial binary toxins (Fig. 1A). Recent progress in understanding the anthrax toxin intracellular uptake and action mechanism is significant but is beyond the limits of this minireview (reviewed in Young and Collier 2007; Collier 2009; Thoren and Krantz 2011; Feld, Brown and Krantz 2012; Liu et al. 2013, 2015; Moayeri et al. 2015). Briefly, the tripartite anthrax toxin is formed by three individually non-toxic proteins that assemble at the host cell surface to form toxin complexes responsible for anthrax symptoms and lethality. Those proteins are two enzymatic A components: lethal factor (LF), a Zn-metalloprotease that cleaves MAP kinase kinases inducing the cell death of macrophages, and edema factor (EF), a Ca2+- and calmodulin-activated adenylyl cyclase, and one translocation/binding B-component (83 kDa protective antigen, or PA). PA, LF and EF associate to form ‘classic’ AB-type binary toxins: lethal toxin (LF + PA) and edema toxin (EF + PA). The anthrax toxin's multistep internalization involves PA binding to the cellular CMG2 and TEM8 receptors, proteolytic cleavage to a 63-kDa form (PA63), oligomerization to create heptameric (Petosa et al. 1997) and/or octameric (Kintzer et al. 2009) ring-shaped prepores offering three (Mogridge, Cunningham and Collier 2002) or four (Kintzer et al. 2009) binding sites for LF and EF, and LF and/or EF biding followed by endocytosis of the oligomeric anthrax toxin complexes. The acidic environment of the endosomes causes conformational changes of the PA63 prepore leading to the oligomer insertion into the endosomal membrane, where it forms a cation-selective ion channel (Blaustein et al. 1989). Recent 2.9-Å cryoelectron microscopy reconstruction of the PA63 pore reveals an elongated ‘flower-on-a-stem’ structure ranging in external diameter from 27 Å to approximately 160 Å with 75-Å long bud and 105-Å long stem (Jiang et al. 2015). PA63 channel then acts as a translocase unfolding and translocating LF and EF inside the cell (Zhang et al. 2004). The endosomal membrane proton gradient (pHendosome < pHcytosol) (Krantz, Finkelstein and Collier 2006), the PA63's phenylalanine residue at position 427 (ϕ-clamp) (Krantz et al. 2005) and the substrate binding α-clamp (Feld et al. 2010; Brown, Thoren and Krantz 2015) were reported as important factors influencing the translocation process. Once in the cytosol, LF and EF perform their catalytic actions (Liu et al. 2013). Figure 1. View largeDownload slide Blocking bacterial toxins at the single-channel level. (A) Illustration of the idea: the binding component of a binary bacterial toxin could be effectively inhibited by an antitoxin blocking the pore. (B) Small-molecule, cyclodextrin- and dendrimer-based inhibitors of the binary bacterial toxins. (C) ( Left) Conductance of PA63, C2IIa and Ib channels in the absence ( top) and presence of AmPrβCD (middle) (Table 3, #3) and AMBnTβCD (bottom) (Table 3, #13). (Right) The PA63 F427A mutant shows much shorter blockages for both AmPrβCD ( middle) and AMBnTβCD (bottom) blockers. Recordings shown at 1-ms time resolution were taken in 1 M KCl at pH 6 and 50 mV applied voltage. Part (C) is reprinted with permission from Bezrukov et al. (2012). Copyright 2012 Biophysical Society. Figure 1. View largeDownload slide Blocking bacterial toxins at the single-channel level. (A) Illustration of the idea: the binding component of a binary bacterial toxin could be effectively inhibited by an antitoxin blocking the pore. (B) Small-molecule, cyclodextrin- and dendrimer-based inhibitors of the binary bacterial toxins. (C) ( Left) Conductance of PA63, C2IIa and Ib channels in the absence ( top) and presence of AmPrβCD (middle) (Table 3, #3) and AMBnTβCD (bottom) (Table 3, #13). (Right) The PA63 F427A mutant shows much shorter blockages for both AmPrβCD ( middle) and AMBnTβCD (bottom) blockers. Recordings shown at 1-ms time resolution were taken in 1 M KCl at pH 6 and 50 mV applied voltage. Part (C) is reprinted with permission from Bezrukov et al. (2012). Copyright 2012 Biophysical Society. Several binary exotoxins secreted by the pathogenic species of Clostridia (Ohishi and Odagiri 1984; Simpson 1984; Aktories et al. 1986; Stiles and Wilkins 1986; Simpson et al. 1987) are structurally and functionally related to the anthrax toxin (reviewed in Popoff and Bouvet 2009; Barth, Stiles and Popoff 2015; Knapp, Benz and Popoff 2015). Those toxins are C2 toxin of C. botulinum, iota toxin of C. perfringens, CDT toxin of C. difficile and CST toxin of C. spiroforme. Clostridial binary toxins are made of two subunits where the enzymatic A subunit acts through mono-ADP-ribosylation of G-actins and the B subunit is believed to bind and translocate the A subunit into cytosol, similarly to PA63 of the anthrax toxin. The B components of anthrax and clostridial binary toxins have high degrees (from 27% to 38%) of amino acid homology and contain four distinct domains involved in cellular receptor binding, oligomerization, pore formation and A subunit binding (Petosa et al. 1997; Schleberger et al. 2006). Similarly to PA63, the proteolytically activated B subunits form ring-shaped prepore heptamers on the surface of eukaryotic cells or in solution (Barth et al. 2000). The cell-bound C2I/C2IIa, Ia/Ib and CDTa/CDTb complexes are internalized by receptor-mediated endocytosis (Blocker et al. 2001; Stiles et al. 2002; Nagahama et al. 2009; Pust, Barth and Sandvig 2010) and A subunits translocate across the endosomal membranes into the cytosol, possibly using the pores formed by B subunits as translocation pathways (Barth et al. 2000; Bachmeyer et al. 2001; Stiles et al. 2002; Blocker et al. 2003; Gibert et al. 2007). Interestingly, in vitro, PA63 is able to bind and translocate His-tagged C2I, while C2II binds, but does not translocate LF and EF (Kronhardt et al. 2011). In mildly acidic conditions (pH < 6.6), the B components of C2 and iota toxins were reported to form cation-selective ion channels (Schmid et al. 1994; Knapp et al. 2002). Similarly to PA63, the phenylalanine clamp (ϕ-clamp) was found to catalyze the unfolding and translocation of the C2I and Ia components across the membrane (Lang et al. 2008; Neumeyer et al. 2008; Knapp et al. 2015). Small-molecule cationic pore blockers Starting from the pioneering work by Alan Finkelstein et al. with tetraalkylammonium ions (Blaustein, Lea and Finkelstein 1990; Blaustein and Finkelstein 1990a, b), a large number of positively charged molecules were shown to reversibly block the K+ current through PA63 (Table 2), C2IIa and Ib channels in the model membranes (Fig. 1C) and to protect cells from the binary toxin action. Thus, chloroquine was reported to reversibly block the PA63 (KDPA = 0.51 μM in 0.1 M KCl) and C2IIa (KDC2IIa = 10 μM in 0.15 M KCl) channels in vitro and prevent transport of the enzymatic C2I subunit of the C2 toxin in cell assay. A weak inhibition by chloroquine observed with the iota toxins’ B subunit, Ib, in the model lipid bilayers (KDIb = 0.22 mM in 0.1 M KCl) was not sufficient to protect cells from iota toxin induced intoxication (Knapp et al. 2002, 2015). Roland Benz et al. have reported that the binding affinity decreased in the order PA63 > C2IIa ≫ Ib which was explained by a decreasing number of the potential binding sites formed by the negatively charged amino acid residues in the cis entrance of these channels (Bachmeyer et al. 2003; Orlik, Schiffler and Benz 2005; Neumeyer et al. 2008). In general, addition of the blockers to membrane bathing electrolyte solutions resulted in the ion current noise increase with the spectral density of the Lorentzian type that is typical for a simple binding-site model. At that, the binding reaction on-rate, which characterizes frequency of the blockages, was dependent on electrolyte ionic strength indicating the involvement of ion–ion electrostatic interactions. The binding reaction off-rate, which characterizes the residence time of a molecule inside a channel, was primarily determined by the structural features of the blocker molecules. Along with the negatively charged residues in the pore lumen, the ϕ-clamp was investigated as a potential site for binding of the PA63, C2IIa (Neumeyer et al. 2008) and Ib (Knapp et al. 2015) pore inhibitors. When a preselected library of 35 available cationic quaternary ammonium and phosphonium ion compounds was tested to compare their blocking activity against the PA63 channel, the more hydrophobic ones possessed higher inhibitory activity (Krantz et al. 2005). Moreover, ϕ-clamp mutations were reported to profoundly affect the binding affinity of hydrophobic cations, suggesting that the ϕ-clamp acts as the binding site. The ϕ-clamp preferred aromatic moieties by 0.7 kcal mol−1 per aromatic ring with the most effective molecules binding at KD < 1 μM. Importantly, the compounds carrying multiple aromatic rings (3 or 4) were reported to have the nM-range binding affinity toward the wt PA63. It was suggested that the ϕ-clamp could be involved in non-specific hydrophobic interactions with blocker molecules while its negative π-clouds could also stabilize the binding via aromatic-aromatic, π-π and cation-π interactions. This fundamental publication has revealed that the effective inhibitors of the channel-forming subunits of the binary toxins are to be designed focusing not only on the charged residues but also on the ϕ-clamp. Similarly, the ϕ-clamp mutations introduced into C2IIa (F428A, F428D, F428Y and F428W) and Ib (F454A) led to a significant decrease in channel's affinity to chloroquine and its analogs (Neumeyer et al. 2008; Knapp et al. 2015). These observations suggest that the cationic molecules decorated with bulky hydrophobic aromatic groups may represent lead compounds suitable for rational optimization as binary toxin inhibitors. With that, several heterocyclic azolopyridinium salts (Table 2, compounds 10 and 11) were recently shown to block the PA63 and C2IIa subunits in the model bilayer membranes in the low-μM concentrations and to fully protect against toxin action in cell assays while having only negligible cytotoxic effects (Beitzinger et al. 2013; Bronnhuber et al. 2014). The authors reported that the pore/blocker binding reaction on-rates were not significantly influenced by the ligand's structure, being close to that of diffusion-controlled processes. Contrarily, the off-rates varied significantly, increasing with the blocker size and decreasing with the number of aromatic groups. Another interesting site for potential investigation is formed by the α-clamp surface (Feld et al. 2010; Brown, Thoren and Krantz 2015). Brown, Thoren and Krantz (2015) have recently described the α-clamp, a deep amphipathic cleft on the surface of the PA oligomer as the polypeptide binding site relevant for the substrate binding (Feld et al. 2010) and translocation. Though the pore-blocking cationic compounds were shown not to interfere with the enzymatic component binding (Nestorovich et al. 2011; Forstner et al. 2014; Roeder et al. 2014), it would be useful to determine if the α-clamp plays any particular role in the PA63/blocker interaction. Table 2. Small molecule cationic blockers of PA63 channel conductance. Note: only molecules with IC50 ≤ 3 μM are shown, see ref. Bezrukov and Nestorovich (2015) for complete list of the antitoxins. #  Blocker  Structure  IC50  Tetraalkylammonium ions (Krantz et al. 2005), (0.1 M KCl)  1  Tetrapropylammonium    350 ± 10 nM  2  Tetrapentylammonium    2 μM  Small aromatic cationic compounds (Krantz et al. 2005), (0.1 M KCl)  3  Tetraphenylphosphonium    46 ± 2 nM  4  Chloroquine    510 ± 30 nM  5  Quinacrine    60 ± 5 nM  6  Benzyltriphenylphosphonium    110 ± 30 nM  7  Butyltriphenylphosphonium    88 ± 5 nM  8  Isoamyltriphenylphosphonium    35 ± 6 nM  9  Methyltriphenylphosphonium    370 ± 60 nM  Azolopyridinium salts (Beitzinger et al. 2013) (0.15 M KCl)  10  HA 1383    1.3 μM  11  HA 1568    2.7 μM  #  Blocker  Structure  IC50  Tetraalkylammonium ions (Krantz et al. 2005), (0.1 M KCl)  1  Tetrapropylammonium    350 ± 10 nM  2  Tetrapentylammonium    2 μM  Small aromatic cationic compounds (Krantz et al. 2005), (0.1 M KCl)  3  Tetraphenylphosphonium    46 ± 2 nM  4  Chloroquine    510 ± 30 nM  5  Quinacrine    60 ± 5 nM  6  Benzyltriphenylphosphonium    110 ± 30 nM  7  Butyltriphenylphosphonium    88 ± 5 nM  8  Isoamyltriphenylphosphonium    35 ± 6 nM  9  Methyltriphenylphosphonium    370 ± 60 nM  Azolopyridinium salts (Beitzinger et al. 2013) (0.15 M KCl)  10  HA 1383    1.3 μM  11  HA 1568    2.7 μM  View Large Table 2. Small molecule cationic blockers of PA63 channel conductance. Note: only molecules with IC50 ≤ 3 μM are shown, see ref. Bezrukov and Nestorovich (2015) for complete list of the antitoxins. #  Blocker  Structure  IC50  Tetraalkylammonium ions (Krantz et al. 2005), (0.1 M KCl)  1  Tetrapropylammonium    350 ± 10 nM  2  Tetrapentylammonium    2 μM  Small aromatic cationic compounds (Krantz et al. 2005), (0.1 M KCl)  3  Tetraphenylphosphonium    46 ± 2 nM  4  Chloroquine    510 ± 30 nM  5  Quinacrine    60 ± 5 nM  6  Benzyltriphenylphosphonium    110 ± 30 nM  7  Butyltriphenylphosphonium    88 ± 5 nM  8  Isoamyltriphenylphosphonium    35 ± 6 nM  9  Methyltriphenylphosphonium    370 ± 60 nM  Azolopyridinium salts (Beitzinger et al. 2013) (0.15 M KCl)  10  HA 1383    1.3 μM  11  HA 1568    2.7 μM  #  Blocker  Structure  IC50  Tetraalkylammonium ions (Krantz et al. 2005), (0.1 M KCl)  1  Tetrapropylammonium    350 ± 10 nM  2  Tetrapentylammonium    2 μM  Small aromatic cationic compounds (Krantz et al. 2005), (0.1 M KCl)  3  Tetraphenylphosphonium    46 ± 2 nM  4  Chloroquine    510 ± 30 nM  5  Quinacrine    60 ± 5 nM  6  Benzyltriphenylphosphonium    110 ± 30 nM  7  Butyltriphenylphosphonium    88 ± 5 nM  8  Isoamyltriphenylphosphonium    35 ± 6 nM  9  Methyltriphenylphosphonium    370 ± 60 nM  Azolopyridinium salts (Beitzinger et al. 2013) (0.15 M KCl)  10  HA 1383    1.3 μM  11  HA 1568    2.7 μM  View Large Multivalent positively charged blockers One of the modern concepts in designing an efficient blocker molecule is attaching multiple copies of its functional groups onto an appropriate scaffold (Vance et al. 2008; Fasting et al. 2012). A number of bacterial protein toxins including the binary bacterial toxins have recently been successfully blocked by novel synthetic multivalent molecules (Branson and Turnbull 2013). Multivalent compound design includes search for a suitable scaffold to attach the ligands which previously were shown to have some activity against the target (Vance et al. 2009). In the discussed case of binary anthrax and clostridial toxins, these moderately active ligands are the cationic and aromatic functional groups. Cyclodextrins In a number of studies, cyclodextrins, the cyclic oligomers of glucose that have a long and successful history of being employed in the pharmaceutical, agrochemical, environmental, cosmetic and food industries (Davis and Brewster 2004), were investigated as potential scaffolds for multivalent binary toxin blockers (Karginov et al. 2005; Nestorovich et al. 2010, 2011; Roeder et al. 2014). In particular, synthetic tailor-made 7-fold symmetrical β-cyclodextrins (d ≈ 15 Å) carrying seven positively charged amino groups covalently linked to the core by hydrophobic linkers (7+βCD) (Table 3) have shown in vitro, in cell cultures, and in vivo (in the case of the anthrax toxin) activity against four binary toxins (anthrax, C2, iota, and CDT), thus demonstrating a potential for the development of universal broad-spectrum blockers (Karginov 2013). The several effective 7+βCD inhibitors of the anthrax toxin were demonstrated to act by inhibiting PA63 single channel conductance with kinetics following the two-state Markov model. Table 3. Polyvalent cationic cyclodextrin blockers of PA63 ion current and LT cytotoxicity. Note: only selected molecules are shown, see Bezrukov and Nestorovich (2015) for complete list of the antitoxins.       Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  Hepta-6-aminoalkyl β-cyclodextrin derivatives (Karginov et al. 2006a)  1  β  -NH2  140 ± 90  20 ± 9  2  β  -S(CH2)2NH2  3.5 ± 0.9  7.8 ± 2.4  3  β  -S(CH2)3NH2  0.57 ± 0.39  2.9 ± 1.0  4  β  -S(CH2)4NH2  1.1 ± 0.5  5.1 ± 2.4  5  β  -S(CH2)5NH2  3.8 ± 1.0  7.5 ± 2.4  7  β  -S(CH2)6NH2  0.97 ± 0.38  0.6 ± 0.3  8  β  -S(CH2)7NH2  4.6 ± 3.2  1.9 ± 1.1  9  β  -S(CH2)8NH2  2.4 ± 0.95  0.3 ± 0.1  10  β  -S(CH2)10NH2  27.0 ± 17.0  2.6 ± 0.7  Hepta-6-guanidinealkyl β-cyclodextrin derivatives (Karginov et al. 2006a;)  11  β    5.3 ± 3.2  8.9 ± 6.0  12  β    12.6 ± 9.0  12.2 ± 2.9  Hepta-6-arylamine β-cyclodextrin derivative (Karginov et al. 2006a; Yannakopoulou et al. 2011)  13  β    0.13 ± 0.10  0.8 ± 0.5  Cationic α- and γ cyclodextrin derivatives (Yannakopoulou et al. 2011)  14  α  -NH2  1200 ± 300  >100  15  γ  -NH2  170 ± 50  12 ± 3  16  α    29 ± 5  45 ± 13  17  γ    2.8 ± 1.3  5.4 ± 0.8        Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  Hepta-6-aminoalkyl β-cyclodextrin derivatives (Karginov et al. 2006a)  1  β  -NH2  140 ± 90  20 ± 9  2  β  -S(CH2)2NH2  3.5 ± 0.9  7.8 ± 2.4  3  β  -S(CH2)3NH2  0.57 ± 0.39  2.9 ± 1.0  4  β  -S(CH2)4NH2  1.1 ± 0.5  5.1 ± 2.4  5  β  -S(CH2)5NH2  3.8 ± 1.0  7.5 ± 2.4  7  β  -S(CH2)6NH2  0.97 ± 0.38  0.6 ± 0.3  8  β  -S(CH2)7NH2  4.6 ± 3.2  1.9 ± 1.1  9  β  -S(CH2)8NH2  2.4 ± 0.95  0.3 ± 0.1  10  β  -S(CH2)10NH2  27.0 ± 17.0  2.6 ± 0.7  Hepta-6-guanidinealkyl β-cyclodextrin derivatives (Karginov et al. 2006a;)  11  β    5.3 ± 3.2  8.9 ± 6.0  12  β    12.6 ± 9.0  12.2 ± 2.9  Hepta-6-arylamine β-cyclodextrin derivative (Karginov et al. 2006a; Yannakopoulou et al. 2011)  13  β    0.13 ± 0.10  0.8 ± 0.5  Cationic α- and γ cyclodextrin derivatives (Yannakopoulou et al. 2011)  14  α  -NH2  1200 ± 300  >100  15  γ  -NH2  170 ± 50  12 ± 3  16  α    29 ± 5  45 ± 13  17  γ    2.8 ± 1.3  5.4 ± 0.8  View Large Table 3. Polyvalent cationic cyclodextrin blockers of PA63 ion current and LT cytotoxicity. Note: only selected molecules are shown, see Bezrukov and Nestorovich (2015) for complete list of the antitoxins.       Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  Hepta-6-aminoalkyl β-cyclodextrin derivatives (Karginov et al. 2006a)  1  β  -NH2  140 ± 90  20 ± 9  2  β  -S(CH2)2NH2  3.5 ± 0.9  7.8 ± 2.4  3  β  -S(CH2)3NH2  0.57 ± 0.39  2.9 ± 1.0  4  β  -S(CH2)4NH2  1.1 ± 0.5  5.1 ± 2.4  5  β  -S(CH2)5NH2  3.8 ± 1.0  7.5 ± 2.4  7  β  -S(CH2)6NH2  0.97 ± 0.38  0.6 ± 0.3  8  β  -S(CH2)7NH2  4.6 ± 3.2  1.9 ± 1.1  9  β  -S(CH2)8NH2  2.4 ± 0.95  0.3 ± 0.1  10  β  -S(CH2)10NH2  27.0 ± 17.0  2.6 ± 0.7  Hepta-6-guanidinealkyl β-cyclodextrin derivatives (Karginov et al. 2006a;)  11  β    5.3 ± 3.2  8.9 ± 6.0  12  β    12.6 ± 9.0  12.2 ± 2.9  Hepta-6-arylamine β-cyclodextrin derivative (Karginov et al. 2006a; Yannakopoulou et al. 2011)  13  β    0.13 ± 0.10  0.8 ± 0.5  Cationic α- and γ cyclodextrin derivatives (Yannakopoulou et al. 2011)  14  α  -NH2  1200 ± 300  >100  15  γ  -NH2  170 ± 50  12 ± 3  16  α    29 ± 5  45 ± 13  17  γ    2.8 ± 1.3  5.4 ± 0.8        Inhibition of conductance,  Inhibition of cytotoxicity,  #  Cyclodextrin  Substituent  IC50, nM (0.1 M KCl)  IC50, μM  Hepta-6-aminoalkyl β-cyclodextrin derivatives (Karginov et al. 2006a)  1  β  -NH2  140 ± 90  20 ± 9  2  β  -S(CH2)2NH2  3.5 ± 0.9  7.8 ± 2.4  3  β  -S(CH2)3NH2  0.57 ± 0.39  2.9 ± 1.0  4  β  -S(CH2)4NH2  1.1 ± 0.5  5.1 ± 2.4  5  β  -S(CH2)5NH2  3.8 ± 1.0  7.5 ± 2.4  7  β  -S(CH2)6NH2  0.97 ± 0.38  0.6 ± 0.3  8  β  -S(CH2)7NH2  4.6 ± 3.2  1.9 ± 1.1  9  β  -S(CH2)8NH2  2.4 ± 0.95  0.3 ± 0.1  10  β  -S(CH2)10NH2  27.0 ± 17.0  2.6 ± 0.7  Hepta-6-guanidinealkyl β-cyclodextrin derivatives (Karginov et al. 2006a;)  11  β    5.3 ± 3.2  8.9 ± 6.0  12  β    12.6 ± 9.0  12.2 ± 2.9  Hepta-6-arylamine β-cyclodextrin derivative (Karginov et al. 2006a; Yannakopoulou et al. 2011)  13  β    0.13 ± 0.10  0.8 ± 0.5  Cationic α- and γ cyclodextrin derivatives (Yannakopoulou et al. 2011)  14  α  -NH2  1200 ± 300  >100  15  γ  -NH2  170 ± 50  12 ± 3  16  α    29 ± 5  45 ± 13  17  γ    2.8 ± 1.3  5.4 ± 0.8  View Large Some preliminary lead compound optimization experiments were conducted. Thus, to test if the nature of the attached positively charged functional groups was important, Karginov et al. (2006a) examined a group of hepta-6-guanidine β-cyclodextrin derivatives, in which positive charges were distributed between two nitrogens of the guanidine moiety (Table 3, compounds 11 and 12). The channel blocking and cell protective activity of these multivalent compounds were decreased only slightly compared to their aminoalkyl analogs. To determine an optimum length of the linkers connecting the positively charged functional groups with the 7+βCD scaffold, a number of hepta-6-thioaminoalkyl derivatives with alkyl spacers of various lengths were investigated (Table 3, compounds 1–10). The alkyl spacers with 3–8 CH2 linkers were shown to be optimal for the PA63 blockage. 7+βCDs with shorter spacers, apparently because of the size and mobility restrictions, were less effective in blocking the pore and protecting the RAW cells against the anthrax toxin. 7+βCDs with longer spacers were cytotoxic and induced instability of the bilayer lipid membranes. The major increase in 7+β-CDs activity was achieved when the chemistry of the spacers carrying amines was modified (Karginov et al. 2006a; Diaz-Moscoso et al. 2011; Yannakopoulou et al. 2011). One of the tested derivatives, per-6-S-(3-aminomethyl)benzylthio-β-cyclodextrin (AMBnTβCD) (Table 3, compound 13), which in addition to the functional amino groups had one phenyl group carried by each thio-hydrocarbon spacer, was selected for more detailed analysis. In the model lipid membranes, AMBnTβCD blocked PA63 with KD = 0.13 ± 0.1 nM (0.1 M KCl, 20 mV) and protected cultured macrophage-like cells from anthrax lethal toxin (PA + LF) intoxication at IC50 = 0.5 ± 0.2 μM. These inhibitory concentrations are the lowest among the reported on the non-peptide PA63 pore blockers. In in vivo experiments, AMBnTβCD completely protected Fisher F344 rats from intoxication with lethal toxin and, in an infection model of anthrax, significantly increased the survival of mice when administered in combination with ciprofloxacin (Moayeri et al. 2008). It was also demonstrated that contrarily to heptameric IB201 inhibition of αHL, the 7-fold symmetry of the blocker molecules complementing the heptameric structure of PA63 channels was not a strict requirement for the effective blockage. Both 6+αCD and 8+γCD were able to block the PA63 (Yannakopoulou et al. 2011) with 6+αCD binding being noticeably weaker and 8+γCD binding being comparable with that of 7+βCD both in planar bilayers and in cell assay (Table 3, compounds 14–17). Analysis of the 7+βCD/pore interactions revealed the contribution of long-range Coulomb forces at moderate and low salt concentrations, whereas at high salt concentrations, the salt-concentration-independent short-range interactions predominate (Bezrukov et al. 2012; Nestorovich et al. 2012). The 7+βCD activity increased in the order Ib < C2IIa < PA63, in parallel with the cationic selectivity of the channels. The observed pattern may indicate that the positive charges carried by the βCD blockers directly bind to the negatively charged residues inside the channels. However, because 7+βCD binding was strongly enhanced by the presence of the aromatic linkers (as discussed with AMBnTβCD) and decreased with the PA63 F428A mutant (Fig. 1C, right), the blockers may also to interact with the conserved ϕ-clamp residues. The authors concluded that a number of Coulomb and salt-concentration-independent short-range interactions acting concurrently within a single binding pocket of the channel lumen are involved in the binding reaction. The particular origins of these forces have not yet been established due to the complications coming from multiple factors. Among those are the hydration state changes of the blocker molecule upon entering the channel pore from the bulk (Crouzy, Berneche and Roux 2001) and an ambiguity related to possible interaction-induced changes in both the blocker and channel structures. Moreover, many different forces such as aromatic–aromatic, π–π and cation–π interactions, hydrogen bonding, van der Waals interactions, etc. might be involved in the blocker binding. With the emerging new structural data on the membrane pore-forming proteins, there is hope that detailed Monte Carlo and molecular dynamics simulations and multiscale modeling combined with the present and forthcoming single-molecule data will cast light upon the relative contribution from the different types of physical forces defining the strength of blocker/channel interactions. PAMAM dendrimers Positively charged multivalent blockers based on a completely different, dendrimer scaffold were also investigated (Forstner et al. 2014). Dendrimers are repeatedly branched polymers with all bonds emanating from a central core where each consecutive growth step represents a new dendrimer ‘generation’ with an increased diameter and doubled number of reactive surface functional groups (Table 4). The well-controlled branched chemical synthesis techniques result in the dendrimers possessing the nanoscale size range, monodispersity, rigid and stable globular polyvalent structure, and highly regulated number of functional groups and surface charges (Wijagkanalan, Kawakami and Hashida 2011). These unique characteristics determine emergent industrial and medical applications of dendrimers which, among multiple others, include usage as antibacterial, antiviral and antiparasitic agents (Helms and Meijer 2006). The commercially available PAMAM dendrimers suggested as the anthrax and C2 toxin blockers are based on an ethylene diamine core and an amidoamine repeat branching structure and come in different generations (G0–G10) varying in size (d = 15–135 Å) and surface charge (z = +4 to +4096). Similarly to the cyclodextrin blockers, the PAMAM dendrimers directly obstructed ion currents through PA63 and C2IIa pores and inhibited channel-facilitated transport of the enzymatic components in cell assay. The in vitro IC50 values of the PAMAM/PA63 binding reaction (0.16–230 nM, depending on the generation) were comparable to that of the rationally designed PA63 β-CD-based inhibitor, AmPrβCD (0.55 nM) (Nestorovich et al. 2010). Table 4. Polyvalent cationic PAMAM dendrimer- and dendron-based blockers of PA63 (Forstner et al. 2014).         Inhibition of conductance,  #  Generation  Measured diameter, Å  NH2 surface groups number  IC50, (0.1 M KCl)  PAMAM-NH2 dendrimers, cis addition  1  0  15  4  128 ± 44 nM  2  1  22  8  5.3 ± 2.6 nM  3  2  29  16  7.15 ± 4.7 nM  4  3  36  32  5.0 ± 1.4 nM  5  4  45  64  2.4 ± 1.3 nM  6  8  97  1024  0.22 ± 0.08 nM  7  10  135  4096  0.16 ± 0.07nM  PAMAM-NH2 dendrimers, trans addition  8  0  15  4  16.5 ± 3.3 μM  9  1  22  8  4.6 ± 1.7 μM  PAMAM-NH2 dendrons, cis addition  10  0 dendron    2  26 ± 7 nM  11  1 dendron    4  4.9 ± 0.7 nM  12  2 dendron    8  4.2 ± 0.9 nM          Inhibition of conductance,  #  Generation  Measured diameter, Å  NH2 surface groups number  IC50, (0.1 M KCl)  PAMAM-NH2 dendrimers, cis addition  1  0  15  4  128 ± 44 nM  2  1  22  8  5.3 ± 2.6 nM  3  2  29  16  7.15 ± 4.7 nM  4  3  36  32  5.0 ± 1.4 nM  5  4  45  64  2.4 ± 1.3 nM  6  8  97  1024  0.22 ± 0.08 nM  7  10  135  4096  0.16 ± 0.07nM  PAMAM-NH2 dendrimers, trans addition  8  0  15  4  16.5 ± 3.3 μM  9  1  22  8  4.6 ± 1.7 μM  PAMAM-NH2 dendrons, cis addition  10  0 dendron    2  26 ± 7 nM  11  1 dendron    4  4.9 ± 0.7 nM  12  2 dendron    8  4.2 ± 0.9 nM  View Large Table 4. Polyvalent cationic PAMAM dendrimer- and dendron-based blockers of PA63 (Forstner et al. 2014).         Inhibition of conductance,  #  Generation  Measured diameter, Å  NH2 surface groups number  IC50, (0.1 M KCl)  PAMAM-NH2 dendrimers, cis addition  1  0  15  4  128 ± 44 nM  2  1  22  8  5.3 ± 2.6 nM  3  2  29  16  7.15 ± 4.7 nM  4  3  36  32  5.0 ± 1.4 nM  5  4  45  64  2.4 ± 1.3 nM  6  8  97  1024  0.22 ± 0.08 nM  7  10  135  4096  0.16 ± 0.07nM  PAMAM-NH2 dendrimers, trans addition  8  0  15  4  16.5 ± 3.3 μM  9  1  22  8  4.6 ± 1.7 μM  PAMAM-NH2 dendrons, cis addition  10  0 dendron    2  26 ± 7 nM  11  1 dendron    4  4.9 ± 0.7 nM  12  2 dendron    8  4.2 ± 0.9 nM          Inhibition of conductance,  #  Generation  Measured diameter, Å  NH2 surface groups number  IC50, (0.1 M KCl)  PAMAM-NH2 dendrimers, cis addition  1  0  15  4  128 ± 44 nM  2  1  22  8  5.3 ± 2.6 nM  3  2  29  16  7.15 ± 4.7 nM  4  3  36  32  5.0 ± 1.4 nM  5  4  45  64  2.4 ± 1.3 nM  6  8  97  1024  0.22 ± 0.08 nM  7  10  135  4096  0.16 ± 0.07nM  PAMAM-NH2 dendrimers, trans addition  8  0  15  4  16.5 ± 3.3 μM  9  1  22  8  4.6 ± 1.7 μM  PAMAM-NH2 dendrons, cis addition  10  0 dendron    2  26 ± 7 nM  11  1 dendron    4  4.9 ± 0.7 nM  12  2 dendron    8  4.2 ± 0.9 nM  View Large All tested dendrimers inhibited PA63 and C2IIa conductance in a concentration-dependent manner. The G1, G2 and G3 dendrimers showed stronger binding per-functional group affinity when compared with the low-generation G0 and high-generation G4, G8 and G10 PAMAM dendrimers. Apparently, the 22-Å G1, 29-Å G2 and 36-Å G3 which have, respectively, 8, 16 and 32 surface primary amines are small enough to enter the pores. The binding was reported to be significantly stronger when the dendrimers were added from the physiologically relevant cis (bud side) compartment (approximately 130 times in the case of G0-NH2 and 870 times in the case of G1-NH2). Not surprisingly, highly positively charged dendrimers of generation 2 and higher exhibited effects on the morphology of the tested cells on their own, decreasing the amount of viable cells. Several ideas to overcome the blocker's toxicity problem were suggested. The more favorable therapeutic window is often achieved with the dendrimers being partially degraded to get the ‘fractured’ or ‘imperfect’ dendrimers (Tang, Redemann and Szoka 1996), possibly because of their increased flexibility. For instance, the channel-blocking activity of the more flexible G1-NH2 carrying four surface primary amines was approximately 26 times higher (IC50 = 4.9 nM) compared with that of the four positively charged G0 dendrimer (IC50 = 128 nM) (Table 4, compounds 10–12). CONCLUDING REMARKS With a staggeringly low number of new therapeutic compounds entering the market annually and with the high-throughput screening approaches frequently generating unsatisfactory results, new promising developments in the drug design are paramount. Modern rational drug discovery is driven by the data on biological targets and by attempts to explore the nature of physical interactions between the designed ligands and their receptors (Lounnas et al. 2013). Ion channels, despite being targeted by about 13% of the marketed drugs, remain underexploited in the current drug discovery programs, especially in the case of pore-forming toxins. Here we have briefly reviewed the recent efforts to rationally design pore-blocking small and multivalent drug molecules of potential pharmaceutical interest that target the so-called pores of virulence—the ion-conductive protein complexes formed by a number of bacterial toxins. Remarkably, in vitro activity of the most potent among these antitoxins compares quite favorably with that of the blockers used for the classical ion-selective channels of neurophysiology. We earlier summarized the representative examples of the most efficient non-peptide inhibitors of the channels of excitable cells (see table 3 in Nestorovich and Bezrukov 2012). Most of the IC50 values reported for the best of these blockers are nearly an order of magnitude higher than that for AMBnTβCD, inhibiting the anthrax PA63 channel with IC50 = 0.13 ± 0.10 nM. The only exceptions we are aware of are the calcium-activated potassium channel KCa2.2 blocker, the bis-quinolinium cyclophane UCL1684, characterized by IC50 = 0.28 nM (Fanger et al. 2001; Wei et al. 2005) and its subsequent modification, UCL 1848, with the aromatic xylyl linkers replaced by aliphatic pentylene groups, exhibiting IC50 of 0.12 nM (Hosseini et al. 2001; Wulff and Zhorov 2008). One of the main challenges in designing inhibitors of ion-selective channels of neurophysiology is to find a drug molecule possessing a high degree of specificity toward a particular channel. In that, the pore-blocking antitoxin design is fundamentally different as it is often aimed at the search for the universal compounds. In this review, we provided an important example of one of the first rationally designed broad-spectrum antitoxin. The universality concept can be easily appreciated. Indeed, a potent, broad-spectrum antidote of tomorrow is expected to protect against more than one virulent agent. The best strategy in search for such a compound is to focus on the shared structural features or functional mechanisms, such as channel-formation and channel-facilitated toxin transport, which could be directionally targeted. The tailor-made seven-positively charged multivalent AMBnTβCD was shown to protect against the cytotoxicity induced by four different toxins: anthrax toxins of B. anthracis (Karginov et al. 2006a), clostridial C2, iota (Nestorovich et al. 2011) and CDT toxins (Roeder et al. 2014). At the same time, the channel-forming protein targeting, at least in inhibiting the binary toxins, has its caveats. Thus, the anthrax toxin's LF component is known to remain active in cells and in animal tissues for days (Moayeri et al. 2013), and LF can be transported not only into the cytosol but also into the lumen of endosomal intraluminal vesicles (Abrami et al. 2013). The vesicles protect LF from proteolytic degradation and later fuse, releasing LF into the cytosol. These findings might suggest that the focus of anthrax antitoxin research should be shifted toward LF as the primary target. This viewpoint is clearly not unfounded; however, the LF-targeting antitoxins would likely forfeit the broad-spectrum aspect by not even being able to protect against the other anthrax toxin—ET. The various antitoxin agents must be developed and stockpiled to provide alternative options in the case of bacterial resistance, low response or dissemination of new bioengineered agents. In addition to their broad-spectrum action, the ideal antidote drug molecules are also expected to possess low toxicity against mammalian cell targets, including ion channels. Despite a relatively low toxicity reported for the β-CD-based antitoxins in cell assays, positively charged multivalent compounds are known to be less biocompatible compared with the neutral and negatively charged agents. There are several reported lead optimization techniques that can diminish the potential cytotoxicity problem. The surface engineering methods can be used to mask the surface positive charges by their partial derivatization with chemically inert groups such as PEG or fatty acids. The cationic molecules could be also encapsulated into liposomes or micelles. For example, the poly(ethylene glycol)-b-poly(aspartic acid) micelles, while being stable at physiological conditions with neutral pH, can disintegrate in the endosomal acidic environment (Zhang et al. 2003; Jang et al. 2005). This interesting feature would allow direct delivery of the encapsulated blockers to the binary toxin channels formed in the endosomal compartments. Therefore, we remain hopeful that the inhibitive action of these tailor-made universal antitoxins could be mostly limited to their intentional targets. FUNDING SMB research is supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. EMN research is supported by NIAID of the NIH under award number 1R15AI099897-01A1. Conflict of interest. None declared. REFERENCES Abrami L Brandi L Moayeri Met al.   Hijacking multivesicular bodies enables long-term and exosome-mediated long-distance action of anthrax toxin Cell Rep  2013 27 986 96 Google Scholar CrossRef Search ADS   Aktories K Barmann M Ohishi Iet al.   Botulinum C2 toxin ADP-ribosylates actin Nature  1986 322 390 2 Google Scholar CrossRef Search ADS PubMed  Alonzo F3rd Torres VJ The bicomponent pore-forming leucocidins of Staphylococcus aureus Microbiol Mol Biol R  2014 78 199 230 Google Scholar CrossRef Search ADS   Alouf JE Pore-forming bacterial toxins: an overview Van der Goot G Pore-Forming Toxins  New York Springer 2001 1 14 Alves GG Machado de Avila RA Chavez-Olortegui CDet al.   Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known Anaerobe  2014 30 102 7 Google Scholar CrossRef Search ADS PubMed  Arbuthnott JP Freer JH Bernheimer AW Physical states of staphylococcal alpha-toxin J Bacteriol  1967 94 1170 7 Google Scholar PubMed  Bachmeyer C Benz R Barth Het al.   Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes and Vero cells: inhibition of channel function by chloroquine and related compounds in vitro and intoxification in vivo FASEB J  2001 15 1658 60 Google Scholar PubMed  Bachmeyer C Orlik F Barth Het al.   Mechanism of C2-toxin inhibition by fluphenazine and related compounds: investigation of their binding kinetics to the C2II-channel using the current noise analysis J Mol Biol  2003 333 527 40 Google Scholar CrossRef Search ADS PubMed  Barth H Aktories K Popoff MRet al.   Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins Microbiol Mol Biol R  2004 68 373 402 Google Scholar CrossRef Search ADS   Barth H Blocker D Behlke Jet al.   Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification J Biol Chem  2000 275 18704 11 Google Scholar CrossRef Search ADS PubMed  Barth H Stiles BG Popoff MR ADP-ribosylating toxins modifying the actin cytoskeleton Alouf JA Ladant D Popoff MR The Comprehensive Sourcebook of Bacterial Protein Toxins  4th edn Amsterdam, Netherlands; Kidlington, Oxford, UK; Waltham, MA, USA Elsevier 2015 397 423 Beitzinger C Bronnhuber A Duscha Ket al.   Designed azolopyridinium salts block protective antigen pores in vitro and protect cells from anthrax toxin PLoS One  2013 8 e66099 Google Scholar CrossRef Search ADS PubMed  Berezhkovskii AM Bezrukov SM On the applicability of entropy potentials in transport problems Eur Phys J-Spec Top  2014 223 3063 77 Google Scholar CrossRef Search ADS PubMed  Bernheimer AW. Some aspects of the history of membrane-damaging toxins Med Microbiol Immun  1996 185 59 63 Google Scholar CrossRef Search ADS   Bernheimer AW Schwartz LL Isolation and composition of staphylococcal alpha toxin J Gen Microbiol  1963 30 455 68 Google Scholar CrossRef Search ADS PubMed  Berube BJ Wardenburg JB Staphylococcus aureus alpha-toxin: nearly a century of intrigue Toxins  2013 5 1140 66 Google Scholar CrossRef Search ADS PubMed  Bezrukov S Nestorovich E Inhibitors of pore-forming toxins Alouf JA Ladant D Popoff MR The Comprehensive Sourcebook of Bacterial Protein Toxins  4th edn Amsterdam, Netherlands; Kidlington, Oxford, UK; Waltham, MA, USA Elsevier 2015 1095 134 Bezrukov SM Liu X Karginov VAet al.   Interactions of high-affinity cationic blockers with the translocation pores of B. anthracis, C. botulinum, and C. perfringens binary toxins Biophys J  2012 103 1208 17 Google Scholar CrossRef Search ADS PubMed  Bezrukov SM Vodyanoy I Parsegian VA Counting polymers moving through a single ion channel Nature  1994 370 279 81 Google Scholar CrossRef Search ADS PubMed  Blaustein RO Finkelstein A Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance J Gen Physiol  1990a 96 905 19 Google Scholar CrossRef Search ADS   Blaustein RO Finkelstein A Diffusion limitation in the block by symmetric tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer membranes J Gen Physiol  1990b 96 943 57 Google Scholar CrossRef Search ADS   Blaustein RO Koehler TM Collier RJet al.   Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers P Natl Acad Sci USA  1989 86 2209 13 Google Scholar CrossRef Search ADS   Blaustein RO Lea EJ Finkelstein A Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Single-channel analysis J Gen Physiol  1990 96 921 42 Google Scholar CrossRef Search ADS PubMed  Blocker D Behlke J Aktories Ket al.   Cellular uptake of the Clostridium perfringens binary iota-toxin Infect Immun  2001 69 2980 7 Google Scholar CrossRef Search ADS PubMed  Blocker D Pohlmann K Haug Get al.   Clostridium botulinum C2 toxin: low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells J Biol Chem  2003 278 37360 7 Google Scholar CrossRef Search ADS PubMed  Branson TR Turnbull WB Bacterial toxin inhibitors based on multivalent scaffolds Chem Soc Rev  2013 42 4613 22 Google Scholar CrossRef Search ADS PubMed  Bronnhuber A Maier E Riedl Zet al.   Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication Toxicology  2014 316 25 33 Google Scholar CrossRef Search ADS PubMed  Brown MJ Thoren KL Krantz BA Role of the alpha clamp in the protein translocation mechanism of anthrax toxin J Mol Biol  2015 427 3340 9 Google Scholar CrossRef Search ADS PubMed  Chesbro WR Heydrick FP Martineau Ret al.   Purification of staphylococcal beta-hemolysin and its action on staphylococcal and streptococcal cell walls J Bacteriol  1965 89 378 89 Google Scholar PubMed  Collier RJ. Membrane translocation by anthrax toxin Mol Aspects Med  2009 30 413 22 Google Scholar CrossRef Search ADS PubMed  Crouzy S Berneche S Roux B Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel J Gen Physiol  2001 118 207 18 Google Scholar CrossRef Search ADS PubMed  Davis ME Brewster ME Cyclodextrin-based pharmaceutics: past, present and future Nat Rev Drug Discov  2004 3 1023 35 Google Scholar CrossRef Search ADS PubMed  Denys J Van de Velde H Sur la production d'une antileucocidine chez les lapin vaccinéscontre le staphylocoque pyogène La Cellule  1895 11 359 72 Diaz-Moscoso A Mendez-Ardoy A Ortega-Caballero Fet al.   Symmetry complementarity-guided design of anthrax toxin inhibitors based on beta-cyclodextrin: synthesis and relative activities of face-selective functionalized polycationic clusters Chem Med Chem  2011 6 181 92 Google Scholar CrossRef Search ADS PubMed  Donahue JA Baldwin JN Hemolysin and leukocidin production by 80/81 strains of Staphylococcus aureus J Infect Dis  1966 116 324 8 Google Scholar CrossRef Search ADS PubMed  Fanger CM Rauer H Neben ALet al.   Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels J Biol Chem  2001 276 12249 56 Google Scholar CrossRef Search ADS PubMed  Fasting C Schalley CA Weber Met al.   Multivalency as a chemical organization and action principle Angew Chem Int Ed  2012 51 10472 98 Google Scholar CrossRef Search ADS   Feld GK Brown MJ Krantz BA Ratcheting up protein translocation with anthrax toxin Protein Sci  2012 21 606 Google Scholar CrossRef Search ADS PubMed  Feld GK Thoren KL Kintzer AFet al.   Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers Nat Struct Mol Biol  2010 17 1383 90 Google Scholar CrossRef Search ADS PubMed  Forstner P Bayer F Kalu Net al.   Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors Biomacromolecules  2014 15 2461 74 Google Scholar CrossRef Search ADS PubMed  Geny B Popoff MR Bacterial protein toxins and lipids: pore formation or toxin entry into cells Biol Cell  2006 98 667 78 Google Scholar CrossRef Search ADS PubMed  Gibert M Marvaud JC Pereira Yet al.   Differential requirement for the translocation of clostridial binary toxins: iota toxin requires a membrane potential gradient FEBS Lett  2007 581 1287 96 Google Scholar CrossRef Search ADS PubMed  Gu LQ Braha O Conlan Set al.   Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter Nature  1999 398 686 90 Google Scholar CrossRef Search ADS PubMed  Gurnev PA Nestorovich EM Channel-forming bacterial toxins in biosensing and macromolecule delivery Toxins  2014 6 2483 540 Google Scholar CrossRef Search ADS PubMed  Helms B Meijer EW Chemistry. dendrimers at work Science  2006 313 929 30 Google Scholar CrossRef Search ADS PubMed  Hosseini R Benton DC Dunn PMet al.   SK3 is an important component of K(+) channels mediating the afterhyperpolarization in cultured rat SCG neurones J Physiol  2001 535 323 34 Google Scholar CrossRef Search ADS PubMed  Jang WD Nishiyama N Zhang GDet al.   Supramolecular nanocarrier of anionic dendrimer porphyrins with cationic block copolymers modified with polyethylene glycol to enhance intracellular photodynamic efficacy Angew Chem Int Ed  2005 44 419 23 Google Scholar CrossRef Search ADS   Jiang J Pentelute BL Collier RJet al.   Atomic structure of anthrax protective antigen pore elucidates toxin translocation Nature  2015 521 545 9 Google Scholar CrossRef Search ADS PubMed  Kaneko J Kamio Y Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes Biosci Biot Bioch  2004 68 981 1003 Google Scholar CrossRef Search ADS   Karginov VA. Cyclodextrin derivatives as anti-infectives Curr Opin Pharmacol  2013 13 717 25 Google Scholar CrossRef Search ADS PubMed  Karginov VA Nestorovich EM Moayeri Met al.   Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design P Natl Acad Sci USA  2005 102 15075 80 Google Scholar CrossRef Search ADS   Karginov VA Nestorovich EM Yohannes Aet al.   Search for cyclodextrin-based inhibitors of anthrax toxins: synthesis, structural features, and relative activities Antimicrob Agents Ch  2006a 50 3740 53 Google Scholar CrossRef Search ADS   Karginov VA Yohannes A Robinson TMet al.   Beta-cyclodextrin derivatives that inhibit anthrax lethal toxin Bioorg Med Chem  2006b 14 33 40 Google Scholar CrossRef Search ADS   Kasianowicz JJ Balijepalli AK Ettedgui Jet al.   Analytical applications for pore-forming proteins Biochim Biophys Acta  2015 pii:s005-2736(15)00308-309 Kintzer AF Thoren KL Sterling HJet al.   The protective antigen component of anthrax toxin forms functional octameric complexes J Mol Biol  2009 392 614 29 Google Scholar CrossRef Search ADS PubMed  Knapp O Benz R Gibert Met al.   Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia J Biol Chem  2002 277 6143 52 Google Scholar CrossRef Search ADS PubMed  Knapp O Benz R Popoff MR Pore-forming activity of clostridial binary toxins Biochim Biophys Acta  2015 pii:s0005-2736(15)00247-3 Knapp O Maier E Benz Ret al.   Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX) Biochim Biophys Acta  2009 1788 2584 93 Google Scholar CrossRef Search ADS PubMed  Knapp O Maier E Waltenberger Eet al.   Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin Cell Microbiol  2015 17 288 302 Google Scholar CrossRef Search ADS PubMed  Krantz BA Finkelstein A Collier RJ Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient J Mol Biol  2006 355 968 79 Google Scholar CrossRef Search ADS PubMed  Krantz BA Melnyk RA Zhang Set al.   A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore Science  2005 309 777 81 Google Scholar CrossRef Search ADS PubMed  Krasilnikov O Ternovsky V Tashmukhamedov B Properties of ion channels induced by alpha-staphylotoxin in bilayer lipid membranes Biofisica  1981 26 271 5 Kronhardt A Rolando M Beitzinger Cet al.   Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells PLoS One  2011 6 e23133 Google Scholar CrossRef Search ADS PubMed  Lang AE Neumeyer T Sun Jet al.   Amino acid residues involved in membrane insertion and pore formation of Clostridium botulinum C2 toxin Biochemistry  2008 47 8406 13 Google Scholar CrossRef Search ADS PubMed  Laventie BJ Potrich C Atmanene Cet al.   p-Sulfonato-calix [n]arenes inhibit staphylococcal bicomponent leukotoxins by supramolecular interactions Biochem J  2013 450 559 71 Google Scholar CrossRef Search ADS PubMed  Lewis M Weaver CD McClain MS Identification of small molecule inhibitors of Clostridium perfringens epsilon-toxin cytotoxicity using a cell-based high-throughput screen Toxins  2010 2 1825 47 Google Scholar CrossRef Search ADS PubMed  Liu S Moayeri M Pomerantsev APet al.   Bacillus anthracis toxins Alouf JA Ladant D Popoff MR The Comprehensive Sourcebook of Bacterial Protein Toxins  4th edn Amsterdam, Netherlands; Kidlington, Oxford, UK; Waltham, MA, USA Elsevier 2015 361 96 Liu S Zhang Y Moayeri Met al.   Key tissue targets responsible for anthrax-toxin-induced lethality Nature  2013 501 63 8 Google Scholar CrossRef Search ADS PubMed  Lounnas V Ritschel T Kelder Jet al.   Current progress in structure-based rational drug design marks a new mindset in drug discovery Comput Struct Biotechnol J  2013 5 e201302011 Google Scholar CrossRef Search ADS PubMed  Majd S Yusko EC Billeh YNet al.   Applications of biological pores in nanomedicine, sensing, and nanoelectronics Curr Opin Biotechnol  2010 21 439 76 Google Scholar CrossRef Search ADS PubMed  Miles G Cheley S Braha Oet al.   The staphylococcal leukocidin bicomponent toxin forms large ionic channels Biochemistry  2001 40 8514 22 Google Scholar CrossRef Search ADS PubMed  Miles G Jayasinghe L Bayley H Assembly of the Bi-component leukocidin pore examined by truncation mutagenesis J Biol Chem  2006 281 2205 14 Google Scholar CrossRef Search ADS PubMed  Miles G Movileanu L Bayley H Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore Protein Sci  2002 11 894 902 Google Scholar CrossRef Search ADS PubMed  Moayeri M Crown D Jiao GSet al.   Small-molecule inhibitors of lethal factor protease activity protect against anthrax infection Antimicrob Agents Ch  2013 57 4139 45 Google Scholar CrossRef Search ADS   Moayeri M Leppla SH Vrentas Cet al.   Anthrax pathogenesis Annu Rev Microbiol  2015 69 185 208 Google Scholar CrossRef Search ADS PubMed  Moayeri M Robinson TM Leppla SHet al.   In vivo efficacy of beta-cyclodextrin derivatives against anthrax lethal toxin Antimicrob Agents Ch  2008 52 2239 41 Google Scholar CrossRef Search ADS   Mogridge J Cunningham K Collier RJ Stoichiometry of anthrax toxin complexes Biochemistry  2002 41 1079 82 Google Scholar CrossRef Search ADS PubMed  Nagahama M Hagiyama T Kojima Tet al.   Binding and internalization of Clostridium botulinum C2 toxin Infect Immun  2009 77 5139 48 Google Scholar CrossRef Search ADS PubMed  Nestorovich EM Bezrukov SM Obstructing toxin pathways by targeted pore blockage Chem Rev  2012 112 6388 430 Google Scholar CrossRef Search ADS PubMed  Nestorovich EM Karginov VA Berezhkovskii AMet al.   Blockage of anthrax PA63 pore by a multicharged high-affinity toxin inhibitor Biophys J  2010 99 134 43 Google Scholar CrossRef Search ADS PubMed  Nestorovich EM Karginov VA Berezhkovskii AMet al.   Kinetics and thermodynamics of binding reactions as exemplified by anthrax toxin channel blockage with a cationic cyclodextrin derivative Proc Natl Acad Sci USA  2012 109 18453 8 Google Scholar CrossRef Search ADS PubMed  Nestorovich EM Karginov VA Bezrukov SM Polymer partitioning and ion selectivity suggest asymmetrical shape for the membrane pore formed by epsilon toxin Biophys J  2010 99 782 9 Google Scholar CrossRef Search ADS PubMed  Nestorovich EM Karginov VA Popoff MRet al.   Tailored ss-cyclodextrin blocks the translocation pores of binary exotoxins from C. botulinum and C. perfringens and protects cells from intoxication PLoS One  2011 6 e23927 Google Scholar CrossRef Search ADS PubMed  Neumeyer T Schiffler B Maier Eet al.   Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel J Biol Chem  2008 283 3904 14 Google Scholar CrossRef Search ADS PubMed  Ohishi I Odagiri Y Histopathological effect of botulinum C2 toxin on mouse intestines Infect Immun  1984 43 54 8 Google Scholar PubMed  Orlik F Schiffler B Benz R Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis Biophys J  2005 88 1715 24 Google Scholar CrossRef Search ADS PubMed  Otto M. Basis of virulence in community-associated methicillin-resistant Staphylococcus aureus Annu Rev Microbiol  2010 64 143 62 Google Scholar CrossRef Search ADS PubMed  Otto M. Methicillin-resistant Staphylococcus aureus infection is associated with increased mortality Future Microbiol  2012 7 189 91 Google Scholar CrossRef Search ADS PubMed  Overington JP Al-Lazikani B Hopkins AL How many drug targets are there? Nat Rev Drug Discov  2006 5 993 6 Google Scholar CrossRef Search ADS PubMed  Petit L Maier E Gibert Met al.   Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers J Biol Chem  2001 276 15736 40 Google Scholar CrossRef Search ADS PubMed  Petosa C Collier RJ Klimpel KRet al.   Crystal structure of the anthrax toxin protective antigen Nature  1997 385 833 8 Google Scholar CrossRef Search ADS PubMed  Popoff MR Bouvet P Clostridial toxins Future Microbiol  2009 4 1021 64 Google Scholar CrossRef Search ADS PubMed  Prevost G Tawk MY Zimmermann-Meisse Get al.   The staphylococcal alpha-toxin and leukotoxins Alouf JA Ladant D Popoff MR The Comprehensive Sourcebook of Bacterial Protein Toxins  4th edn Amsterdam, Netherlands; Kidlington, Oxford, UK; Waltham, MA, USA Elsevier 2015 739 72 Pust S Barth H Sandvig K Clostridium botulinum C2 toxin is internalized by clathrin- and Rho-dependent mechanisms Cell Microbiol  2010 12 1809 20 Google Scholar CrossRef Search ADS PubMed  Ragle BE Bubeck Wardenburg J Anti-alpha-hemolysin monoclonal antibodies mediate protection against Staphylococcus aureus pneumonia Infect Immun  2009 77 2712 8 Google Scholar CrossRef Search ADS PubMed  Roeder M Nestorovich EM Karginov VAet al.   Tailored cyclodextrin pore blocker protects mammalian cells from Clostridium difficile binary toxin CDT Toxins  2014 6 2097 114 Google Scholar CrossRef Search ADS PubMed  Schleberger C Hochmann H Barth Het al.   Structure and action of the binary C2 toxin from Clostridium botulinum J Mol Biol  2006 364 705 15 Google Scholar CrossRef Search ADS PubMed  Schmid A Benz R Just Iet al.   Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine J Biol Chem  1994 269 16706 11 Google Scholar PubMed  Scott C Griffin SD Viroporins: structure, function and potential as antiviral targets J Gen Virol  2015 96 2000 27 Google Scholar CrossRef Search ADS PubMed  Simpson LL. Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin J Pharmacol Exp Ther  1984 230 665 9 Google Scholar PubMed  Simpson LL Stiles BG Zepeda HHet al.   Molecular basis for the pathological actions of Clostridium perfringens iota toxin Infect Immun  1987 55 118 22 Google Scholar PubMed  Song L Hobaugh MR Shustak Cet al.   Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore Science  1996 274 1859 66 Google Scholar CrossRef Search ADS PubMed  Stiles BG Barth G Barth Het al.   Clostridium perfringens epsilon toxin: a malevolent molecule for animals and man? Toxins  2013 5 2138 60 Google Scholar CrossRef Search ADS PubMed  Stiles BG Hale ML Marvaud JCet al.   Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex Biochem J  2002 367 801 8 Google Scholar CrossRef Search ADS PubMed  Stiles BG Wilkins TD Purification and characterization of Clostridium perfringens iota toxin: dependence on two nonlinked proteins for biological activity Infect Immun  1986 54 683 8 Google Scholar PubMed  Tang MX Redemann CT Szoka FCJr In vitro gene delivery by degraded polyamidoamine dendrimers Bioconjugate Chem  1996 7 703 14 Google Scholar CrossRef Search ADS   Thoren KL Krantz BA The unfolding story of anthrax toxin translocation Mol Microbiol  2011 80 588 95 Google Scholar CrossRef Search ADS PubMed  Van de Velde H Etude sur le mécanisme de la virulence du staphylocoque pyogène La Cellule  1894 10 403 60 Vance D Martin J Patke Set al.   The design of polyvalent scaffolds for targeted delivery Adv Drug Deliv Rev  2009 61 931 9 Google Scholar CrossRef Search ADS PubMed  Vance D Shah M Joshi Aet al.   Polyvalency: a promising strategy for drug design Biotechnol Bioeng  2008 101 429 34 Google Scholar CrossRef Search ADS PubMed  Wei AD Gutman GA Aldrich Ret al.   International union of pharmacology. LII. Nomenclature and molecular relationships of calcium-activated potassium channels Pharmacol Rev  2005 57 463 72 Google Scholar CrossRef Search ADS PubMed  Wickenden A Priest B Erdemli G Ion channel drug discovery: challenges and future directions Future Med Chem  2012 4 661 79 Google Scholar CrossRef Search ADS PubMed  Wijagkanalan W Kawakami S Hashida M Designing dendrimers for drug delivery and imaging: pharmacokinetic considerations Pharm Res  2011 28 1500 19 Google Scholar CrossRef Search ADS PubMed  Wioland L Dupont JL Bossu JLet al.   Attack of the nervous system by Clostridium perfringens Epsilon toxin: from disease to mode of action on neural cells Toxicon  2013 75 122 35 Google Scholar CrossRef Search ADS PubMed  Wulff H Zhorov BS K + channel modulators for the treatment of neurological disorders and autoimmune diseases Chem Rev  2008 108 1744 73 Google Scholar CrossRef Search ADS PubMed  Yamashita K Kawai Y Tanaka Yet al.   Crystal structure of the octameric pore of staphylococcal gamma-hemolysin reveals the beta-barrel pore formation mechanism by two components P Natl Acad Sci USA  2011 108 17314 9 Google Scholar CrossRef Search ADS   Yannakopoulou K Jicsinszky L Aggelidou Cet al.   Symmetry requirements for effective blocking of pore-forming toxins: comparative study with alpha-, beta-, and gamma-cyclodextrin derivatives Antimicrob Agents Ch  2011 55 3594 7 Google Scholar CrossRef Search ADS   Young JA Collier RJ Anthrax toxin: receptor binding, internalization, pore formation, and translocation Annu Rev Biochem  2007 76 243 65 Google Scholar CrossRef Search ADS PubMed  Zhang GD Harada A Nishiyama Net al.   Polyion complex micelles entrapping cationic dendrimer porphyrin: effective photosensitizer for photodynamic therapy of cancer J Control Release  2003 93 141 50 Google Scholar CrossRef Search ADS PubMed  Zhang S Udho E Wu Zet al.   Protein translocation through anthrax toxin channels formed in planar lipid bilayers Biophys J  2004 87 3842 9 Google Scholar CrossRef Search ADS PubMed  © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
TI - Inhibiting bacterial toxins by channel blockage
JF - Pathogens and Disease
DO - 10.1093/femspd/ftv113
DA - 2016-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/inhibiting-bacterial-toxins-by-channel-blockage-6XMw3xcd0q
SP - ftv113
VL - 74
IS - 2
DP - DeepDyve
ER -