Inhibiting bacterial toxins by channel blockage.

Pathogens and Disease Advance Access published December 9, 2015

Inhibiting Bacterial Toxins by Channel Blockage

Sergey M. Bezrukov1* and Ekaterina M. Nestorovich2* 1

Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human

Development, National Institutes of Health, Bethesda, MD, U.S.A. 2

Department of Biology, The Catholic University of America, Washington, DC, U.S.A.

*Address correspondence to: Sergey M. Bezrukov, Program in Physical Biology, Eunice Kennedy

Bethesda, MD, U.S.A. ; Phone 301-402-4701, E-Mail: [email protected] and Ekaterina M. Nestorovich, Department of Biology, The Catholic University of America, Washington, DC, U.S.A.; Phone 202-319-6723, E-Mail: [email protected]

ABSTRACT 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.

Key Words Rational drug design, structure-based drug discovery, channel-blocking antitoxins, single molecule/protein interaction, multivalent interactions, physical forces of efficient blockage

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Shriver National Institute of Child Health and Human Development, National Institutes of Health,

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. In 2001, Joseph Alouf 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 (Alouf, 2001). 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-

(Geny & 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, membraneperforating 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 et al., 2006) producing approximately US$12 billion in worldwide sales (Wickenden et al., 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 & 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 & Nestorovich, 2015). The progress in the field is supported by theoretical studies that include

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called AB-type toxins act in the cytosol, enzymatically modifying their specific intracellular substrates

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 & 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.

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 -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; 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 et al., 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 H., 1894; Denys & Van de Velde, 1895; Bernheimer & Schwartz, 1963; Chesbro et al., 1965; Donahue & Baldwin, 1966; Arbuthnott et al., 1967) have recently prompted special attention (reviewed in (Prevost et al., 2015)) because of the wide spread of a multi-drug resistant strain of the bacterium, the so-called methicillin-resistant S. aureus, or MRSA (Otto, 2010; Otto, 2012) responsible for several difficult-to-treat infections in humans. Amongst multiple

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INHIBITING MEMBRANE-PERFORATING BACTERIAL TOXINS

toxins, S. aureus secretes several that form pores in host membranes, which include -hemolysin (αHL), -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 & Bubeck Wardenburg, 2013)). More than thirty years ago, Oleg Krasilnikov and co-authors successfully reconstituted αHL into a model lipid bilayer membrane and reported formation of large, slightly anion selective pores of ~ 1 nS conductance (1 M KCl, room temperature) (Krasilnikov et al., 1981). Fifteen years later, a 1.9-Å resolution crystal structure of the αHL channel has shown a hollow mushroom-like 100 Å×100 Å heptamer consisting of the stem, cap, and rim domains and two constriction zones with radii of 0.9 nm and 0.6 – 0.7 nm (Song et al., 1996). The αHL molecular sensing properties, stability, and structural robustness have determined its

molecules to macromolecules and polymers (recently reviewed in (Gurnev & 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 -cyclodextrin (CD) 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 CD 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, CD’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., 2006a; Ragle & Bubeck Wardenburg, 2009; Yannakopoulou et al., 2011). One of the newly designed 7-positively charged hepta-6-substituted -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 αHLmediated 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+CD inhibitors showed the effect similar to

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wide usage for the stochastic resistive-pulse sensing of different types of molecules, ranging from small

IB201, it is too early to speculate on the particular molecular interactions involved in the αHL pore inhibition. In contrast to the homogeneous αHL oligomers, a family of bicomponent Staphylococcal cytolysins (reviewed in (Alonzo & Torres, 2014)), -hemolysin (Hlg), leukocidin (Luk), and Panton-Valentine leukocidin (PVL), are formed via interaction of two distinct class F and class S polypeptides (Kaneko & Kamio, 2004; Yamashita et al., 2011). In the model bilayer membranes, the bi-component octameric leukocidin, Luk was reported to form large, ~ 2.5 nS in 1 M KCl, water-filled pores (Miles et al., 2001; Miles et al., 2002; Miles et al., 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 Downloaded from http://femspd.oxfordjournals.org/ by guest on January 6, 2016

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 refs. (Stiles et al., 2013; Wioland et al., 2013; Alves et al., 2014)), the major virulence factor produced by B and D types of Clostridium 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 anionselective pores with a single-channel conductance ranging between 440 – 640 pS in 1 M KCl (Petit et al., 2001; Knapp et al., 2009). Polyethylene glycol (PEG) partitioning experiments (Nestorovich et al., 2010a) suggest that the channel is asymmetrical with the trans opening of the ETX pore being wider (~1.0 nm) compared to its cis opening (~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 towards

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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 et al., 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

action mechanism is significant but is beyond the limits of this minireview (reviewed in (Young & Collier, 2007; Collier, 2009; Thoren & Krantz, 2011; Feld et al., 2012; Liu et al., 2013; Liu et al., 2015; Liu et al., 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 (MEKK) 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 et al., 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 cryo-electron microscopy reconstruction of the PA63 pore reveals an elongated “flower-on-a-stem” structure ranging in external diameter from 27 Å to ~ 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 et al., 2006), the PA63’s phenylalanine residue at position 427 (-clamp) (Krantz et al., 2005) and the substrate binding α-clamp (Feld et al., 2010; Brown et al.,

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binary toxins (Fig. 1A). Recent progress in understanding the anthrax toxin intracellular uptake and

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). Several binary exotoxins secreted by the pathogenic species of Clostridia (Ohishi & Odagiri, 1984; Simpson, 1984; Aktories et al., 1986; Stiles & Wilkins, 1986; Simpson et al., 1987) are structurally and functionally related to the anthrax toxin (reviewed in (Popoff & Bouvet, 2009; Barth et al., 2015; Knapp et al., 2015)). Those toxins are C2 toxin of Clostridium botulinum, iota toxin of Clostridium perfringens, CDT toxin of Clostridium difficile, and CST toxin of Clostridium spiroforme. Clostridial binary toxins are made of two subunits where the enzymatic A subunit acts through mono-ADP-ribosylation of Gactins 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

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 et al., 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 (-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 and colleagues with tetraalkylammonium ions (Blaustein & Finkelstein, 1990a; Blaustein & Finkelstein, 1990b; Blaustein et al., 1990), 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. 1B) and to protect cells from the binary toxin action. Thus, chloroquine was reported to reversibly block the PA63 (KDPA = 0.51 M in 0.1 M KCl) and C2IIa (KDC2IIa = 10 M in 0.15 M KCl) channels in vitro and prevent transport of the enzymatic C2I

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27% to 38%) of amino acid homology and contain four distinct domains involved in cellular receptor

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; Knapp et al., 2015). Roland Benz and colleagues 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 et al., 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 negatively charged residues in the pore lumen, the -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, -clamp mutations were reported to profoundly affect the binding affinity of hydrophobic cations, suggesting that the -clamp acts as the binding site. The -clamp preferred aromatic moieties by 0.7 kcal/mol per aromatic ring with the most effective molecules binding at KD < 1M. Importantly, the compounds carrying multiple aromatic rings (3 or 4) were reported to have the nM-range binding affinity towards the wt PA63. It was suggested that the -clamp could be involved in nonspecific hydrophobic interactions with blocker molecules while its negative -clouds could also stabilize the binding via aromatic-aromatic, -, and cation- 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 -clamp. Similarly, the -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 10,11) were recently shown to block the PA63 and C2IIa subunits in the model bilayer membranes in the low-M 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

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channel, was primarily determined by the structural features of the blocker molecules. Along with 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 et al., 2015). Bryan Krantz and colleagues 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 (Brown et al., 2015). 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.

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 & 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 & Brewster, 2004), were investigated as potential scaffolds for multivalent binary toxin blockers (Karginov et al., 2005; Nestorovich et al., 2010b; Nestorovich et al., 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. Some preliminary lead compound optimization experiments were conducted. Thus to test if the nature of the attached positively charged functional groups was important, the authors examined a group

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Multivalent positively charged blockers

of hepta-6-guanidine β-cyclodextrin derivatives, in which positive charges were distributed between two nitrogens of the guanidine moiety (Table 3, compounds 11-12) (Karginov et al., 2006b). 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., 2006b; Diaz-Moscoso (3-aminomethyl)benzylthio-β-cyclodextrin (AMBnTβCD), which in addition to the functional aminogroups 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+CD and 8+CD were able to block the PA63 (Yannakopoulou et al., 2011) with 6+CD binding being noticeably weaker and 8+CD 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+CD/pore interactions revealed the contribution of long-range Coulomb forces at moderate and low salt concentrations, whereas at high salt concentrations, the salt-concentrationindependent short-range interactions predominate (Bezrukov et al., 2012; Nestorovich et al., 2012). The 7+CD activity increased in the order Ib < C2IIa < PA63, in parallel with the cationic selectivity of the channels (Fig. 1C). The observed pattern may indicate that the positive charges carried by the CD blockers directly bind to the negatively charged residues inside the channels. However, because 7+CD 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 were also shown to interact with

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et al., 2011; Yannakopoulou et al., 2011) (Table 3, compound 13). One of the tested derivatives, per-6-S-

the conserved -clamp residues. The authors concluded that a number of Coulomb and saltconcentration-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 et al., 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, -, cation- 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 MonteCarlo and molecular dynamics simulations and multi-scale modeling combined with the present and forthcoming single-molecule data will cast light upon the relative contribution from the different types of

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 et al., 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 & 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 to 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 -CD based inhibitor, AmPrCD (0.55 nM) (Nestorovich et al., 2010b). 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

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physical forces defining the strength of blocker/channel interactions.

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 (~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 et al., 1996), possibly because of their increased flexibility. For instance, the channel-blocking activity of the more flexible G1-NH2 carrying 4 surface primary amines was ~ 26 times higher (IC50 = 4.9 nM) compared with that of the 4 positively charged G0 dendrimer (IC50 = 128 nM) (Table 4, compounds 10-12).

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 poreblocking small and multivalent drug molecules of potential pharmaceutical interest that target the socalled “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 ref. (Nestorovich & 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 & Zhorov, 2008).

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CONCLUDING REMARKS

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 7-positively charged multivalent AMBnTβCD was shown to protect against the cytotoxicity induced by four different toxins: anthrax toxins of Bacillus anthracis (Karginov et al., 2006b), clostridial C2, iota (Nestorovich et al., 2011), and CDT toxins (Roeder et al.,

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 towards 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 - edema factor. 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 -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,

13


2014).

we remain hopeful that the inhibitive action of these tailor-made universal antitoxins could be mostly limited to their intentional targets.

Acknowledgements: 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 statement: none declared.

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Figure 1.

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Wijagkanalan W, Kawakami S & Hashida M (2011) Designing dendrimers for drug delivery and imaging: pharmacokinetic considerations Pharm Res 28: 1500-1519.

A

B

1. Small molecule blockers

Chloroquine Table 2, #4

Isoamyltriphenylphosphonium, Table 2, #8

2. Multivalent cyclodextrin blockers

AmPrCD Table 3, #3

AMBnTCD Table 3, #13

3. Multivalent PAMAM dendrimer blockers G0-G3 Table 4, #1-4

Figure Legends Figure 1. 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 effective 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 AmPrCD (middle) (Table 3, #3) and AMBnTCD (bottom) (Table 3, #13). (Right) The PA63 F427A mutant shows much shorter blockages for both AmPrCD (middle) and AMBnTCD (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.

22


C

Table 1. α-, -, and -CD blockers of αHL (Yannakopoulou et al., 2011)

#

Cyclodextrin

NH

1

Inhibition of conductance,

Inhibition of cytotoxicity,

IC50, nM (0.1 M KCl)

IC50, M

NH3Cl

>5000

>100

NH3Cl

~50

3.3+2.3

>5000

>100

Substituent

NHBoc

 O

NH

2

NHBoc

 O

3

NHBoc



NH3Cl O

Table 2. Small molecule cationic blockers of PA63 channel conductance. Note: only molecules with IC50 ≤ 3 µM are shown, see ref. (Bezrukov & 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 M

Small aromatic cationic compounds (Krantz et al., 2005), (0.1 M KCl) 3

Tetraphenylphosphonium

46 ± 2 nM

23


NH

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


4

Azolopyridinium Salts (Beitzinger et al., 2013) (0.15 M KCl) 10

HA 1383

1.3 M

11

HA 1568

2.7 M

24

Table 3. Polyvalent cationic cyclodextrin blockers of PA63 ion current and LT cytotoxicity. Note: only selected molecules are shown, see ref. (Bezrukov & Nestorovich, 2015) for complete list of the antitoxins.

#

Cyclodextrin

Substituent

Inhibition of conductance, IC50, nM (0.1 M KCl)

Inhibition of cytotoxicity, IC50, M

Hepta-6-aminoalkyl -cyclodextrin derivatives (Karginov et al., 2006) 

-NH2

140 ± 90

20 ± 9

2



-S(CH2)2NH2

3.5 + 0.9

7.8 + 2.4

3



-S(CH2)3NH2

0.57 + 0.39

2.9 + 1.0

4



-S(CH2)4NH2

1.1 + 0.5

5.1 + 2.4

5



-S(CH2)5NH2

3.8 + 1.0

7.5 + 2.4

7



-S(CH2)6NH2

0.97 + 0.38

0.6 + 0.3

8



-S(CH2)7NH2

4.6 + 3.2

1.9 + 1.1

9



-S(CH2)8NH2

2.4 + 0.95

0.3 + 0.1

10



-S(CH2)10NH2

27.0 + 17.0

2.6 + 0.7


1

Hepta-6-guanidinealkyl -cyclodextrin derivatives (Karginov et al., 2006) NH2

11



-S(CH2)2

12



-S(CH2)3

N H

NH

5.3+3.2

8.9+6.0

12.6+9.0

12.2+2.9

NH2 N H

NH

Hepta-6-arylamine -cyclodextrin derivative (Karginov et al., 2006; Yannakopoulou et al., 2011)

13



NH3Cl

0.13+0.10

0.8+0.5

S

25

Cationic - and  cyclodextrin derivatives (Yannakopoulou et al., 2011) 14



-NH2

1200 ± 300

>100

15



-NH2

170 ± 50

12 ± 3

16



NH3Cl

29+5

45+13

NH3Cl

2.8+1.3

5.4+0.8

S



17

S


Table 4. Polyvalent cationic PAMAM dendrimer- and dendron-based blockers of PA63 (Forstner et al., 2014)

#

Generation

Measured diameter, Å

NH2 surface groups number

Inhibition of conductance, 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 M

9

1

22

8

4.61.7 M

26

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


27

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