The Comprehensive Sourcebook of Bacterial Protein Toxins

Inhibitors of pore-forming 38 toxins Sergey M. Bezrukov1 and Ekaterina M. Nestorovich2 1Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.; 2Department of Biology, The Catholic University of America, Washington, DC, USA. Introduction A significant number of bacteria, including the emerging multidrug-resistant “super- bugs,” such as Pseudomonas aeruginosa, Clostridium difficile, Staphylococcus aureus, and Escherichia coli, secrete highly potent exotoxins with no antitoxins cur- rently available to disable them. Several bacterial toxins, such as the botulinum neu- rotoxin (BoNT) of Clostridium botulinum, can be aerosolized and used as biological weapons directly. With some bacterial diseases, such as anthrax, flu-like symptoms appear only after bacteria have proliferated inside the host and have started to produce toxins that ultimately cause death [1]. Even though aggressive antibacterial treatment effectively inhibits Bacillus anthracis propagation, patients can still die because of intoxication [2]. Furthermore, several engineered strains of B. anthracis, which are resistant to the existing antibacterial agents, have already been developed [3,4]. Therefore, the discovery and development of small-molecule antitoxins represent a high-priority task in drug design, medicinal chemistry, and biodefense [4,5]. In the last ten years, a number of biophysical studies have focused on finding ways to disable those bacterial toxins which form ion channels in the host’s cell membranes. This chapter focuses on the existing efforts to design antitoxins that counteract channel- forming bacterial toxins. Idea of toxin inhibition by channel blockage is suggested by nature As evolution has progressed, nature has created various neurotoxins, which are small molecule or peptide-based toxins that exclusively target ion-selective channels of excitable cells [6–8]. Humanity has had many interactions with neurotoxins over his- tory, much to its detriment. Regardless of which version of Cleopatra’s self-poisoning is factual, toxin-triggered alteration of channel functioning was likely involved. She could have either suffered from intoxication following blockage of her ligand- gated channels by venom of the Egyptian cobra or from the fatal action of the chloride channel inhibitor extracted from cicuta. The Comprehensive Sourcebook of Bacterial Protein Toxins. DOI: http://dx.doi.org/10.1016/B978-0-12-800188-2.00038-0 © 2015 Elsevier Ltd. All rights reserved. 1096 Clinical Aspects, Applications of Bacterial Protein Toxins in Cell Biology and Therapy These days, due to research on the biological functions of neurotoxins, these compounds are used not only to understand the structural and functional aspects of ion channels of excitable membranes, but also to advance the pharmaceutical approaches to use these toxins in different therapeutic applications. Contrariwise, among the great variety of virulence factors secreted by different organisms, including bacteria, there is a significant group of toxins, which instead of blocking the host ion-selective channels, form ion-conductive pores in cell membranes. For most of these toxins, no effective blockers have been developed and approved for animal or human use. However, one of the modern ideas to target the bacterial exotoxins is obvious and simple. Following nature, which generated pore-blocking toxins, we can rationally design powerful antitoxin agents that will specifically block the conductive pathways of the pore-forming virulence factors. This idea has already seen its medical application within the past 50 years. In 1965, amantadine, the small-molecule blocker of the tetrameric proton-selective M2 channel from the viral envelope of influenza A virus [9–11] had been approved for human use. Despite the developed resistance of the virus to amantadine, the M2 channel is still one of the main targets for virtual screening and rational design of prospective drugs to treat influenza. A number of different viroporins, including p7 of hepatitis C virus and viral protein U (Vpu) of HIV-1, were also explored as potential targets for the development of pore-blocking agents. Therefore, the approach originally created by nature to modify the channel’s functions by the selectively acting pore-blocking agents enjoys well-deserved attention. Despite not being able to replicate the same evolutionary path which nature has previously taken, it is now possible to use modern-day single-channel and single-molecule biophysical methodologies to investigate channel-forming toxins and to rationally design their potential inhibitors by exploring the roles of various interactions involved in channel-facilitated transport. Pore-forming bacterial toxins and their inhibitors Interactions with solute define channel transport properties Transport properties of a channel are governed by its interactions with solute molecules. In addition to Coulomb, van der Waals, hydrogen bonding, and other short-range forces, an important role belongs to entropic interactions. Indeed, ion- and metabolite-specific channels restrict translational freedom of solutes that they transport by confining the molecules within their water-filled pores. In addition, the geometry of practically every known channel pore is not that of a regular cylinder, but is often characterized by constrictions along the channel axis. During the past decade, an analytical approach was developed [12–18], which allows one to calculate the major transport parameters such as the probability and characteristic times of solute translocation. The approach is based on the description of the solute particle diffusion in the channel in terms of the particle Green’s function G(x,t;x0). It is defined as the = probability density to find the particle at point x at time t if it was at x0 at t 0 and Inhibitors of pore-forming toxins 1097 has not escaped from the channel during that time. The Green’s function obeys the Smoluchowski equation: ∂Gxtx(,; ) ∂ ⎪⎧ ∂ ⎪⎫ 0 ⎨Dx()exp (ββ Ux ()) [ exp (Ux ())(,; Gxtx )]⎬, (38.1) ∂ ∂ ⎪ ch ∂ 0 ⎪ tx⎩⎪ t ⎭⎪ = δ − < < with the initial condition G(x,0;x0) (x x0), 0 x L, and the radiation boundary conditions at the channel openings at x = 0 and x = L ∂ k [(,;)Gxtxexpxp (β Ux ())] 5 0 Gt(,;00xU ) e (β ()), ∂ 0 0 x x50 Dch ()0 (38.2) ∂ k 2 [(,;)Gxtxexpxp (β Ux ())] 5 L GLtx(,; ) e (β UL ()). ∂ 0 0 x xL5 DLch () β = Here, 1/(kBT), with kB and T having their usual meanings of the Boltzmann constant and absolute temperature, respectively; and Dch(x) is the particle diffusion coefficient inside the channel. The potential of mean force U(x) includes, in the general case, contributions not only from actual physical forces, but also entropic potential accounting for the varying channel geometry. The rate constants k0 and kL relate the one-dimensional (1D) problem described by Eqs. (38.1) and (38.2) with the three-dimensional (3D) case of interest by characterizing the escape efficiency. For the channel of a complex (including asymmetric) shape, but with identical circular openings of radius R on both ends, these rate constants are [12–14]: 4D kkk555 b , (38.3) 0 L π R where Db is the particle diffusion coefficient in the bulk. In the general case, it is impossible to solve Eqs. (38.1)–(38.3) analytically; however, the most important characteristics of the particle dynamics in the channel can be calculated explicitly. Among them, the particle translocation probability is of key interest. In the absence of the external fields, U(0) = U(L), and with Eq. (38.3) satis- fied, the probabilities to translocate from the left to the right, Ptr(0), and from the right to the left, Ptr(L), are equal to each other and are given by [13] 1 PPL()0 ϭϭ () . (38.4) tr tr 4D L exp(())βUx 2 1 b ∫ dx π 0 R Dxch () It is seen that with the increasing attractive interactions (that is, a deep potential well, −βU(x) ≫ 1), the translocation probability increases to its maximal value of ½ for the diffusion in the absence of the external driving force. 1098 Clinical Aspects, Applications of Bacterial Protein Toxins in Cell Biology and Therapy Other important parameters are different characteristic times spent by the particle in the channel. Among them are the mean translocation time, return times (for a particle which enters and leaves the channel from the same side), and lifetimes of the particle in the channel. For the translocation time, it was found that, surprisingly, it is τ = τ = direction-independent, tr(0) tr(L) ttr, and given by [14] L ⎡ x β ⎤ ⎡ L β ⎤ ⎢ 112exp(())Uy ⎥ ⎢ βΔ exp( UUy()) ⎥ 2β ∫ ⎢11k0 ∫ dy⎥ ⎢ kL exp() U ∫ dexpyUxdx⎥ (()) 0 ⎣ 0 Dy() ⎦ ⎣ x Dy() ⎦ τ 5 ch ch , tr L β 121βΔ 2βΔ exp(())Ux kk0 L exp() Ukk0 L exxp()U ∫ dx 0 Dxch () (38.5) where ΔU = U(L) − U(0). Moreover, not only the mean value, but also the probability density of the translocation time is direction independent [18]. However, the mean lifetimes in the channel, depend on the side from which the particle enters the channel. Even for U(0) = U(L), they are still side dependent [14]: L ⎡ L β ⎤ ⎢ + exp(Uy ( )) ⎥ −β ∫ ⎢1 kL ∫ dy⎥ exp (( U x)) dx 0 ⎣ x Dy() ⎦ τ()0 = ch L β +++ exp(Uy ( )) k0 kkkLL0 ∫ dy 0 Dych () (38.6) L ⎡ x β ⎤ ⎢ + exp(Uy ( )) ⎥ −β ∫ ⎢1 k0 ∫ dyUxdx⎥ exp ( ( )) 0 ⎣ 0 Dy() ⎦ τ()L = ch L β ++ exp(Uy ( )) kkkk00LL∫ ddy 0 Dych () Eqs. (38.5) and (38.6) give the mean times for arbitrary dependences of the potential of mean force, U(x), and particle diffusion coefficient, Dch(x), on the particle coor- dinate in the channel, x. More general formulas for the translocation probability and characteristic times are available in [14,18]. For a simplified case of a symmetric cylindrical channel with a rectangular potential well of depth U0 occupying the entire channel and a position-independent dif- = fusion coefficient Dch(x) Dch, the expressions for the translocation probability and average lifetime in the channel simplify and take the form 1 P 5 (38.7) tr 4DL 2 12b exp( βU ) π 0 Dch R and π τ = RL β (38.8) exp(U0 ).

Load more