toxins Review Inhibition of Pore-Forming Proteins Neža Omersa, Marjetka Podobnik and Gregor Anderluh * Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; [email protected] (N.O.); [email protected] (M.P.) * Correspondence: [email protected]; Tel.: +386-1-476-02-61 Received: 28 June 2019; Accepted: 10 September 2019; Published: 19 September 2019 Abstract: Perforation of cellular membranes by pore-forming proteins can affect cell physiology, tissue integrity, or immune response. Since many pore-forming proteins are toxins or highly potent virulence factors, they represent an attractive target for the development of molecules that neutralize their actions with high efficacy. There has been an assortment of inhibitors developed to specifically obstruct the activity of pore-forming proteins, in addition to vaccination and antibiotics that serve as a plausible treatment for the majority of diseases caused by bacterial infections. Here we review a wide range of potential inhibitors that can specifically and effectively block the activity of pore-forming proteins, from small molecules to more specific macromolecular systems, such as synthetic nanoparticles, antibodies, antibody mimetics, polyvalent inhibitors, and dominant negative mutants. We discuss their mechanism of inhibition, as well as advantages and disadvantages. Keywords: pore-forming proteins; pore-forming toxins; anthrax toxin; lipid membranes; pore formation; inhibitor Key Contribution: The manuscript reviews current possibilities for inhibition of pore-forming proteins with an emphasis on alternative approaches. 1. Introduction to Toxic Pore-Forming Proteins 1.1. Different Modes of Creating a Pore in Cellular Membranes Plasma as well as organelle membranes are vital for cells. They protect cells from the environment, including invading organisms, enable exchange of substances either between cells and their surroundings or between different cellular compartments, cell adhesion, transport, metabolism, and flow of information via cell signaling. Thus, interfering with the integrity of membranes can disturb cellular processes and can, in extreme cases, be detrimental. During evolution, organisms from all kingdoms of life have evolved mechanisms to form pores in membranes, in order to attack other organisms or defend against them, to digest their prey, or as a part of the immune system to remove unwanted cells. Excellent reviews are available describing diverse modes of transmembrane pore formation by proteins [1–8]. Pore-forming proteins (PFPs) are generally secreted by cells as soluble monomers that assemble into structured oligomeric complexes at the target membrane surface. Upon binding to the lipid membrane, monomers oligomerize on its surface to form structured assemblies called prepores and undergo conformational changes in order to expose hydrophobic surfaces, leading to spontaneous insertion into the lipid bilayer, pore formation, and membrane permeabilization. Pores made by PFPs are largely diverse in inner diameter, ranging from 0.7 nm as in the case of colicins [9] to the largest known pores of cholesterol-dependent cytolysins (CDCs) with diameters of 25–40 nm [8]. Depending on the size of the pore, different substances pass through, such as ions (e.g., Ca2+, K+), small molecules (e.g., adenosine triphosphate (ATP)), or large molecules (e.g., proteins) [10]. Toxins 2019, 11, 545; doi:10.3390/toxins11090545 www.mdpi.com/journal/toxins Toxins 2019, 11, 545 2 of 22 Toxins 2018, 10, x FOR PEER REVIEW 2 of 22 Structurally, PFPs are divided into two major classes based on secondary structure elements that divided into two major classes based on secondary structure elements that frame the transmembrane frame the transmembrane channel of their pores either with α-helices (i.e., α-PFPs) [11] or β-barrels channel of their pores either with α-helices (i.e., α-PFPs) [11] or β-barrels (β-PFPs) [1,2,12] (Figure 1). (β-PFPs) [1,2,12] (Figure1). Three dimensional structures of soluble monomeric PFPs, prepores and Three dimensional structures of soluble monomeric PFPs, prepores and pores from different families pores from di erent families have been known to date [13–22] and several excellent reviews describe have been knownff to date [13–22] and several excellent reviews describe their features [10,23,24]. Thesetheir features structural [10 ,models23,24]. Theseprovide structural a valuable models insight provide into athe valuable mechanism insight of intoaction the by mechanism PFPs and of cruciallyaction by contribute PFPs and cruciallyto a rational contribute design toof atheir rational potential design inhibitors. of their potential The shapes inhibitors. of pores The are shapesquite diverse.of pores Some are quite PFPs diverse. form matrix-type Some PFPs toroidal form matrix-type pores, where toroidal the transmembrane pores, where the protein transmembrane units are interspersedprotein units by are lipids interspersed [3], such by aslipids actinoporins [3], such [25,26 as actinoporins], colicins [27], [25 ,and26], proteins colicins [ 27from], and the proteinsBcl-2 family from ofthe apoptotic Bcl-2 family proteins of apoptotic [28]. In contrast proteins to [28 toroidal]. In contrast formations, to toroidal pore walls formations, can also pore be completely walls can also built be α α ofcompletely proteins, builtforming of proteins, either compact forming α either-barrels compact as in the-barrels case of α as-PFP in the cytolysin case of A-PFP from cytolysin Escherichia A fromcoli (FigureEscherichia 1), colior (Figureβ-barrels,1), orformedβ-barrels, by β formed-PFPs such by β -PFPsas α- suchand asγ-toxinα- and fromγ-toxin Staphylococcus from Staphylococcus aureus, membraneaureus, membrane attack attackcomplex/perforin complex/perforin (MACPF)/ch (MACPF)olesterol-dependent/cholesterol-dependent cytolysins cytolysins (CDCs) (CDCs) protein protein superfamily, or or aerolysin-like aerolysin-like proteins proteins [2]. [2]. β-barrelsβ-barrels are are also also formed formed by bybacterial bacterial secretion secretion systems systems of typeof type III and III and IV [1,29] IV [1 ,and29] andbinary binary AB type AB toxins, type toxins, where where the B component the B component is pore-forming is pore-forming and allows and translocationallows translocation of A subunits of A subunits that possess that ca possesstalytic catalyticactivity (e.g., activity diphtheria; (e.g., diphtheria; anthrax; α anthrax;-, ε-, ι-, αand-, " C2-, ι -, toxins)and C2 [14,30–32] toxins) [14 (Figure,30–32] 1). (Figure In the1 ).case In theof β case-barrel of proteinsβ-barrel such proteins as MACPF/CDCs, such as MACPF arc-type/CDCs, toroidal arc-type porestoroidal partly pores lined partly with lined lipids with as lipids well as as well multimeric as multimeric pores pores can canalso also be bebuilt built in inaddition addition to to fully fully proteinaceous ring-shaped ring-shaped pores pores [3]. [3]. The The majority majority of of work work regarding regarding PFP PFP inhibitors inhibitors has has been been done done on the B component (protective (protective antigen antigen or or PA) PA) of of the the anthrax anthrax toxin toxin from from BacillusBacillus anthracis anthracis, ,hence hence a a short description of of this this toxin toxin follows. follows. For For an an in in-depth-depth understanding understanding of of individual individual PFPs PFPs and and their their characteristics, an an interested interested reader reader can can choose choose from from recent recent reviews reviews about about that that topic topic [1–3,7,8,33–38]. [1–3,7,8,33–38]. Figure 1. Two major classes of pore-forming proteins (PFPs) based on the structural element present Figurein the final1. Two pore, majorα-helical classes PFPs of pore-forming exemplified proteins by the cytolysin (PFPs) based A from on Escherichiathe structural coli element(PDB ID present 2WCD) α inon the the final left, pore, and β -barrel-helical PFPs PFPs exemplified exemplified by by the the anthrax cytolysin toxin A from protective Escherichia antigen coli pore(PDB from ID 2WCD)Bacillus β onanthracis the left,(PDB and ID -barrel 3J9C) on PFPs the right.exemplified Ribbon by representations the anthrax toxin of proteins protective are drawnantigen by pore using from PyMOL Bacillus [39 ]. anthracisA single (PDB protomer ID 3J9C) in the on pore the right. is shown Ribbon in pink. represen Thetations approximate of proteins position are drawn of the lipidby using membrane PyMOL is [39].shown A single in brown. protomer in the pore is shown in pink. The approximate position of the lipid membrane is shown in brown. 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