Artificial transmembrane ion transporters as potential therapeutics

Abstract

Artificial transmembrane ion transporters are attractive in the context of drug discovery since, in principle, they may be used as novel therapeutics by promoting apoptosis (programmed cell death), interfering with autophagic processes, or inducing an antibiotic response via disrupting cellular ion homeostasis or dissipating intracellular pH gradients. Considerable effort has been devoted into the design of artificial transporters with selective ion recognition and transport. However, only limited progress has been made in terms of translating fundamental research involving transport mechanisms into systems that function in vitro. In this review, we summarize recent progress in the area of artificial ion transporters with a focus on their application as potential therapeutics. The associated discussion of accomplishments and remaining challenges is designed to hasten the day when research on artificial transmembrane ion transporters is translated into bona fide clinical benefits. Various artificial transmembrane transporters, designed to function through mobile carrier or channel mechanisms, have been developed in the past decade. With the aid of structural manipulation and by employing either discrete chemical entities or self-assembled nanostructures, progress has been made in achieving the selective recognition and transmembrane transport of key ions. The ability to perturb intracellular pH or disrupt intracellular ion homeostasis makes transmembrane ion transporters of interest as potential therapeutics that might see use as cancer treatments or as antibacterial agents. In this review, recent progress in the area of artificial transmembrane ion transporter research is summarized with an emphasis on applications involving anticancer research and antibiotic applications. The examples chosen for highlights are meant to be illustrative of key themes involving synthetic ion transport rather than comprehensive. Nevertheless, it is anticipated that this review will provide a useful entry point for the general reader and set the stage for further progress in the area. Various artificial transmembrane transporters, designed to function through mobile carrier or channel mechanisms, have been developed in the past decade. With the aid of structural manipulation and by employing either discrete chemical entities or self-assembled nanostructures, progress has been made in achieving the selective recognition and transmembrane transport of key ions. The ability to perturb intracellular pH or disrupt intracellular ion homeostasis makes transmembrane ion transporters of interest as potential therapeutics that might see use as cancer treatments or as antibacterial agents. In this review, recent progress in the area of artificial transmembrane ion transporter research is summarized with an emphasis on applications involving anticancer research and antibiotic applications. The examples chosen for highlights are meant to be illustrative of key themes involving synthetic ion transport rather than comprehensive. Nevertheless, it is anticipated that this review will provide a useful entry point for the general reader and set the stage for further progress in the area. Cellular membranes serve as barriers for the exchange of most substrates into and out of cells. However, the exchange of solutes between cells and their extracellular milieu is crucial for maintaining the normal operation of biological systems.1Szostak J.W. Bartel D.P. Luisi P.L. Synthesizing life.Nature. 2001; 409: 387-390Google Scholar, 2Dubyak G.R. Ion homeostasis, channels, and transporters: an update on cellular mechanisms.Adv. Physiol. Educ. 2004; 28: 143-154Google Scholar, 3Bröer S. Amino acid transport across mammalian intestinal and renal epithelia.Physiol. 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Artificial transmembrane ion transporters, which can induce apoptosis (so-called programmed cell death) in cancer cells by changing the intracellular pH or disrupting intracellular ion homeostasis, could provide one such alternative approach.24Hosogi S. Kusuzaki K. Inui T. Wang X. Marunaka Y. Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell.J. Cell. Mol. Med. 2014; 18: 1124-1133Google Scholar, 25Busschaert N. Park S.H. Baek K.H. Choi Y.P. Park J. Howe E.N.W. Hiscock J.R. Karagiannidis L.E. Marques I. Félix V. et al.A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations.Nat. Chem. 2017; 9: 667-675Google Scholar, 26Smith B.A. Daschbach M.M. Gammon S.T. Xiao S. Chapman S.E. Hudson C. et al.In vivo cell death mediated by synthetic ion channels.Chem. Commun. 2011; 47: 7977-7979Google Scholar, 27Park S.H. Park S.H. Howe E.N.W. Hyun J.Y. Chen L.J. Hwang I. 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Rev. 2010; 39: 1448-1456Google Scholar For instance, polyether ionophores, complex natural products that can transport cations across biological membranes, often display antimicrobial activity, a finding that has inspired the design and synthesis of artificial ion transporters as antibacterial agents.34Kevin II, D.A. Meujo D.A. Hamann M.T. Polyether ionophores: broad-spectrum and promising biologically active molecules for the control of drug-resistant bacteria and parasites.Expert Opin. Drug Discov. 2009; 4: 109-146Google Scholar Since a variety of ion transporters have been reported to date, the discussion will be classified according to the type of ions involved, with particular emphasis being given to systems that show promise as transporters of chloride, potassium, and sodium. This focus reflects not only the physiological importance of these ions but also the fact that they have been the target of most artificial ion transport development work reported to date. Additionally, the examples chosen in this review represent a selection of anion transporters reported in the literature that have been studied most extensively in biological systems. To provide the readers with an introductory guideline to this field, a brief summary of synthetic ion transporters is given in this section. The term ion carrier applies to a transporter that shuttles across the bilayer and, in doing so, brings about the translocation of ions (Figure 1). In contrast, ion channels are scaffolds that can mediate ion transport across membrane bilayers without undergoing appreciable motion and without disturbing the lipid bilayer supra-structures. Synthetic ion channels include both unimolecular channels and self-assembled channels. Unimolecular synthetic ion channels are typically macrocyclic molecules that are long enough to insert into lipid bilayer membranes and serve as guides for transmembrane ion transport. An advantage of unimolecular channels is their stability, reflecting the fact that they comprise single molecules that remain intact inside lipid bilayers. However, the synthesis of unimolecular channels is often difficult and time consuming. Therefore, the majority of synthetic ion channels reported to date have been created in situ via the controlled self-assembly of monomers within the lipid bilayers. However, constructing channels with stable structures and selective binding sites via the rational self-assembly of smaller building blocks remains a challenge. To differentiate an ion channel from a carrier-type ion transporter, black lipid membrane (BLM) experiments in planar bilayers are typically performed (Figure 2A).35Matile S. Sakai N. The characterization of synthetic ion channels and pores.in: Schalley C.A. Analytical Methods in Supramolecular Chemistry. Second Edition. Wiley-VCH, Weinheim, Germany2012: 711-742Google Scholar Dynamic “open-closed” conductance is generally taken as evidence of ion channel formation (Figure 2B). Moreover, some key parameters of the ion channels could be inferred from these studies, such as the conductance g, the lifetime τ1, and the open probability Po. The construction of so-called Hill plots is another tool used to distinguish carrier- from channel-based transport mechanisms.36Litvinchuk S. Bollot G. Mareda J. Som A. Ronan D. Shah M.R. Perrottet P. Sakai N. Matile S. Thermodynamic and kinetic stability of synthetic multifunctional rigid-rod β-barrel pores: evidence for supramolecular catalysis.J. Am. Chem. Soc. 2004; 126: 10067-10075Google Scholar The slope of a Hill plot is equal to the Hill coefficient for the interactions between the agent in question and the membrane under study. A Hill plot with a slope greater than 1.0 is taken as an indication that there is a synergistic concentration dependence (channel mechanism), whereas a slope less than 1.0 indicates competitive concentration dependence (carrier mechanism) (Figure 3A). These limiting regimes are generally interpreted in terms of channel and carrier mechanisms, respectively. However, it is to be appreciated that Hill analyses of synthetic ion channels are not fully developed, and appropriate caution must be exercised in terms of drawing concrete mechanistic conclusions.Figure 3(A) Typical Hill plot for synthetic ion transporters. Reprinted with permission from Matile and Sakai,35Matile S. Sakai N. The characterization of synthetic ion channels and pores.in: Schalley C.A. Analytical Methods in Supramolecular Chemistry. Second Edition. Wiley-VCH, Weinheim, Germany2012: 711-742Google Scholar copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.Show full caption(B) Standard configuration of the so-called HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) assay. Reprinted with permission from Wu et al.,37Wu X. Howe E.N.W. Gale P.A. Supramolecular transmembrane anion transport: new assays and insights.Acc. Chem. Res. 2018; 51: 1870-1879Google Scholar copyright 2018 American Chemical Society.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (B) Standard configuration of the so-called HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) assay. Reprinted with permission from Wu et al.,37Wu X. Howe E.N.W. Gale P.A. Supramolecular transmembrane anion transport: new assays and insights.Acc. Chem. Res. 2018; 51: 1870-1879Google Scholar copyright 2018 American Chemical Society. A synthetic ion transporter involved in mediating the transport of only a single ion or molecule across a lipid membrane is termed as uniporter, and the process is referred to as uniport. Transporters that move two molecules or ions in the same direction across the membrane are called symporters. If two molecules are moved in opposite directions across the bilayer, the process is called antiport. Transporters involved in the movement of specific ions are called ionophores. If the action of a transporter in moving ions across a membrane results in a net change in charge, the process is described as electrogenic; if there is no net change in charge, the transport is described as electroneutral. The primary method used to characterize putative ion transporters under cell-free conditions is the liposomal assay.37Wu X. Howe E.N.W. Gale P.A. Supramolecular transmembrane anion transport: new assays and insights.Acc. Chem. Res. 2018; 51: 1870-1879Google Scholar In general, fluorescence spectroscopy is used to monitor fluorophore-containing large unilamellar vesicles (LUVs). Depending on the design of the setup and the fluorescent probes employed, such analyses can provide insights into the influx or efflux of various types of ions. Often, LUVs are prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and then encapsulated with either the pH-sensitive ratiometric fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) or the halide-sensitive fluorophore lucigenin (Figure 3B). The fluorescence intensities of HPTS and lucigenin are calibrated to the pH and chloride concentrations, respectively. This allows the optical-based monitoring of H+ or Cl− transport processes. Other methods for monitoring changes in ion concentrations, such as NMR spectroscopy and ion-selective electrodes, have also been used in liposomal transport assays. To test whether a synthetic compound can act as an ion transporter in cells, its ability to induce cytosolic ion concentration changes needs to be evaluated. Taking a potential chloride transporter as an example, changes in Cl− concentration in the intracellular matrix are typically monitored using a chloride-selective fluorescent probe, such as N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), which undergoes fluorescence quenching in the presence of chloride ions. Therefore, upon preincubation of cells with an effective transporter or synthetic channel, quenching of MQAE fluorescence would be expected due to a mediated increase in the intracellular Cl− concentration.38Verkman A.S. Sellers M.C. Chao A.C. Leung T. Ketcham R. Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications.Anal. Biochem. 1989; 178: 355-361Google Scholar A related strategy involves the use of Fischer rat thyroid epithelial (FRT) cells that express a mutant yellow fluorescent protein (YFP) whose fluorescence is sensitively quenched by chloride ions.39Jayaraman S. Haggie P. Wachter R.M. Remington S.J. 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Transmembrane ion channels are essential for cell proliferation and appear to play a role in the development of cancer. This was initially noted in the case of potassium channels but has been suggested for other cation channels and chloride channels.44Kunzelmann K. Ion channels and cancer.J. Membr. Biol. 2005; 205: 159-173Google Scholar Thus, over the past decades, there has been considerable effort devoted to creating artificial ion transporters that can help understand and mimic the functions of natural ion channels. In fact, a number of synthetic ion transporters, designed to promote the through-membrane transport of specific ions, such as chloride, sodium, and potassium, have been developed to date.45Benke B.P. Aich P. Kim Y. Kim K.L. Rohman M.R. Hong S. Hwang I.C. Lee E.H. Roh J.H. Kim K. Iodide-selective synthetic ion channels based on shape-persistent organic cages.J. Am. Chem. Soc. 2017; 139: 7432-7435Google Scholar, 46Lang C. Li W. Dong Z. Zhang X. Yang F. 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