Halide-selective, proton-coupled anion transport by phenylthiosemicarbazones.

Phenylthiosemicarbazones (PTSCs) are proton-coupled anion transporters with pH-switchable behaviour known to be regulated by an imine protonation equilibrium. Previously, chloride/nitrate exchange by PTSCs was found to be inactive at pH 7.2 due to locking of the thiourea anion binding site by an intramolecular hydrogen bond, and switched ON upon imine protonation at pH 4.5. The rate-determining process of the pH switch, however, was not examined. We here develop a new series of PTSCs and demonstrate their conformational behaviour by X-ray crystallographic analysis and pH-switchable anion transport properties by liposomal assays. We report the surprising finding that the protonated PTSCs are extremely selective for halides over oxyanions in membrane transport. Owing to the high chloride over nitrate selectivity, the pH-dependent chloride/nitrate exchange of PTSCs originates from the rate-limiting nitrate transport process being inhibited at neutral pH.

Photomodulation of Transmembrane Transport and Potential by Stiff-Stilbene Based Bis(thio)ureas.

Membrane transport proteins fulfill important regulatory functions in biology with a common trait being their ability to respond to stimuli in the environment. Various small-molecule receptors, capable of mediating transmembrane transport, have been successfully developed. However, to confer stimuli-responsiveness on them poses a fundamental challenge. Here we demonstrate photocontrol of transmembrane transport and electric potential using bis(thio)ureas derived from stiff-stilbene. UV-vis and 1H NMR spectroscopy are used to monitor E-Z photoisomerization of these bis(thio)ureas and 1H NMR titrations reveal stronger binding of chloride to the (Z)-form than to the (E)-form. Additional insight into the binding properties is provided by single crystal X-ray crystallographic analysis and DFT geometry optimization. Importantly, the (Z)-isomers are much more active in transmembrane transport than the respective (E)-isomers as shown through various assays. As a result, both membrane transport and depolarization can be modulated upon irradiation, opening up new prospects toward light-based therapeutics as well as physiological and optopharmacological tools for studying anion transport-associated diseases and to stimulate neuronal activity, respectively.

Synthetic Na+/K+ exchangers promote apoptosis by disturbing cellular cation homeostasis.

A number of artificial cation ionophores (or transporters) have been developed for basic research and biomedical applications. However, their mechanisms of action and the putative correlations between changes in intracellular cation concentrations and induced cell death remain poorly understood. Here, we show that three hemispherand-strapped calix[4]pyrrole-based ion-pair receptors act as efficient Na+/K+ exchangers in the presence of Cl- in liposomal models and promote Na+ influx and K+ efflux (Na+/K+ exchange) in cancer cells to induce apoptosis. Mechanistic studies reveal that these cation exchangers induce endoplasmic reticulum (ER) stress in cancer cells by perturbing intracellular cation homeostasis, promote generation of reactive oxygen species, and eventually enhance mitochondria-mediated apoptosis. However, they neither induce osmotic stress nor affect autophagy. This study provides support for the notion that synthetic receptors, which perturb cellular cation homeostasis, may provide new small molecules with potentially useful apoptotic activity.

Tetrapodal Anion Transporters.

Synthetic anion transporters that facilitate chloride transport are promising candidates for channelopathy treatments. However, most anion transporters exhibit an undesired side effect of facilitating proton transport via interacting with fatty acids present in the membrane. To address the limitation, we here report the use of a new tetrapodal scaffold to maximize the selective interaction with spherical chloride over binding the carboxylate headgroup of fatty acids. One of the new transporters demonstrated a high selectivity for chloride uniport over fatty acid-induced proton transport while being >10 times more active in chloride uniport than strapped calixpyrroles that were previously the only class of compounds known to possess similar selectivity properties.

Stimuli-Responsive Cycloaurated "OFF-ON" Switchable Anion Transporters.

Anion transporters have shown potential application as anti-cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are "switched on" in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concentrations in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.

Development of a Library of Thiophene-Based Drug-Like Lego Molecules: Evaluation of Their Anion Binding, Transport Properties, and Cytotoxicity.

The anion-binding and transport properties of an extensive library of thiophene-based molecules are reported. Seventeen bis-urea positional isomers, with different binding conformations and lipophilicities, have been synthesized by appending α- or ß-thiophene or α-, ß-, or γ-benzo[b]thiophene moieties to an ortho-phenylenediamine central core, yielding six subsets of positional isomers. Through 1 H NMR, X-ray crystallography, molecular modelling, and anion efflux studies, it is demonstrated that the most active transporters adopt a pre-organized binding conformation capable of promoting the recognition of chloride, using urea and C-H binding groups in a cooperative fashion. Additional large unilamellar vesicle-based assays, carried out under electroneutral and electrogenic conditions, together with N-methyl-d-glucamine chloride assays, have indicated that anion efflux occurs mainly through an H+ /Cl- symport mechanism. On the other hand, the most efficient anion transporter displays cytotoxicity against tumor cell lines, while having no effects on a cystic fibrosis cell line.

Fatty Acid Fueled Transmembrane Chloride Transport.

Generation of chemical gradients across biological membranes of cellular compartments is a hallmark of all living systems. Here we report a proof-of-concept prototype transmembrane pumping system in liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addition of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochemical H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl- cotransport. Further addition of fatty acid efficiently fuels the system to continuously drive chloride transport against the concentration gradient, up to [Cl-]in 65 mM | [Cl-]out 100 mM, and is 1400 times more efficient than using an external fuel. Based on our findings from dissecting the H+/Cl- flux process with the use of different liposomal fluorescence assays, and supported by additional liposome-based 13C NMR and DLS studies; we proposed that the presence of an anionophore can induce asymmetric distribution of fatty acid, and contribute to another Cl- flux mechanism in this system.

Investigating the Influence of Steric Hindrance on Selective Anion Transport.

A series of symmetrical and unsymmetrical alkyl tren based tris-thiourea anion transporters were synthesised and their anion binding and transport properties studied. Overall, increasing the steric bulk of the substituents resulted in improved chloride binding and transport abilities. Including a macrocycle in the scaffold enhanced the selectivity of chloride transport in the presence of fatty acids, by reducing the undesired H⁺ flux facilitated by fatty acid flip-flop. This study demonstrates the benefit of including enforced steric hindrance and encapsulation in the design of more selective anion receptors.

Author Correction: Squaramide-based synthetic chloride transporters activate TFEB but block autophagic flux.

In the version of this article originally submitted, it was stated that the first three authors (Shaoyi_ Than, Yan Wang, Wei Xie) had contributed equally. However, in the published version this information was missing.

Squaramide-based synthetic chloride transporters activate TFEB but block autophagic flux.

Cystic fibrosis is a disease caused by defective function of a chloride channel coupled to a blockade of autophagic flux. It has been proposed to use synthetic chloride transporters as pharmacological agents to compensate insufficient chloride fluxes. Here, we report that such chloride anionophores block autophagic flux in spite of the fact that they activate the pro-autophagic transcription factor EB (TFEB) coupled to the inhibition of the autophagy-suppressive mTORC1 kinase activity. Two synthetic chloride transporters (SQ1 and SQ2) caused a partially TFEB-dependent relocation of the autophagic marker LC3 to the Golgi apparatus. Inhibition of TFEB activation using a calcium chelator or calcineurin inhibitors reduced the formation of LC3 puncta in cells, yet did not affect the cytotoxic action of SQ1 and SQ2 that could be observed after prolonged incubation. In conclusion, the squaramide-based synthetic chloride transporters studied in this work (which can also dissipate pH gradients) are probably not appropriate for the treatment of cystic fibrosis yet might be used for other indications such as cancer.

Fluorinated synthetic anion carriers: experimental and computational insights into transmembrane chloride transport.

A series of fluorinated tripodal tris-thioureas function as highly active anion transporters across lipid bilayers and cell membranes. Here, we investigate their mechanism of action using anion transport assays in cells and synthetic vesicles and molecular modelling of transporter-lipid interactions. When compared with non-fluorinated analogues, fluorinated compounds demonstrate a different mechanism of membrane transport because the free transporter cannot effectively diffuse through the membrane. As a result, in H+/Cl- cotransport assays, fluorinated transporters require the presence of oleic acid to form anionic oleate complexes for recycling of the transporter, whereas non-fluorinated analogues readily diffuse through the membrane as free transporters and show synergistic transport with the proton transporter gramicidin. Molecular dynamics simulations revealed markedly stronger transporter-lipid interactions for fluorinated compounds compared with non-fluorinated analogues and hence, higher energy barriers for fluorinated compounds to cross the membrane as free transporters. With use of appropriate proton transporters to ensure measurement of the correct rate-limiting steps, the transport rates determined in synthetic vesicle assays show excellent agreement with the anion transport rates determined in cell-based assays. We conclude that integration of computational and experimental methods provides a strategy to optimise transmembrane anion transporter design for biomedical applications.

Determinants of Ion-Transporter Cancer Cell Death.

Recently, we showed that synthetic anion transporters DSC4P-1 and SA-3 had activity related to cancer cell death. They were found to increase intracellular chloride and sodium ion concentrations. They were also found to induce apoptosis (DSC4P-1) and both induce apoptosis and inhibit autophagy (SA-3). However, determinants underlying these phenomenological findings were not elucidated. The absence of mechanistic understanding has limited the development of yet-improved systems. Here, we show that three synthetic anion transporters, DSC4P-1, SA-3, and 8FC4P, induce osmotic stress in cells by increasing intracellular ion concentrations. This triggers the generation of reactive oxygen species via a sequential process and promotes caspase-dependent apoptosis. In addition, two of the transporters, SA-3 and 8FC4P, induce autophagy by increasing the cytosolic calcium ion concentration promoted by osmotic stress. However, they eventually inhibit the autophagy process as a result of their ability to disrupt lysosome function through a transporter-mediated decrease in a lysosomal chloride ion concentration and an increase in the lysosomal pH.

Supramolecular Transmembrane Anion Transport: New Assays and Insights.

Transmembrane anion transport has been the focus of a number of supramolecular chemistry research groups for a number of years. Much of this research is driven by the biological relevance of anion transport and the search to find new treatments for diseases such as cystic fibrosis, which is caused by genetic problems leading to faulty cystic fibrosis transmembrane conductance regulator (CFTR) channels, which in turn lead to reduced chloride and bicarbonate transport through epithelial cell membranes. Considerable effort has been devoted to the development of new transporters, and our group along with others have been searching for combinations of organic scaffolds and anion binding groups that produce highly effective transporters that work at low concentration. These compounds may be used in the future as "channel replacement therapies", restoring the flux of anions through epithelial cell membranes and ameliorating the symptoms of cystic fibrosis. Less effort has been put into gaining a fundamental understanding of anion transport processes. Over the last 3 years, our group has developed a number of new transport assays that allow anion transport mechanisms to be determined. This Account covers the latest developments in this area, providing a concise review of the new techniques we can use to study anion transport processes individually without resorting to measurement of exchange processes and the new insights that these assays provide. The Account provides an overview of the effects of anion transporters on cells and an explanation of why many systems perturb pH gradients within cells in addition to transporting chloride. We discuss assays to determine whether anionophores facilitate chloride or HCl transport and how this latter assay can be modified to determine chloride versus proton selectivity in small-molecule anion receptors. We show how molecular design can be used to produce receptors that are capable of transporting chloride without perturbing pH gradients. We cover the role that anion transporters in the presence of fatty acids play in dissipating pH gradients across lipid bilayer membranes and the effect that this process has on chloride-selective transport. We also discuss how coupling of anion transport to cation transport by natural cationophores can be used to determine whether anion transport is electrogenic or electroneutral. In addition, we compare these new assays to the previously used chloride/nitrate exchange assay and show how this exchange assay can underestimate the chloride transport ability of certain receptors that are rate-limited by nitrate transport.

Full elucidation of the transmembrane anion transport mechanism of squaramides using in silico investigations.

A comprehensive experimental and theoretical investigation of the transmembrane chloride transport promoted by four series of squaramide derivatives, with different degrees of fluorination, number of convergent N-H binding units and conformational shapes, is reported. The experimental chloride binding and transport abilities of these small synthetic molecules in liposomes were rationalised with quantum descriptors and molecular dynamics simulations in POPC bilayers. The tripodal tren-based compounds, with three squaramide binding motifs, have high chloride affinity, isolating the anion from water molecules within the membrane model and preventing its release to the aqueous phase, in agreement with the absence of experimental transport activity. In contrast, the symmetrical mono-squaramides, with moderate chloride binding affinity, are able to bind and release chloride either in the aqueous phase or at the membrane interface level, in line with experimentally observed high transport activity. The PMF profiles associated with the diffusion of these free transporters and their chloride complexes across phospholipid bilayers show that the assisted chloride translocation is thermodynamically favoured.

New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(thio)ureas.

The anion transport properties of a series of previously reported tren-based anionophores have been revisited using new assays designed to measure anion uniport. This study provides new insights into the transport mechanism and selectivity of this important class of transporters. Specifically, we report the chloride and nitrate transport selectivity of these systems and quantify sulfate transport to determine EC50 values for sulfate transport for the first time. Two new assays were developed to study bicarbonate transport allowing accurate quantification of chloride/bicarbonate exchange.

Fluorescent squaramides as anion receptors and transmembrane anion transporters.

A series of squaramide-based anion transporters functionalised with the 1,8-naphthalimide fluorophore has been developed for improved ionophoric activity and fluorescent imaging in cells.

Fluorinated tripodal receptors for potentiometric chloride detection in biological fluids.

Fluorinated tripodal compounds were recently reported to be efficient transmembrane transporters for a series of inorganic anions. In particular, this class of receptors has been shown to be suitable for the effective complexation of chloride, nitrate, bicarbonate and sulfate anions via hydrogen bonding. The potentiometric properties of urea and thiourea-based fluorinated tripodal receptors are explored here for the first time, in light of the need for reliable sensors for chloride monitoring in undiluted biological fluids. The ion selective electrode (ISE) membranes with tren-based tris-urea bis(CF3) tripodal compound (ionophore I) were found to exhibit the best selectivity for chloride over major lipophilic anions such as salicylate ( [Formula: see text] ) and thiocyanate ( [Formula: see text] ). Ionophore I-based ISEs were successfully applied for chloride determination in undiluted human serum as well as artificial serum sample, the slope of the linear calibration at the relevant background of interfering ions being close to Nernstian (49.8±1.7mV). The results of potentiometric measurements were confirmed by argentometric titration. Moreover, the ionophore I-based ISE membrane was shown to exhibit a very good long-term stability of potentiometric performance over the period of 10 weeks. Nuclear magnetic resonance (NMR) titrations, potentiometric sandwich membrane experiments and density functional theory (DFT) computational studies were performed to determine the binding constants and suggest 1:1 complexation stoichiometry for the ionophore I with chloride as well as salicylate.

Indole-based perenosins as highly potent HCl transporters and potential anti-cancer agents.

Prodigiosin is one of the most potent anion transporters in lipid bilayer membranes reported to date. Inspired by the structure of this natural product, we have recently designed and synthesised a new class of H+/Cl- cotransporters named 'perenosins'. Here we report a new library of indole-based perenosins and their anion transport properties. The new transporters demonstrated superior transmembrane transport efficiency when compared to other indole-based transporters, due to favourable encapsulating effects from the substituents on the perenosin backbone. Anion transport assays were used to determine the mechanism of chloride transport revealing that the compounds function as 'strict' HCl cotransporters. Cell viability studies showed that some compounds specifically trigger late-onset cell death after 72 h with a unique correlation to the position of alkyl chains on the perenosins. Further investigations of cell death mechanism showed a mixture of cell cycle arrest and apoptosis was responsible for the observed decrease in cell viability.

A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations.

Perturbations in cellular chloride concentrations can affect cellular pH and autophagy and lead to the onset of apoptosis. With this in mind, synthetic ion transporters have been used to disturb cellular ion homeostasis and thereby induce cell death; however, it is not clear whether synthetic ion transporters can also be used to disrupt autophagy. Here, we show that squaramide-based ion transporters enhance the transport of chloride anions in liposomal models and promote sodium chloride influx into the cytosol. Liposomal and cellular transport activity of the squaramides is shown to correlate with cell death activity, which is attributed to caspase-dependent apoptosis. One ion transporter was also shown to cause additional changes in lysosomal pH, which leads to impairment of lysosomal enzyme activity and disruption of autophagic processes. This disruption is independent of the initiation of apoptosis by the ion transporter. This study provides the first experimental evidence that synthetic ion transporters can disrupt both autophagy and induce apoptosis.

Cyclic peptide unguisin A is an anion receptor with high affinity for phosphate and pyrophosphate.

Unguisin A (1) is a marine-derived, GABA-containing cyclic heptapeptide. The biological function of this flexible macrocycle is obscure. Here we show that compound 1 lacks any detectable activity in antimicrobial growth inhibition assays, a result that runs contrary to a previous report. However, we find that 1 functions as a promiscuous host molecule in a variety of anion-binding interactions, with high affinity particularly for phosphate and pyrophosphate. We also show that a series of rigidified, backbone-fluorinated analogues of 1 displays altered affinity for chloride ions.