Development of a Systematic Strategy toward Promotion of α-Synuclein Aggregation Using 2-Hydroxyisophthalamide-Based Systems.

Establishing a potent scheme against α-synuclein aggregation involved in Parkinson's disease has been evaluated as a promising route to identify compounds that either inhibit or promote the aggregation process of α-synuclein. In the last two decades, this perspective has guided a dramatic increase in the efforts, focused on developing potent drugs either for retardation or promotion of the self-assembly process of α-synuclein. To address this issue, using a chemical kinetics platform, we developed a strategy that enabled a progressively detailed analysis of the molecular events leading to protein aggregation at the microscopic level in the presence of a recently synthesized 2-hydroxyisophthalamide class of small organic molecules based on their binding affinity. Furthermore, qualitatively, we have developed a strategy of disintegration of α-synuclein fibrils in the presence of these organic molecules. Finally, we have shown that these organic molecules effectively suppress the toxicity of α-synuclein oligomers in neuron cells.

Molecular Self-Assembly as a Tool to Construct Transmembrane Supramolecular Ion Channels.

Self-assembly has become a powerful tool for building various supramolecular architectures with applications in material science, environmental science, and chemical biology. One such area is the development of artificial transmembrane ion channels that mimic naturally occurring channel-forming proteins to unveil various structural and functional aspects of these complex biological systems, hoping to replace the defective protein channels with these synthetically accessible moieties. This account describes our recent approaches to construct supramolecular ion channels using synthetic molecules and their applications in medicinal chemistry.

Esterase-Activatable Synthetic M+/Cl- Channel Induces Apoptosis and Disrupts Autophagy in Cancer Cells.

The formation of a supramolecular synthetic M+/Cl- channel in the membrane phospholipid bilayer has been reported upon activation of a methyl pivalate-linked N1,N3-dialkyl-2-hydroxyisophthalamide by esterases. The channel formation induces apoptosis in cancer cells via the intrinsic pathway. Interestingly, the supramolecular channel was also shown to disrupt autophagy in cancer cells by causing alkalization of lysosomes - a feature that has been confirmed at the cellular and protein level.

A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels.

Cancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4-nitrobenzenesulfonyl (DNS) protected 2-hydroxyisophthalamide system that exploits GSH for its activation into free 2-hydroxyisophthalamide forming supramolecular M+ /Cl- channels. Better permeation of the DNS protected compound into MCF-7 cells compared to the free 2-hydroxyisophthalamide and GSH-activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH-activatable transport-mediated cell death was further validated in rat insulinoma cells (INS-1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF-7 cells with efficiency similar to that of the anticancer drug doxorubicin.

Phototriggered Release of a Transmembrane Chloride Carrier from an o-Nitrobenzyl-Linked Procarrier.

While there have been many studies on synthetic chloride carriers and a recent application for apoptotic cell death, so far, the proposed huge potential of these systems in targeting cancer has not been realized due to their cytotoxicity to healthy cells. Herein, we describe the development of an indole-2-carboxamide receptor as an efficient membrane chloride carrier while the corresponding o-nitrobenzyl-linked derivative is a procarrier of the ion. Photoirradiation of the procarrier in liposomes results in release of the active carrier with up to 90 % transport efficiency. Such photorelease of the carrier also works within cancer cells, resulting in efficient cell killing. Such photocleavable procarriers have great potential as a photodynamic therapy to combat various types of cancers.

Chloride Transport across Liposomes and Cells by Nontoxic 3-(1H-1,2,3-Triazol-1-yl)benzamides.

Synthetic anion transmembrane transporters are adding new aspirations for treating channelopathies by replacing defective ion channels. The availability of such suitable candidates is still infrequent due to the associated toxicity. Here, we report 3-(1H-1,2,3-triazol-1-yl)benzamides as transmembrane anion carriers, nontoxic to cells. The selective and electrogenic chloride transport activity was established by fluorescence and ion selective electrode-based assays. MQAE assay confirmed the chloride uptake into the cells by the nontoxic compounds.

A Pyridyl-Linked Benzimidazolyl Tautomer Facilitates Prodigious H+/Cl- Symport through a Cooperative Protonation and Chloride Ion Recognition.

We report two pyridyl-linked benzimidazolyl hydrazones as HCl cotransporters that are 5 and 2 times superior to prodigiosin, a natural product whose transport efficiency has never been routed by synthetic molecules. These hydrazones provide a suitable HCl binding site through a cooperative protonation and chloride ion recognition. HCl transport by the most active compound induces lysosome deacidification. Viability assays confirmed that the compounds induce cytotoxicity toward human breast cancer MCF-7 cells but are relatively nontoxic toward noncancerous HEK293T cells.

Apoptosis-inducing activity of a fluorescent barrel-rosette M+/Cl- channel.

Synthetic transmembrane ion transport systems are emerging as new tools for anticancer therapy. Here, a series of 2-hydroxy-N 1,N 3-diarylisophthalamide-based fluorescent ion channel-forming compounds are reported. Ion transport studies across large unilamellar vesicles confirmed that the compound with two 3,5-bis(trifluoromethyl)phenyl arms is the most efficient transporter among the series and it facilitates M+/Cl- symport. The compound formed supramolecular ion channels with a single-channel conductance of 100 ± 2 pS, a diameter of 5.06 ± 0.16 Å and a permeability ratio, P Cl- /P K+ , of 8.29 ± 1. The molecular dynamics simulations of the proposed M2.11 channel (i.e. 11 coaxial layers of a dimeric rosette) with K+ and Cl- in the preequilibrated POPC lipid bilayer with water molecules illustrated various aspects of channel formation and ion permeation. Cell viability assay with the designed compounds indicated that cell death is being induced by the individual compounds which follow the order of their ion transport activity and chloride and cations play roles in cell death. The inherent fluorescence of the most active transporter was helpful to monitor its permeation in cells by confocal microscopy. The apoptosis-inducing activity upon perturbation of intracellular ionic homeostasis was established by monitoring mitochondrial membrane depolarization, generation of reactive oxygen species, cytochrome c release, activation of the caspase 9 pathway, and finally the uptake of the propidium iodide dye in the treated MCF7 cells.

Anion Selective Ion Channel Constructed from a Self-Assembly of Bis(cholate)-Substituted Fumaramide.

The self-assembled ion channel constructed from bis(cholate)-substituted fumaramide is reported. Such ion channel formation was favored by intermolecular hydrogen bonding interactions among fumaramide moieties. Fluorescence kinetics experiments and planar bilayer conductance measurements confirmed the selective chloride transport through the channel. Theoretical calculations were done to confirm the hydrogen bonded self-assembly of fumaramides and chloride recognition within the cavity.

Self-assembly of small-molecule fumaramides allows transmembrane chloride channel formation.

This study reports the formation of self-assembled transmembrane anion channels by small-molecule fumaramides. Such artificial ion channel formation was confirmed by ion transport across liposomes and by planar bilayer conductance measurements. The geometry-optimized model of the channel and Cl- ion selectivity within the channel lumen was also illustrated.