Roberto Quesada

Using small molecules to facilitate exchange of bicarbonate and chloride anions across liposomal membranes

Bicarbonate is involved in a wide range of biological processes, which include respiration, regulation of intracellular pH and fertilization. In this study we use a combination of NMR spectroscopy and ion-selective electrode techniques to show that the natural product prodigiosin, a tripyrrolic molecule produced by microorganisms such as Streptomyces and Serratia, facilitates chloride/bicarbonate exchange (antiport) across liposomal membranes. Higher concentrations of simple synthetic molecules based on a 4,6-dihydroxyisophthalamide core are also shown to facilitate this antiport process. Although it is well known that proteins regulate Cl(-)/HCO(3)(-) exchange in cells, these results suggest that small molecules may also be able to regulate the concentration of these anions in biological systems.

Anion Transporters and Biological Systems

In this Account, we discuss the development of new lipid bilayer anion transporters based on the structure of anionophoric natural products (the prodigiosins) and purely synthetic supramolecular systems. We have studied the interaction of these compounds with human cancer cell lines, and, in general, the most active anion transporter compounds possess the greatest anti-cancer properties. Initially, we describe the anion transport properties of synthetic molecules that are based on the structure of the family of natural products known as the prodiginines. Obatoclax, for example, is a prodiginine derivative with an indole ring that is currently in clinical trials for use as an anti-cancer drug. The anion transport properties of the compounds were correlated with their toxicity toward small cell human lung cancer GLC4 cells. We studied related compounds with enamine moieties, tambjamines, that serve as active transporters. These molecules and others in this series could depolarize acidic compartments within GLC4 cells and trigger apoptosis. In a study of the variation of lipophilicity of a series of these compounds, we observed that, as log P increases, the anion transport efficiency reaches a peak and then decreases. In addition, we discuss the anion transport properties of series of synthetic supramolecular anion receptor species. We synthesized trisureas and thioureas based on the tren backbone, and found that the thiourea compounds effectively transport anions. Fluorination of the pendant phenyl groups in this series of compounds greatly enhances the transport properties. Similar to our earlier results, the most active anion transporters reduced the viability of human cancer cell lines by depolarizing acidic compartments in GLC4 cells and triggering apoptosis. In an attempt to produce simpler transporters that obey Lipinskis Rule of Five, we synthesized simpler systems containing a single urea or thiourea group. Once again the thiourea systems, and in particular a thiourea with a pendant indole group, transported anions efficiently. A series of related compounds containing a pendant trifluoromethyl group showed enhanced transport and significant anticancer properties. Researchers still need to determine of the exact mechanism of how these compounds depolarize acidic organelles within cancer cells. However, this work shows that these transporters based upon both natural products and purely synthetic supramolecular systems transport anions, depolarize acidic compartments within cancer cells and trigger apoptosis.

Octafluorocalix[4]pyrrole: a chloride/bicarbonate antiport agent

meso-Octamethyloctafluorocalixpyrrole, a simple tetrapyrrolic macrocycle, has been shown to function as both a chloride/nitrate and a chloride/bicarbonate antiport agent for lipid bilayer transmembrane anion transport. This is the first example of a synthetic macrocyclic pyrrole-based receptor capable of transmembrane bicarbonate transport.

meso-Octamethylcalix[4]pyrrole: an old yet new transmembrane ion-pair transporter

The first example of lipid bilayer membrane transport of a salt by a calix[4]pyrrole is reported.

Pyrrolylamidourea based anion receptors

The anion binding behaviour of a number of pyrrolylamidourea and thiourea compounds have been studied in DMSO solution. Mono-amidothioureapyrrole compounds were found to be deprotonated by basic anions such as fluoride, acetate, benzoate or dihydrogenphosphate with the structure of the deprotonated species elucidated by X-ray crystallography. 2,5-Bis-amidoureapyrroles were synthesized and found to be effective anion receptors for a range of putative anionic guests.

Synthetic Prodiginine Obatoclax (GX15‐070) and Related Analogues: Anion Binding, Transmembrane Transport, and Cytotoxicity Properties

Synthetic prodiginine obatoclax shows promise as a potential anticancer drug. This compound promotes apoptosis of cancer cells, although the mechanism of action is unclear. To date, only the inhibition of BCL-2 proteins has been proposed as a mechanism of action. To gain insight into other possible modes of action, we have studied the anion-binding properties of obatoclax and related analogues in solution, in the solid state, and by means of density functional theory calculations. These compounds are well suited to interact with anions such as chloride and bicarbonate. The anion-transport properties of the compounds synthesized were assayed in model phospholipid liposomes by using a chloride-selective-electrode technique and (13)C NMR spectroscopy. The results demonstrated that these compounds are efficient anion exchangers that promote chloride, bicarbonate, and nitrate transport through lipid bilayers at very low concentrations. In vitro studies on small-cell lung carcinoma cell line GLC4 showed that active ionophores are able to discharge pH gradients in living cells and the cytotoxicity of these compounds correlates well with ionophoric activity.

The role of lipophilicity in transmembrane anion transport

The transmembrane anion transport activity of a series of synthetic molecules inspired by the structure of tambjamine alkaloids can be tuned by varying the lipophilicity of the receptor, with carriers within a certain log P range performing best.

Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: Prodigiosin vs. obatoclax

The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modeling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

Biological activity of synthetic ionophores: ion transporters as prospective drugs?

The control of ion transport and homeostasis is a critical function of living organisms. In this perspective, an overview of different synthetic systems capable of facilitating the transmembrane transport of ions along with the biological activity exerted by these compounds is presented. Examples of both cation selective and anion selective transporters are highlighted. The potential future applications of these systems in the treatment of conditions derived from the dysregulation of natural ion transport mechanisms and the development of new antimicrobials and anticancer drugs are discussed.