Situating the phosphonated calixarene-cytochrome C association by molecular dynamics simulations.

Protein-calixarenes binding plays an increasingly central role in many applications, spanning from molecular recognition to drug delivery strategies and protein inhibition. These ligands obey a specific bio-supramolecular chemistry, which can be revealed by computational approaches, such as molecular dynamics simulations. In this paper, we rely on all-atom, explicit-solvent molecular dynamics simulations to capture the electrostatically driven association of a phosphonated calix-[4]-arene with cytochome-C, which critically relies on surface-exposed paired lysines. Beyond two binding sites identified in direct agreement with the x-ray structure, the association has a larger structural impact on the protein dynamics. Then, our simulations allow a direct comparison to analogous calixarenes, namely, sulfonato, similarly reported as "molecular glue." Our work can contribute to a robust in silico predictive tool to assess binding sites for any given protein of interest for crystallization, with the specificity of a macromolecular cage whose endo/exo orientation plays a role in the binding.

GOx-assisted synthesis of pillar[5]arene based supramolecular polymeric nanoparticles for targeted/synergistic chemo-chemodynamic cancer therapy.

BACKGROUND: Cancer is the most serious world's health problems on the global level and various strategies have been developed for cancer therapy. Pillar[5]arene-based supramolecular therapeutic nano-platform (SP/GOx NPs) was constructed successfully via orthogonal dynamic covalent bonds and intermolecular H-bonds with the assistance of glucose oxidase (GOx) and exhibited efficient targeted/synergistic chemo-chemodynamic cancer therapy. METHODS: The morphology of SP/GOx NPs was characterized by DLS, TEM, SEM and EDS mapping. The cancer therapy efficinecy was investigated both in vivo and in vitro. RESULTS: SP/GOx NPs can load drug molecules (Dox) and modify target molecule (FA-Py) on its surface conveniently. When the resultant FA-Py/SP/GOx/Dox NPs enters blood circulation, FA-Py will target it to cancer cells efficiently, where GOx can catalyst the overexpressed glucose to generate H2O2. Subsequently, the generated H2O2 in cancer cells catalyzed by ferrocene unit to form •OH, which can kill cancer cells. Furthermore, the loaded Dox molecules released under acid microenvironment, which can further achieve chemo-therapy. CONCLUSION: All the experiments showed that the excellent antitumor performance of FA-Py/SP/GOx/Dox NPs, which provided an new method for pillar[5]arene-based supramolecular polymer for biomedical applications.

Coassembly of hypoxia-sensitive macrocyclic amphiphiles and extracellular vesicles for targeted kidney injury imaging and therapy.

BACKGROUND: Hypoxia is a major contributor to global kidney diseases. Targeting hypoxia is a promising therapeutic option against both acute kidney injury and chronic kidney disease; however, an effective strategy that can achieve simultaneous targeted kidney hypoxia imaging and therapy has yet to be established. Herein, we fabricated a unique nano-sized hypoxia-sensitive coassembly (Pc/C5A@EVs) via molecular recognition and self-assembly, which is composed of the macrocyclic amphiphile C5A, the commercial dye sulfonated aluminum phthalocyanine (Pc) and mesenchymal stem cell-excreted extracellular vesicles (MSC-EVs). RESULTS: In murine models of unilateral or bilateral ischemia/reperfusion injury, MSC-EVs protected the Pc/C5A complex from immune metabolism, prolonged the circulation time of the complex, and specifically led Pc/C5A to hypoxic kidneys via surface integrin receptor α4ß1 and αLß2, where Pc/C5A released the near-infrared fluorescence of Pc and achieved enhanced hypoxia-sensitive imaging. Meanwhile, the coassembly significantly recovered kidney function by attenuating cell apoptosis, inhibiting the progression of renal fibrosis and reducing tubulointerstitial inflammation. Mechanistically, the Pc/C5A coassembly induced M1-to-M2 macrophage transition by inhibiting the HIF-1α expression in hypoxic renal tubular epithelial cells (TECs) and downstream NF-κB signaling pathway to exert their regenerative effects. CONCLUSION: This synergetic nanoscale coassembly with great translational potential provides a novel strategy for precise kidney hypoxia diagnosis and efficient kidney injury treatment. Furthermore, our strategy of coassembling exogenous macrocyclic receptors with endogenous cell-derived membranous structures may offer a functional platform to address multiple clinical needs.

Strapped calix[4]pyrroles: from syntheses to applications.

Supramolecular chemistry is a central topic in modern chemistry. It touches on many traditional disciplines, such as organic chemistry, inorganic chemistry, physical chemistry, materials chemistry, environmental chemistry, and biological chemistry. Supramolecular hosts, inter alia macrocyclic hosts, play critical roles in supramolecular chemistry. Calix[4]pyrroles, non-aromatic tetrapyrrolic macrocycles defined by sp3 hybridized meso bridges, have proved to be versatile receptors for neutral species, anions, and cations, as well as ion pairs. Compared to the parent system, octamethylcalix[4]pyrrole and its derivatives bearing simple appended functionalities, strapped calix[4]pyrroles typically display enhanced binding affinities and selectivities. In this review, we summarize advances in the design and synthesis of strapped calix[4]pyrroles, as well as their broad utility in molecular recognition, supramolecular extraction, separation technology, ion transport, and as agents capable of inhibiting cancer cell proliferation. Future challenges within this sub-field are also discussed.

Dioxygen Activation and Pyrrole α-Cleavage with Calix[4]pyrrolato Aluminates: Enzyme Model by Structural Constraint.

The present work describes the reaction of triplet dioxygen with the porphyrinogenic calix[4]pyrrolato aluminates to alkylperoxido aluminates in high selectivity. Multiconfigurational quantum chemical computations disclose the mechanism for this spin-forbidden process. Despite a negligible spin-orbit coupling constant, the intersystem crossing (ISC) is facilitated by singlet and triplet state degeneracy and spin-vibronic coupling. The formed peroxides are stable toward external substrates but undergo an unprecedented oxidative pyrrole α-cleavage by ligand aromatization/dearomatization-initiated O-O σ-bond scission. A detailed comparison of the calix[4]pyrrolato aluminates with dioxygen-related enzymology provides insights into the ISC of metal- or cofactor-free enzymes. It substantiates the importance of structural constraint and element-ligand cooperativity for the functions of aerobic life.

Cooperative Binding and Stepwise Encapsulation of Drug Molecules by Sulfonylcalixarene-Based Metal-Organic Supercontainers.

The cooperative binding behavior of a face-directed octahedral metal-organic supercontainer featuring one endo cavity and six exo cavities was thoroughly examined in chloroform solution through ultraviolet-visible (UV-Vis) titration technique using two representative drug molecules as the guests. The titration curves and their nonlinear fit to Hill equation strongly suggest the efficient encapsulation of the guest molecules by the synthetic host, which exhibit interesting cooperative and stepwise binding behavior. Based on the control experiments using tetranuclear complex as a reference, it is clear that two equivalents of the guest molecules are initially encapsulated inside the endo cavity, followed by the trapping of six additional equivalents of the drug molecules through six exo cavities (1 eq. per exo cavity), and the remaining guests are entrapped by the external pockets. The results provide an in-depth understanding of the cooperative binding behavior of metal-organic supercontainers, which opens up new opportunities for designing synthetic receptors for truly biomimetic functional applications.

Host-Guest Complexation of Oxaliplatin and Para-Sulfonatocalix[n]Arenes for Potential Use in Cancer Therapy.

P-sulfonatocalix[n]arenes have demonstrated a great potential for encapsulation of therapeutic drugs via host-guest complexation to improve solubility, stability, and bioavailability of encapsulated drugs. In this work, guest-host complexes of a third-generation anticancer drug (oxaliplatin) and p-4-sulfocalix[n]arenes (n = 4 and 6; p-SC4 and p-SC6, respectively) were prepared and investigated, using 1H NMR, UV, Job's plot analysis, and DFT calculations, for use as cancer therapeutics. The peak amplitude of the prepared host-guest complexes was linearly proportional to the concentration of oxaliplatin in the range of 1.0 × 10-5 M-1 to 2.1 × 10-4 M-1. The reaction stoichiometry between either p-SC4 or p-SC6 and oxaliplatin in the formed complexes was 1:1. The stability constants for the complexes were 5.07 × 104 M-1 and 6.3 × 104 M-1. These correspond to complexation free energy of -6.39 and -6.52 kcal/mol for p-SC4 and p-SC6, respectively. Complexation between oxaliplatin and p-SC4 or p-SC6 was found to involve hydrogen bonds. Both complexes exhibited enhanced biological and high cytotoxic activities against HT-29 colorectal cells and MCF-7 breast adenocarcinoma compared to free oxaliplatin, which warrants further investigation for cancer therapy.

Calixarene-based phosphinic acids as inhibitors of protein tyrosine phosphatases.

In the present work, the derivatives of calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene bearing four methylene(phenyl)phosphinic acid groups on the upper rim of the macrocycle were synthesized and studied as inhibitors of human protein tyrosine phosphatases. The inhibitory capacities of the three compounds towards PTP1B were higher than those for protein tyrosine phosphatases TC-PTP, MEG1, MEG2, and SHP2. The most potent sulfonylcalix[4]arene phosphinic acid displayed Ki value of 32 nM. The thiacalix[4]arene phosphinic acid was found to be a low micromolar inhibitor of PTP1B with selectivity over the other PTPs. The kinetic experiments showed that the inhibitors compete with the substrate for the active site of the enzyme. Molecular docking was performed to explain possible binding modes of the calixarene-based phosphinic inhibitors of PTP1B.

Synthetic Receptors for the Recognition and Discrimination of Post-Translationally Methylated Lysines.

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology, including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side-chain methylation of lysine. For the analysis and discrimination of differently methylated lysines, antibodies are widely used, although methylated peptide and protein targets are known to be particularly difficult to differentiate by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation procedure and is not suited for working in cells. The desire to overcome the above-mentioned challenges has promoted the development of synthetic receptor molecules that recognise and bind methyllysines. Such "artificial antibodies" are of interest for a number of applications, for example, as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines, including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbiturils and acyclic receptors.

An Electron-Rich Calix[4]arene-Based Receptor with Unprecedented Binding Affinity for Nitric Oxide.

Calixarenes have found widespread application as building blocks for the design and synthesis of functional materials in host-guest chemistry. The ongoing desire to develop a detailed understanding of the nature of NO bonding to multichromophoric π-stacked assemblies led us to develop an electron-rich methoxy derivative of calix[4]arene (3), which we show exists as a single conformer in solution at ambient temperature. Here, we examine the redox properties of this derivative, generate its cation radical (3+. ) using robust chemical oxidants, and determine the relative efficacy of its NO binding in comparison with model calixarenes. We find that 3/3+. is a remarkable receptor for NO+ /NO, with unprecedented binding efficacy. The availability of precise experimental structures of this calixarene derivative and its NO complex, obtained by X-ray crystallography, is critically important both for developing novel functional NO biosensors, and understanding the role of stacked aromatic donors in efficient NO binding, which may have relevance to biological NO transport.

Galectin Targeted Therapy in Oncology: Current Knowledge and Perspectives.

The incidence and mortality of cancer have increased over the past decades. Significant progress has been made in understanding the underpinnings of this disease and developing therapies. Despite this, cancer still remains a major therapeutic challenge. Current therapeutic research has targeted several aspects of the disease such as cancer development, growth, angiogenesis and metastases. Many molecular and cellular mechanisms remain unknown and current therapies have so far failed to meet their intended potential. Recent studies show that glycans, especially oligosaccharide chains, may play a role in carcinogenesis as recognition patterns for galectins. Galectins are members of the lectin family, which show high affinity for ß-galactosides. The galectin-glycan conjugate plays a fundamental role in metastasis, angiogenesis, tumor immunity, proliferation and apoptosis. Galectins' action is mediated by a structure containing at least one carbohydrate recognition domain (CRD). The potential prognostic value of galectins has been described in several neoplasms and helps clinicians predict disease outcome and determine therapeutic interventions. Currently, new therapeutic strategies involve the use of inhibitors such as competitive carbohydrates, small non-carbohydrate binding molecules and antibodies. This review outlines our current knowledge regarding the mechanism of action and potential therapy implications of galectins in cancer.

The Investigation on Resorcinarenes towards either Inhibiting or Promoting Insulin Fibrillation.

Different tail-engineered resorcinarenes have been examined for insulin fibrillation by experimental and computational studies. The resorcinarene showed a promising effect on the inhibition of insulin fibrillation, studied using a ThT assay, circular dichroism spectroscopy, and atomic force microscopy. Both the ThT assay and computational results indicate the tail from the resorcinarene has an impact on insulin fibrillation by either inhibition or promotion because of the resident position on insulin. These observations have significant biological implications in the design of drug molecules as well as the development of potential therapeutic strategies.

Complete tetraglycosylation of a calix[4]arene by a chemo-enzymatic approach.

Polyglycosylated calixarenes are efficient and selective multivalent ligands for lectins. However, the chemical decoration of these macrocyclic scaffolds with saccharides of increasing complexity is hampered by the highly complex chemistry of carbohydrates. An alternative to the conventional approach is the enzymatic diversification of simple glycocluster-presented glycans. In this work, we present a highly efficient chemo-enzymatic approach to tetra-N-acetyl-lactosaminylcalix[4]arene via glycan extension catalyzed by a human ß-1,4-galactosyltransferase. This demonstrates that calixarenes can be exhaustively processed by enzymatic glycosyl transfer despite the heavy steric crowding, paving the way to the design and achievement of multivalent ligands based on these macrocyclic scaffolds having complex branched glycans.

Phosphorylation-Responsive Membrane Transport of Peptides.

Phosphorylation and dephosphorylation of peptides by kinases and phosphatases is essential for signal transduction in biological systems, and many diseases involve abnormal activities of these enzymes. Herein, we introduce amphiphilic calixarenes as key components for supramolecular, phosphorylation-responsive membrane transport systems. Dye-efflux experiments with liposomes demonstrated that calixarenes are highly active counterion activators for established cell-penetrating peptides, with EC50 values in the low nanomolar range. We have now found that they can even activate membrane transport of short peptide substrates for kinases involved in signal transduction, whereas the respective phosphorylated products are much less efficiently transported. This allows regulation of membrane transport activity by protein kinase A (PKA) and protein kinase C (PKC), as well as monitoring of their activity in a label-free kinase assay.

Synthetic Channel Specifically Inserts into the Lipid Bilayer of Gram-Positive Bacteria but not that of Mammalian Erythrocytes.

A series of tubular molecules with different lengths have been synthesized by attaching Trp-incorporated peptides to the pillar[5]arene backbone. The tubular molecules are able to insert into the lipid bilayer to form unimolecular transmembrane channels. One of the channels has been revealed to specifically insert into the bilayer of the Gram-positive bacteria. In contrast, this channel cannot insert into the membranes of the mammalian rat erythrocytes even at the high concentration of 100 µm. It was further demonstrated that, as a result of this high membrane selectivity, the channel exhibits efficient antimicrobial activity for the Gram-positive bacteria and very low hemolytic toxicity for mammalian erythrocytes.

Supramolecular modeling of mono-copper enzyme active sites with calix[6]arene-based funnel complexes.

Supramolecular bioinorganic chemistry is a natural evolution in biomimetic metallic systems since it constitutes a further degree of complexity in modeling. The traditional approach consisting of mimicking the first coordination sphere of metal sites proved to be very efficient, because valuable data are extracted from these examples to gain insight in natural systems mechanisms. But it does not reproduce several specific aspects of enzymes that can be mimicked by the implementation of a cavity embedding the labile active site and thus controlling the properties of the metal ion by noncovalent interactions. This Account reports on a strategy aimed at reproducing some supramolecular aspects encountered in the natural systems. The cavity complexes described herein display a coordination site constructed on a macrocycle. Thanks to a careful design of the cavity-based ligands, complexes orienting their labile site specifically toward the inside of the macrocycle were obtained. The supramolecular systems are based on the flexible calix[6]arene core that surrounds the metal ion labile site, thereby constraining exogenous molecules to pass through the conic funnel to reach the metal center. Such an architecture confers to the metal ion very unusual properties and behaviors, which in many aspects are biologically relevant. Three generations of calix[6]-based ligands are presented and discussed in the context of modeling the monocopper sites encountered in some enzymes. A wide range of phenomena are highlighted such as the impact that the size and shape of the access channel to the metal center have on the selectivity and rate of the binding process, the possible remote control of the electronics through small modifications operated on the cavity edges, induced-fit behavior associated with host-guest association (shoe-tree effect) that affects the redox properties of the metal ion and the electron exchange pathway, consequences of forbidden associative ligand exchange allowing a redox switch to drive an "antithermodynamic" ligand exchange, drastic effects of the full control of the second coordination sphere, and dioxygen activation in a confined chamber conducted to a selective and unusual four-electron redox process. All these findings bring new clues for better understanding the control exerted by the proteic environment on a metal center, allow the identification of new reaction pathways, and lead to new proposals for enzymatic catalytic cycle (such as the formation of an alkylhydroperoxide intermediate for mononuclear Cu-hydroxylases). The supramolecular systems may also be exploited for designing highly selective and sensitive probes for molecules of particular function and shape or to design new selective catalysts.

Interaction of a calix[4]arene derivative with a DOPC bilayer: biomolecular simulations towards chloride transport.

The ability of a calix[4]arene derivative (CX-1), bearing four protonated NH3(+) groups located in the upper rim and aliphatic tails in the lower rim, to interact with a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) model bilayer and promote transmembrane chloride transport was investigated by molecular dynamics (MD) simulations. Unconstrained MD simulations show that the interaction of CX-1 with DOPC occurs via the NH3(+) groups, which are able to establish electrostatic interactions and multiple hydrogen bonds with the DOPC phosphate groups, while the aliphatic tails point towards the water phase (when CX-1 starts from the water phase) or to the membrane (when CX-1 is initially positioned within the bilayer). The interaction does not induce any relevant perturbation on the biophysical properties of the bilayer system (area per lipid, thickness, and hydration) apart from a systematic increase in the order parameter of the C2 carbon atom of the sn-1 lipid tail, meaning that the bilayer conserves its integrity. Since total internalization of CX-1 was not observed in the unconstrained MD time-scale, constant velocity steered molecular dynamics (SMD) simulations were performed in order to simulate the CX-1 permeation across the bilayer. At pulling velocities lower than 0.0075 nmps(-1), chloride transport was observed. The Potential of Mean Force (PMF), calculated with the weighted histogram analysis method, indicates a barrier of ca. 58kJmol(-1) for this mobile carrier to cross the membrane.

Supramolecular Ligands for Histone Tails by Employing a Multivalent Display of Trisulfonated Calix[4]arenes.

Post-translational modification of histone tails plays critical roles in gene regulation. Thus, molecules recognizing histone tails and controlling their epigenetic modification are desirable as biochemical tools to elucidate regulatory mechanisms. There are, however, only a few synthetic ligands that bind to histone tails with substantial affinity. We report CA2 and CA3, which exhibited sub-micromolar affinity to histone tails (especially tails with a trimethylated lysine). Multivalent display of trisulfonated calix[4]arene was important for strong binding. CA2 was applicable not only to synthetic tail peptides but also to endogenous histone proteins, and was successfully used to pull-down endogenous histones from nuclear extract. These findings indicate the utility of these supramolecular ligands as biochemical tools for studying chromatin regulator protein and as a targeting motif in ligand-directed catalysis to control epigenetic modifications.

Synthesis, biological evaluation and molecular docking of calix[4]arene-based ß-diketo derivatives as HIV-1 integrase inhibitors.

In this publication, we design and report the synthesis of calix[4]arene-based ß-diketo derivatives as novel HIV-1 integrase (IN) inhibitors. The target compounds were obtained using Claisen condensation, and their structures were characterized by NMR and ESI-MS. Preliminary bioassays showed that calix[4]arene-based ß-diketo derivatives inhibit strand transfer (ST) with IC50 values between 5.9 and 21.2 µM. Docking studies revealed the predominant binding modes that were distinct from the binding modes of raltegravir, which suggests a novel binding region in the IN active site. Moreover, these compounds are predicted not to interact with some of the key amino acids (GLN148 and ASN155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors.

Indicator displacement assays inside live cells.

The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells.