Comparative analyses of site-directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor.

Melatonin is an indoleamine neurohormone made by the pineal gland. Its receptors, MTNR1A and MTNR1B, are members of the G-protein-coupled receptor (GPCR) family and are involved in sleep, circadian rhythm, and mood disorders, and in the inhibition of cancer growth. These receptors, therefore, represent significant molecular targets for insomnia, circadian sleep disorders, and cancer. The yeast Saccharomyces cerevisiae is an attractive host for assaying agonistic activity for human GPCR. We previously constructed a GPCR-based biosensor employing a high-sensitivity yeast strain that incorporated both a chimeric yeast-human Gα protein and a bright fluorescent reporter gene (ZsGreen). Similar approaches have been used for simple and convenient measurements of various GPCR activities. In the current study, we constructed a fluorescence-based yeast biosensor for monitoring the signaling activation of human melatonin receptors. We used this system to analyze point mutations, including previously unreported mutations of the consensus sequences of MTNR1A and MTNR1B melatonin receptors and compared their effects. Most mutations in the consensus sequences significantly affected the signaling capacities of both receptors, but several mutations showed differences between these subtype receptors. Thus, this yeast biosensor holds promise for revealing the functions of melatonin receptors.

Microenvironment pH-Induced Selective Cell Death for Potential Cancer Therapy Using Nanofibrous Self-Assembly of a Peptide Amphiphile.

Self-assembly of synthetic molecules has been drawing broad attention as a novel emerging approach in drug discovery. Here, we report selective cell death induced by a novel peptide amphiphile that self-assembles to form entangled nanofibers (hydrogel) based on intracellular pH (pHi). We found that a palmitoylated hexapeptide (C16-VVAEEE) formed a hydrogel below pH 7. The formation of the nanofibrous self-assembly was responsive to a small pH change around pH 7. The cytotoxicity of C16-VVAEEE was correlated with pHi of cells. Microscope observation demonstrated the self-assembly of C16-VVAEEE inside HEK293 cells. In vivo experiments revealed that the transcutaneous administration of C16-VVAEEE showed remarkable anti-tumor activity. This study proposes that distinct microenvironment inside living cells can be used as a trigger for the intracellular self-assembly of a peptide amphiphile, which provide a new clue to drug discovery.

One-Step Biotinylation of Cellulose Paper by Polymer Coating to Prepare a Paper-Based Analytical Device.

Cellulose paper has strong potential as an analytical platform owing to its unique characteristics. In the present study, we investigated a procedure for functionalizing the surface of cellulose paper by dip-coating a mixture of a functional polymer and a perfluoroalkylated surfactant (surfactant 1). The functional polymer comprised a mixture of methyl methacrylate and poly(ethylene glycol) methacrylate monomers. The monomer ratio in the functional polymer affected the hydrophilicity and water absorbance of the cellulose paper after dip-coating. Furthermore, the presence of surfactant 1 during dip-coating promoted the surface segregation of poly(ethylene glycol) (PEG) moieties in the polymer, which enhanced the hydrophilicity, prevented nonspecific protein adsorption, and maintained the water absorbance of the dip-coated cellulose paper. Dip-coating with another functional polymer containing biotin groups produced a cellulose paper with a biotin-decorated surface in a one-step procedure. The displayed biotin groups immobilized avidin on the surface, and the PEG moieties in the polymer prevented nonspecific protein adsorption. We then immobilized a thrombin-binding DNA aptamer on the avidin-immobilized cellulose paper to prepare a paper-based analytical device. It is possible to visualize thrombin in model solutions and serum using the paper-based analytical device.

Controlling Surface Segregation of a Polymer To Display Carboxy Groups on an Outermost Surface Using Perfluoroacyl Groups.

Controlling the surface properties of solid polymers is important for practical applications. We here succeeded in controlling the surface segregation of polymers to display carboxy groups on an outermost surface, which allowed the covalent immobilization of functional molecules via the carboxy groups on a substrate surface. Random methacrylate-based copolymers containing carboxy groups, which were protected with perfluoroacyl (Rf) groups, were dip-coated on acrylic substrate surfaces. X-ray photoelectron spectroscopy and contact-angle measurements revealed that the Rf groups were segregated to the outermost surface of the dip-coated substrates. The Rf groups were removed by hydrolysis of the Rf esters in the copolymers, resulting in the display of carboxy groups on the surface. The quantification of carboxy groups on a surface revealed that the carboxy groups were reactive to a water-soluble solute in an aqueous solution. The surface segregation was affected by the molecular structure of the copolymer used for dip-coating.

Short Oligopeptides for Biocompatible and Biodegradable Supramolecular Hydrogels.

Short Phe-rich oligopeptides, consisting of only four and five amino acids, worked as effective supramolecular hydrogelators for buffer solutions at low gelator concentrations (0.5-1.5 wt %). Among 10 different oligopeptides synthesized, peptide P1 (Ac-Phe-Phe-Phe-Gly-Lys) showed high gelation ability. Transmission electron microscopy observations suggested that the peptide molecules self-assembled into nanofibrous networks, which turned into gels. The hydrogel of peptide P1 showed reversible thermal gel-sol transition and viscoelastic properties typical of a gel. Circular dichroism spectra revealed that peptide P1 formed a ß-sheetlike structure, which decreased with increasing temperature. The self-assembly of peptide P1 occurred even in the presence of nutrients in culture media and common surfactants. Escherichia coli and yeast successfully grew on the hydrogel. The hydrogel exhibited low cytotoxicity to animal cells. Finally, we demonstrated that functional compounds can be released from the hydrogel in different manners based on the interaction between the compounds and the hydrogel.

In Situ Synthesis of a Supramolecular Hydrogelator at an Oil/Water Interface for Stabilization and Stimuli-Induced Fusion of Microdroplets.

Supramolecular hydrogels are expected to have applications as novel soft materials in various fields owing to their designable functional properties. Herein, we developed an in situ synthesis of supramolecular hydrogelators, which can trigger gelation of an aqueous solution without the need for temperature change. This was achieved by mixing two precursors, which induced the synthesis of a supramolecular gelator and its instantaneous self-assembly into nanofibers. We then performed the in situ synthesis of this supramolecular gelator at an oil/water interface to produce nanofibers that covered the surfaces of the oil droplets (nanofiber-stabilized oil droplets). External stimuli induced fusion of the droplets owing to disassembly of the gelator molecules. Finally, we demonstrated that this stimuli-induced droplet fusion triggered a synthetic reaction within the droplets. This means that the confined nanofiber-stabilized droplets can be utilized as stimuli-responsive microreactors.

Cancer cell death induced by the intracellular self-assembly of an enzyme-responsive supramolecular gelator.

We report cancer cell death initiated by the intracellular molecular self-assembly of a peptide lipid, which was derived from a gelator precursor. The gelator precursor was designed to form nanofibers via molecular self-assembly, after cleavage by a cancer-related enzyme (matrix metalloproteinase-7, MMP-7), leading to hydrogelation. The gelator precursor exhibited remarkable cytotoxicity to five different cancer cell lines, while the precursor exhibited low cytotoxicity to normal cells. Cancer cells secrete excessive amounts of MMP-7, which converted the precursor into a supramolecular gelator prior to its uptake by the cells. Once inside the cells, the supramolecular gelator formed a gel via molecular self-assembly, exerting vital stress on the cancer cells. The present study thus describes a new drug where molecular self-assembly acts as the mechanism of cytotoxicity.

Surfactant-induced polymer segregation to produce antifouling surfaces via dip-coating with an amphiphilic polymer.

We propose a rational strategy to control the surface segregation of an amphiphilic copolymer in its dip-coating with a low-molecular-weight surfactant. We synthesized a water-insoluble methacrylate-based copolymer containing oligo(ethylene glycol) (OEG) (copolymer 1) and a perfluoroalkylated surfactant (surfactant 1) containing OEG. The dip-coating of copolymer 1 with surfactant 1 resulted in the segregation of surfactant 1 on the top surface of the dip-coated layer due to the high hydrophobicity of its perfluoroalkyl group. OEG moieties of surfactant 1 were accompanied by those of copolymer 1 in its segregation, allowing the OEG moieties of copolymer 1 to be located just below the top surface of the dip-coated layer. The removal of surfactant 1 produced the surface covered by the OEG moieties of the copolymer that exhibited antifouling properties. Using this strategy, we also succeeded in the introduction of carboxy groups on the dip-coated surface and demonstrated that the carboxy groups were available for the immobilization of functional molecules on the surface.

Quantification of Amino Groups on Solid Surfaces Using Cleavable Fluorescent Compounds.

We quantified amino groups displayed on inorganic and organic surfaces in aqueous solution using different types of cleavable fluorescent compounds and an aldehyde dye. The cleavable fluorescent compounds were designed to bind covalently to amino groups and then liberated under specific conditions. Among the investigated materials, cleavable coumarin was most appropriate for the quantification of amino groups on silica and resin surfaces. The developed method can measure small amounts (∼pmol/cm(2)) of amino groups on a flat polymeric surface, detecting only amino groups that are exposed to aqueous solution and available for surface immobilization of ligands and biomolecules.

Supramolecular gelators based on benzenetricarboxamides for ionic liquids.

Supramolecular gelators comprising 1,3,5-benzenetricarboxylic acids and amino acid methyl esters (glycine, L-alanine, L-valine, L-leucine, L-methionine, and L-phenylalanine) for ionic liquids were developed. Ten types of ionic liquids were gelated using the above-mentioned gelators at relatively low concentrations. Field emission-scanning electron microscopy and confocal laser scanning microscopy analyses revealed that these gelators self-assembled into an entangled fibrous structure in ionic liquids, leading to the gelation of the ionic liquids. Comparison studies, involving compounds analogous to the gelators, and Fourier transform infrared spectroscopy measurements suggested that hydrogen bonding played a key role in the self-assembly of the gelator molecules. The ionogels displayed reversible thermal transition characteristics and viscoelastic properties typical of a gel. The gelation of the ionic liquids studied under a wide range of gelator concentrations did not affect the intrinsic conductivity of the ionic liquids.

Scattering/Fluorescence Dual-Mode Imaging in MnO2-Coated Silicon Nanospheres for Cancer Cell Detection.

A tumor microenvironment (TME)-responsive nanoprobe composed of a fluorescent dye-decorated silicon (Si) nanosphere core and a thin MnO2 shell is proposed for simple and intelligent detection of cancer cells. The Si nanosphere core with diameters of 100-200 nm provides environment-independent Mie scattering imaging, while, simultaneously, the MnO2 shell provides the capability to switch the on/off state of the dye fluorescence reacted to the glutathione (GSH) and/or H2O2 levels in a cancer cell. Si-MnO2 core-shell nanosphere probes are fabricated in a solution-based process from crystalline Si nanosphere cores. The fluorescence switching under exposure to GSH is demonstrated, and the mechanism is discussed based on detailed optical characterizations including single-particle spectroscopy. Different types of human cells are incubated with the nanoprobes, and a proof of concept experiment is performed. From the combination of the robust scattering images and GSH- and H2O2-sensitive fluorescence images, the feasibility of cancer cell detection by the multimodal nanoprobes is demonstrated.

Molecular Aggregation Strategy for Inhibiting DNases.

This study highlights the novel potential of molecular aggregates as inhibitors of a disease-related protein. Enzyme inhibitors have been studied and developed as molecularly targeted drugs and have been applied for cancer, autoimmune diseases, and infections. In many cases, enzyme inhibitors that are used for therapeutic applications interact directly with enzymes in a molecule-to-molecule manner. We found that the aggregates of a small compound, Mn007, inhibited bovine pancreatic DNase I. Once Mn007 molecules formed aggregates, they exhibited inhibitory effects specific to DNases that require divalent metal ions. A DNase secreted by Streptococcus pyogenes causes streptococcal toxic shock syndrome (STSS). STSS is a severe infectious disease with a fatality rate exceeding 30% in patients, even in this century. S. pyogenes disrupts the human barrier system against microbial infections through the secreted DNase. Until now, the discovery/development of a DNase inhibitor has been challenging. Mn007 aggregates were found to inhibit the DNase secreted by S. pyogenes, which led to the successful suppression of S. pyogenes growth in human whole blood. To date, molecular aggregation has been outside the scope of drug discovery. The present study suggests that molecular aggregation is a vast area to be explored for drug discovery and development because aggregates of small-molecule compounds can inhibit disease-related enzymes.

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Preparation of affinity membranes using thermally induced phase separation for one-step purification of recombinant proteins.

We synthesized several surfactant-like ligands and prepared affinity membranes by introducing them into porous polymeric membranes using the thermally induced phase separation method. The ligands (nitrilotriacetate, iminodiacetate, and glutathione) were successfully displayed on the surfaces of cellulose diacetate membranes. Membranes functionalized with nitrilotriacetate and glutathione captured and released hexahistidine-tagged enhanced green fluorescent protein (His-tag GFP) and glutathione S-transferase (GST) selectively under appropriate conditions. The affinity membranes also enabled highly selective purification of target proteins (GFP and GST) from cell lysates. The protein-binding capacity was 15 µg/cm(2) for His-tag GFP and 13 µg/cm(2) for GST. The application-specific membranes described in this work will aid high-throughput screening and high-throughput analysis of recombinant proteins.

Display of amino groups on substrate surfaces by simple dip-coating of methacrylate-based polymers and its application to DNA immobilization.

The implementation of a reactive functional group onto a material surface is of great importance. Reactive functional groups (e.g., an amino group and a hydroxyl group) are usually hydrophilic, which makes it difficult to display them on a dry polymer surface. We here propose a novel method for displaying amino groups on the surfaces of polymeric substrates through dip-coating of a methacrylate-based copolymer. We synthesized copolymers composed of methyl methacrylate and 2-aminoethyl methacrylate with different protecting groups or ion-complexes on their amino groups, then dip-coated the copolymers onto a poly(methyl methacrylate) (PMMA) substrate. Evaluation using a cleavable fluorescent compound, which was synthesized in the present study to quantify a small amount (pmol/cm(2)) of amino groups on a solid surface, revealed that the protection of amino groups affected their surface segregation in the copolymer coating. p-Toluenesulfonate ion-complex and tert-butoxycarbonyl (Boc) protection of amino groups were found to effectively display amino groups on the surface (more than 70 pmol/cm(2)). The density of amino groups displayed on a surface can be easily controlled by mixing the copolymer and PMMA before dip-coating. Dip-coating of the copolymer with Boc protection on various polymeric substrates also successfully displayed amino groups on their surfaces. Finally, we demonstrated that the amino groups displayed can be utilized for the immobilization of a DNA oligonucleotide on a substrate surface.

Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry.

We report a method to prepare a DNA-enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K(d) was approximately 10⁻6 (M⁻¹) toward a His-tag present on a recombinant protein via the complexation of Ni²âº. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²âº complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni²âº complex. The DNA-enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.

Versatile supramolecular gelators that can harden water, organic solvents and ionic liquids.

We developed novel supramolecular gelators with simple molecular structures that could harden a broad range of solvents: aqueous solutions of a wide pH range, organic solvents, edible oil, biodiesel, and ionic liquids at gelation concentrations of 0.1-2 wt %. The supramolecular gelators were composed of a long hydrophobic tail, amino acids and gluconic acid, which were prepared by liquid-phase synthesis. Among seven types of the gelators synthesized, the gelators containing L-Val, L-Leu, and L-Ile exhibited high gelation ability to various solvents. These gelators were soluble in aqueous and organic solvents, and also in ionic liquids at high temperature. The gelation of these solvents was thermally reversible. The microscopic observations (TEM, SEM, and CLSM) and small-angle X-ray scattering (SAXS) measurements suggested that the gelator molecules self-assembled to form entangled nanofibers in a large variety of solvents, resulting in the gelation of these solvents. Molecular mechanics and density functional theory (DFT) calculations indicated the possible molecular packing of the gelator in the nanofibers. Interestingly, the gelation of an ionic liquid by our gelator did not affect the ionic conductivity of the ionic liquid, which would provide an advantage to electrochemical applications.

Rewritable Surface on a Plastic Substrate Using Fluorous Affinity.

Fluorous chemistry has unique features and high potential applicability, which are distinct from those of nonfluorinated organic compounds. However, there are limited reports detailing the applications of fluorous-fluorous interactions (fluorophilicity or fluorous affinity), likely because these interactions are not found in nature. In the present study, we describe the rewritable surface functionalization of a plastic substrate based on fluorous affinity. Plastic substrates were dip-coated with a series of methacrylate-based fluoropolymers to generate fluorous surfaces. Fluorous-tagged small molecules [perfluoroalkyl (Rf) amines] were immobilized on the fluorous surfaces via fluorous-fluorous interactions, thereby introducing reactive functional groups (amino moieties) on the surface. The amino groups displayed on the surface (accessible by a reactant) were successfully quantified using a reactive fluorophore, which enabled quantitative analysis of the Rf-amines immobilized on the fluorous surface that were available for the subsequent reaction. The effects of the molecular structures of the fluoropolymers and Rf-amines on the surface immobilization of Rf-amines were also investigated quantitatively. The surface coated with a fluoropolymer containing -C8F17 most effectively immobilized an Rf-amine comprising two -C6F13 chains. The adhered Rf-amines were easily removed by washing the surface with methanol, and then, they could successfully be re-immobilized on the surface. Finally, the presented approach enabled the rewritable micropatterning of an Rf-tagged biomolecule on a plastic surface through microcontact printing.

In Situ Synthesis of an Anticancer Peptide Amphiphile Using Tyrosine Kinase Overexpressed in Cancer Cells.

Cell-selective killing using molecular self-assemblies is an emerging concept for cancer therapy. Reported molecular self-assemblies are triggered by hydrolysis of well-designed molecules inside or outside cancer cells. This hydrolysis can occur in cancer and normal cells because of the abundance of water in living systems. Here, we report the in situ synthesis of a self-assembling molecule using a tyrosine kinase overexpressed in cancer cells. We designed a tyrosine-containing peptide amphiphile (C16-E4Y) that is transformed into a phosphorylated peptide amphiphile (C16-E4pY) by the overexpressed tyrosine kinase. Phosphorylation of C16-E4Y promoted self-assembly to form nanofibers in cancer cells. C16-E4Y exhibited selective cytotoxicity toward cancer cells overexpressing the tyrosine kinase. Self-assembled C16-E4pY induced endoplasmic reticulum stress that caused apoptotic cell death. Animal experiments revealed that C16-E4Y has antitumor activity. These results show that an enzyme overexpressed in cancer cells is available for intracellular synthesis of an antitumor self-assembling drug that is cell-selective.

Inhibition of Melittin Activity Using a Small Molecule with an Indole Ring.

We investigated d-amino acids as potential inhibitors targeting l-peptide toxins. Among the l- and d-amino acids tested, we found that d-tryptophan (d-Trp) acted as an inhibitor of melittin-induced hemolysis. We then evaluated various Trp derivatives and found that 5-chlorotryptamine (5CT) had the largest inhibitory effect on melittin. The indole ring, amino group, and steric hindrance of an inhibitor played important roles in the inhibition of melittin activity. Despite the small size and simple molecular structure of 5CT, its IC50 was approximately 13 µg/mL. Fluorescence quenching, circular dichroism measurements, and size-exclusion chromatography revealed that 5CT interacted with Trp19 in melittin and affected the formation of the melittin tetramer involved in hemolysis. Molecular dynamics simulation of melittin also indicated that the interaction of 5CT with Trp19 in melittin affected the formation of the tetramer.