Intracellular protein-responsive supramolecules: protein sensing and in-cell construction of inhibitor assay system.

Supramolecular nanomaterials responsive to specific intracellular proteins should be greatly promising for protein sensing and imaging, controlled drug release or dynamic regulation of cellular processes. However, valid design strategies to create useful probes are poorly developed, particularly for proteins inside living cells as targets. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent probes consisting of a protein ligand and a fluorophore on the live cell surface, as well as in test tube settings. Herein, we discovered that our self-assembled supramolecular probes having a rhodamine derivative (tetramethylrhodamine or rhodamine-green) can incorporate and stay as less-fluorescent aggregates inside the living cells, so as to sense the protein activity in a reversible manner. Using the overexpressed model protein (dihydrofolate reductase), we demonstrated that this turn-on/off mode is controlled by selective ligand-protein recognition inside the live cells. Not only such a model protein, but also endogenous human carbonic anhydrase and heat shock protein 90 were specifically visualized in living mammalian cells, by use of the similar ligand-tethered supramolecular probes. Furthermore, such reversibility allowed us to intracellularly construct a unique system to evaluate the inhibitors affinity toward specific endogenous proteins in live cells, highlighting the potential of dynamic supramolecules as novel intelligent biomaterials.

Specific detection and imaging of enzyme activity by signal-amplifiable self-assembling (19)F MRI probes.

Specific turn-on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal-amplifiable self-assembling (19) F NMR/MRI probe for turn-on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are "silent" when aggregated, but exhibit a disassembly driven turn-on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these (19) F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by (19) F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by (19) F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn-on (19) F probes may serve as a screening platform for the activity of MMPs.

Specific cell surface protein imaging by extended self-assembling fluorescent turn-on nanoprobes.

Visualization of tumor-specific protein biomarkers on cell membranes has the potential to contribute greatly to basic biological research and therapeutic applications. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore in test tube settings. This method is based on recognition-driven disassembly of the nanoprobes, which induces a clear turn-on fluorescent signal. In the present study, we have successfully extended the range of applicable fluorophores to the more hydrophilic ones such as fluorescein or rhodamine by introducing a hydrophobic module near the fluorophore. Increasing the range of available fluorophores allowed selective imaging of membrane-bound proteins under live cell conditions. That is, overexpressed folate receptor (FR) or hypoxia-inducible membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific biomarkers were fluorescently visualized using the designed supramolecular nanoprobes in the turn-on manner. Moreover, a cell-based inhibitor-assay platform for CA on a live cell surface was constructed, highlighting the potential applicability of the self-assembling turn-on probes.

Substrate/Product-targeted NMR monitoring of pyrimidine catabolism and its inhibition by a clinical drug.

We report the application of one-dimensional triple-resonance NMR to metabolic analysis and thereon-based evaluation of drug activity. Doubly (13)C/(15)N-labeled uracil ([(15)N1,(13)C6]-uracil) was prepared. Its catabolic (degradative) conversion to [(13)C3,(15)N4]-ß-alanine and inhibition thereof by gimeracil, a clinical co-drug used with the antitumor agent 5-fluorouracil, in mouse liver lysates were monitored specifically using one-dimensional triple-resonance ((1)H-{(13)C-(15)N}) NMR, but not double-resonance ((1)H-{(13)C}) NMR, in a ratiometric manner. The administration of labeled uracil to a mouse resulted in its non-selective distribution in various organs, with efficient catabolism to labeled ß-alanine exclusively in the liver. The co-administration of gimeracil inhibited the catabolic conversion of uracil in the liver. In marked contrast to in vitro results, however, gimeracil had practically no effect on the level of uracil in the liver. The potentiality of triple-resonance NMR in the analysis of in vivo pharmaceutical activity of drugs targeting particular metabolic reactions is discussed.

Systematic study of protein detection mechanism of self-assembling 19F NMR/MRI nanoprobes toward rational design and improved sensitivity.

(19)F NMR/MRI probe is expected to be a powerful tool for selective sensing of biologically active agents owing to its high sensitivity and no background signals in live bodies. We have recently reported a unique supramolecular strategy for specific protein detection using a protein ligand-tethered self-assembling (19)F probe. This method is based on a recognition-driven disassembly of the nanoprobes, which induced a clear turn-on signal of (19)F NMR/MRI. In the present study, we conducted a systematic investigation of the relationship between structure and properties of the probe to elucidate the mechanism of this turn-on (19)F NMR sensing in detail. Newly synthesized (19)F probes showed three distinct behaviors in response to the target protein: off/on, always-on, and always-off modes. We clearly demonstrated that these differences in protein response could be explained by differences in the stability of the probe aggregates and that "moderate stability" of the aggregates produced an ideal turn-on response in protein detection. We also successfully controlled the aggregate stability by changing the hydrophobicity/hydrophilicity balance of the probes. The detailed understanding of the detection mechanism allowed us to rationally design a turn-on (19)F NMR probe with improved sensitivity, giving a higher image intensity for the target protein in (19)F MRI.

Disassembly-driven turn-on fluorescent nanoprobes for selective protein detection.

"Switchable" fluorescent probes, which induce changes in the fluorescence properties (e.g., intensity and/or wavelength) only at the intended target protein, are particularly useful for selective protein detection or imaging. However, the strategy for designing such smart probes remains very limited. We report herein a novel mechanism for generating protein-specific "turn-on" fluorescent probes. Our approach uses an amphiphilic, self-assembling compound consisting of a fluorophore and a protein ligand. In the absence of target protein, the probe forms self-assembled aggregates in aqueous solution and displays almost no fluorescence because of efficient quenching. On the other hand, it emits bright fluorescence in response to the target protein through recognition-induced disassembly of the probe. On the basis of this strategy, we successfully developed three types of fluorescent probes that allow the detection of carbonic anhydrase, avidin, and trypsin via turn-on emission signals. It is anticipated that the present supramolecular approach may facilitate the development of new protein-specific switchable fluorescent probes that are useful for a wide range of applications, such as diagnosis and molecular imaging.

Synthesis of puromycin derivatives with backbone-elongated substrates and associated translation inhibitory activities.

We have synthesized a series of 5'-phosphorylated and 5'-cytidylyl-(3'-5')-cytidylyl-(3'-5')-puromycin derivatives that have backbone-elongated substrates. All the synthesized puromycin derivatives showed good solubility in water and were applied to translation inhibitory assay in a reconstituted Escherichia coli translation system.

Unexpected preference of the E. coli translation system for the ester bond during incorporation of backbone-elongated substrates.

There have been recent advances in the ribosomal synthesis of various molecules composed of nonnatural ribosomal substrates. However, the ribosome has strict limitations on substrates with elongated backbones. Here, we show an unexpected loophole in the E. coli translation system, based on a remarkable disparity in its selectivity for beta-amino/hydroxy acids. We challenged beta-hydroxypropionic acid (beta-HPA), which is less nucleophilic than beta-amino acids but free from protonation, to produce a new repertoire of ribosome-compatible but main-chain-elongated substrates. PAGE analysis and mass-coupled S-tag assays of amber suppression experiments using yeast suppressor tRNAPheCUA confirmed the actual incorporation of beta-HPA into proteins/oligopeptides. We investigated the side-chain effects of beta-HPA and found that the side chain at position alpha and R stereochemistry of the beta-substrate is preferred and even notably enhances the efficiency of incorporation as compared to the parent substrate. These results indicate that the E. coli translation machinery can utilize main-chain-elongated substrates if the pKa of the substrate is appropriately chosen.

Efforts toward codon table engineering: expansion of unnatural substrates acceptable by the E. coli ribosome.

The ribosome catalyzes oligo/polymerization of amino acids. We designed an allowable modification of amino acid backbone, based on the hypothesized mechanism of peptidyl transfer reaction. Nonsense suppression method was used to investigate the acceptability of these substrates in the prokaryotic ribosomal system. The E.coli ribosome showed a restricted tolerance to the backbone modification, especially for main-chain elongation. However, our designed homologous beta-hydroxyalkanoic acid with elongated methylene (backbone) chain-length was revealed to be a possible substrate for the E.coli ribosome.