Delivery of external proteins into the cytoplasm using protein capsules modified with IgG on the surface, created from the amphiphilic two helix-bundle protein OLE-ZIP.

This study explores a new method for delivering therapeutic proteins into specific cells using OLE-ZIP capsules that present IgG. OLE-ZIP capsules is a spherical caspules prepared from amphihilic dimetic coiled-coil peptide, OLE-ZIP. Upon presenting cetuximab, these capsules showed preferential uptake in A431 cells and increased cytotoxicity when loaded with RNase A.

Structure-Based Identification of Potent Lysine-Specific Demethylase 1 Inhibitor Peptides and Temporary Cyclization to Enhance Proteolytic Stability and Cell Growth-Inhibitory Activity.

Peptides are attractive drug candidates, but their utility is greatly limited by their inherent susceptibility to proteolytic degradation and their inability to pass through the cell membrane. Here, we employ a strategy of temporary cyclization to develop a cell-active lysine-specific demethylase 1 (LSD1/KDM1A) inhibitor peptide. We first identified a highly potent LSD1-inhibitory linear peptide, with the assistance of X-ray crystal structure data of inhibitor peptide-bound LSD1·CoREST. The peptide was converted to a redox-activatable cyclic peptide incorporating cell-penetrating peptide (CPP), expecting selective activation under intracellular reducing conditions. The cyclic peptide moiety exhibited enhanced stability to protease and was converted to the linear, unmodified LSD1 inhibitor peptide under reducing conditions. The cyclic peptide with CPP inhibited the proliferation of human acute myeloid leukemia cells (HL-60) in the low micromolar concentration range.

Specific effects of antitumor active norspermidine on the structure and function of DNA.

We compared the effects of trivalent polyamines, spermidine (SPD) and norspermidine (NSPD), a chemical homologue of SPD, on the structure of DNA and gene expression. The chemical structures of SPD and NSPD are different only with the number of methylene groups between amine groups, [N-3-N-4-N] and [N-3-N-3-N], respectively. SPD plays vital roles in cell function and survival, including in mammals. On the other hand, NSPD has antitumor activity and is found in some species of plants, bacteria and algae, but not in humans. We found that both polyamines exhibit biphasic effect; enhancement and inhibition on in vitro gene expression, where SPD shows definitely higher potency in enhancement but NSPD causes stronger inhibition. Based on the results of AFM (atomic force microscopy) observations together with single DNA measurements with fluorescence microscopy, it becomes clear that SPD tends to align DNA orientation, whereas NSPD induces shrinkage with a greater potency. The measurement of binding equilibrium by NMR indicates that NSPD shows 4-5 times higher affinity to DNA than SPD. Our theoretical study with Monte Carlo simulation provides the insights into the underlying mechanism of the specific effect of NSPD on DNA.

Stable Iron Porphyrin Intramolecularly Coordinated by Alcoholate Anion: Synthesis and Evaluation of Axial Ligand Effect of Alcoholate on Spectroscopy and Catalytic Activity.

We synthesized intramolecularly aliphatic alcoholate-coordinated iron porphyrins (1a, 1b) that retain their axial coordination in the presence of another ligand or oxidant. The electron-donative character of alcoholate was less than that of thiolate, and the coordination ability of a sixth ligand to 1a and 1b was very much lower than in the case of the thiolate-coordinated compounds. Density functional theory calculations indicated that the marked difference in coordination ability could be explained in terms of thermodynamic and steric factors. The catalytic oxidizing ability of the thiolate-coordinated compound, SR complex, was much higher than that of 1a.

Design and synthesis of a 4-aminoquinoline-based molecular tweezer that recognizes protoporphyrin IX and iron(iii) protoporphyrin IX and its application as a supramolecular photosensitizer.

We report on the design and synthesis of a new type of 4-aminoquinoline-based molecular tweezer 1 which forms a stable host-guest complex with protoporphyrin IX (PPIX) via multiple interactions in a DMSO and HEPES buffer (pH 7.4) mixed solvent system. The binding constant for the 1 : 1 complex (K 11) between 1 and PPIX is determined to be 4 × 106 M-1. Furthermore, 1 also forms a more stable complex with iron(iii) protoporphyrin IX (Fe(iii)PPIX), the K 11 value for which is one order of magnitude greater than that for PPIX, indicating that 1 could be used as a recognition unit of a synthetic heme sensor. On the other hand, the formation of the stable PPIX·1 complex (supramolecular photosensitizer) prompted us to apply it to photodynamic therapy (PDT). Cell staining experiments using the supramolecular photosensitizer and evaluations of its photocytotoxicity indicate that the PDT activity of PPIX is improved as the result of the formation of a complex with 1.

Inhibition of FAD-dependent lysine-specific demethylases by chiral polyamine analogues.

Lysine-specific demethylases 1 and 2 (LSD1 and LSD2) are flavoenzyme demethylases, and their inhibitors are considered as potential chemical tools and anticancer agents. Here we report polyamine-based inhibitors of LSD1 and LSD2. In the initial screening, partially constrained polyamine 2 which contains three trans-cyclopentane units with a total of six stereogenic centers, showed the most potent LSD1-inhibitory activity. We then prepared a set of optical isomers of 2 and evaluated their inhibitory activities toward LSD1, LSD2, monoamine oxidases A and B (MAO-A and MAO-B). Optical isomers of 2 showed LSD1-inhibitory activity with K i values of 2.2 to 6.4 µM, and LSD2-inhibitory activity with K i values of 4.4 to 39 µM; there was a general preference for LSD1 to LSD2. All of them showed weak to negligible inhibition of MAO-A and MAO-B. This selectivity seemed to reflect the differences in the size and shape of the catalytic cavity of target enzymes, and our strategy of employing a set of optical isomers appears to be an effective approach for exploring the structural features of this family of enzymes. Polyamine 9 showed most potent LSD1-inhibitory activity (K i = 2.2 µM in vitro), and it also inhibited the proliferation of HL-60 cells (IC50 = 49 µM). On the other hand, 12 was the most potent inhibitors of LSD2 with in vitro K i values of 4.4 µM.

Active site geometry of a novel aminopropyltransferase for biosynthesis of hyperthermophile-specific branched-chain polyamine.

Branched-chain polyamines are found exclusively in thermophilic bacteria and Euryarchaeota and play essential roles in survival at high temperatures. In the present study, kinetic analyses of a branched-chain polyamine synthase from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-BpsA) were conducted, showing that N4 -bis(aminopropyl)spermidine was produced by sequential additions of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine, through bifunctional catalytic action. Tk-BpsA catalyzed the aminopropylation of the linear-chain polyamines spermidine, spermine, norspermidine, and the tertiary-branched polyamines N4 -aminopropylspermidine and N4 -aminopropylnorspermidine, but not of short-chain diamines, putrescine, and cadaverine, suggesting that Tk-BpsA does not catalyze the aminopropylation of primary amino groups of diamines. X-ray structural analyses of Tk-BpsA in the presence or absence of the substrates spermidine and dcSAM revealed that a large, negatively charged cavity is responsible for the binding of branched-chain substrates. The binding is different from that in the active site of linear polyamine spermidine/spermine synthases, and loop-closures occur upon the binding of spermidine. Based on structural analyses, further kinetic studies were carried out for various mutants, revealing that Asp159, positioned between the reactive secondary amino group of the substrate polyamine and a sulfur atom of the product 5'-methylthioadenosine and in a Gly-Asp-Asp-Asp motif, functions as a catalytic center, with reactions proceeding via a ping-pong mechanism. Our study provides a novel aminopropyltransfer reaction mechanism, distinct from the SN 2 displacement mechanism found in other known linear spermidine/spermine synthases. DATABASE: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with PDB codes 5XNF for apo-Tk-BpsA, 5XNH for the binary complex, and 5XNC for the ternary complex.

Development and crystallographic evaluation of histone H3 peptide with N-terminal serine substitution as a potent inhibitor of lysine-specific demethylase 1.

Lysine-specific demethylase 1 (LSD1/KDM1A) is a flavoenzyme demethylase, which removes mono- and dimethyl groups from histone H3 Lys4 (H3K4) or Lys9 (H3K9) in complexes with several nuclear proteins. Since LSD1 is implicated in the tumorigenesis and progression of various cancers, LSD1-specific inhibitors are considered as potential anti-cancer agents. A modified H3 peptide with substitution of Lys4 to Met [H3K4M] is already known to be a potent competitive inhibitor of LSD1. In this study, we synthesized a series of H3K4M peptide derivatives and evaluated their LSD1-inhibitory activities in vitro. We found that substitutions of the N-terminal amino acid with amino acids having a larger side chain were generally not tolerated, but substitution of Ala1 to Ser unexpectedly resulted in more potent inhibitory activity toward LSD1. X-ray crystallographic analysis of H3K4M derivatives bound to the LSD1·CoREST complex revealed the presence of additional hydrogen bonding between the N-terminal Ser residue of the H3 peptide derivative and LSD1. The present structural and biochemical findings will be helpful for obtaining more potent peptidic inhibitors of LSD1.

Activation of lysine-specific demethylase 1 inhibitor peptide by redox-controlled cleavage of a traceless linker.

We have previously employed cyclization of a linear peptide as a strategy to modulate peptide function and properties, but cleavage to regenerate the linear peptide left parts of the linker structure on the peptide, interfering with its activity. Here, we focused on cyclization of a linear peptide via a "traceless" disulfide-based linkage that would be cleaved and completely removed in a reducing environment, regenerating the original linear peptide without any linker-related structure. Thus, the linker would serve as a redox switch that would be activated in the intracellular environment. We applied this strategy to a lysine-specific demethylase 1 (LSD1) inhibitor peptide 1. The resulting cyclic peptide 2 exhibited approximately 20 times weaker LSD1-inhibitory activity than peptide 1. Upon addition of reducing reagent, the linker was completely removed to regenerate the linear peptide 1, with full restoration of the LSD1-inhibitory activity. In addition, the cyclic peptide was far less susceptible to proteolysis than the linear counterpart. Thus, this switch design not only enables control of functional activity, but also improves stability. This approach should be applicable to a wide range of peptides, and may be useful in the development of peptide pharmaceuticals.

Design of New Extraction Surfactants for Membrane Proteins from Peptide Gemini Surfactants.

The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native-PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins.

Facile synthesis of peptide-porphyrin conjugates: Towards artificial catalase.

A facile synthetic method for peptide-porphyrin conjugates containing four peptide units on one porphyrin was developed using chemoselective reactions. The key building blocks, 5,10,15,20-tetrakis(3-azidophenyl)porphyrin 1 and 5,10,15,20-tetrakis(5-azido-3-pyridyl)porphyrin 2, were efficiently synthesized and used as substrates for two well-known chemoselective reactions, traceless Staudinger ligation and copper-catalyzed azide alkyne cycloaddition (so-called click chemistry). Both reactions gave the desired compounds, and click chemistry was superior for our purpose. To confirm the value of the established methodology, nine peptide-porphyrin conjugates were synthesized, and their catalase- and peroxidase-like activity in water was evaluated. Our synthetic strategy is expected to be valuable for the preparation of artificial heme protein models.

X-ray radiation causes electromagnetic interference in implantable cardiac pacemakers.

BACKGROUND: X-rays are not thought to cause electromagnetic interference (EMI) in implantable cardiac pacemakers. However, x-ray radiation during computed tomography (CT) scanning has been reported to cause EMI in some implantable cardiac pacemakers. The objectives of this study were to identify the location within the pacemakers where x-ray radiation causes EMI and to investigate the association of EMI with the x-ray radiation conditions. METHODS: We verified the location where x-ray radiation caused EMI using a CT scanner and conventional radiographic x-ray equipment. An inhibition test and an asynchronous test were performed using five types of implantable cardiac pacemakers. RESULTS: X-ray radiation inhibited the pacing pulses of four types of implantable cardiac pacemakers when the body of each implantable cardiac pacemaker, containing a complementary metal-oxide semiconductor (CMOS), was scanned using a CT scanner. We confirmed that x-ray-induced EMI depends on the x-ray radiation conditions, that is, the tube voltage, tube current, x-ray dose, and direction of x-ray radiation, as well as the sensing thresholds of the implantable cardiac pacemakers. CONCLUSIONS: X-ray radiation caused EMI in some implantable cardiac pacemakers, probably because the CMOS component was irradiated. The occurrence of EMI depended on the pacemaker model, sensing threshold of the pacemaker, and x-ray radiation conditions.

Structurally designed trans-2-phenylcyclopropylamine derivatives potently inhibit histone demethylase LSD1/KDM1 .

Lysine-specific demethylase 1 (LSD1/KDM1) demethylates histone H3, in addition to tumor suppressor p53 and DNA methyltransferase 1 (Dnmt1), thus regulating eukaryotic gene expression by altering chromatin structure. Specific inhibitors of LSD1 are desired as anticancer agents, because LSD1 aberrations are associated with several cancers, and LSD1 inhibition restores the expression of abnormally silenced genes in cancerous cells. In this study, we designed and synthesized several candidate compounds to inhibit LSD1, based on the structures of LSD1 and monoamine oxidase B (MAO-B), in complex with an antidepressant tranylcypromine (2-PCPA) derivative. Compound S2101 exhibited stronger LSD1 inhibition than tranylcypromine and the known small LSD1 inhibitors in LSD1 demethylation assays, with a k(inact)/K(I) value of 4560 M(-1) s(-1). In comparison with tranylcypromine, the compound displayed weaker inhibition to the monoamine oxidases. The inhibition modes of the two 2-PCPA derivatives, 2-PFPA and S1201, were identified by determination of the inhibitor-bound LSD1 structures, which revealed the enhanced stability of the inhibitor-FAD adducts by their interactions with the surrounding LSD1 residues. These molecules are potential pharmaceutical candidates for cancer or latent virus infection, as well as research tools for LSD1-related biological investigations.

Inhibition of gamma-secretase activity by helical beta-peptide foldamers.

Alzheimer's disease (AD) is a neurodegenerative disorder pathologically characterized by extensive extracellular deposition of amyloid-beta (Abeta) peptides as senile plaques, and inhibition of "amyloidogenic" amyloid precursor protein (APP) processing by gamma-secretase is an important strategy for prevention and treatment of AD. Here we show that beta-peptide foldamers designed to adopt a 12-helical conformation in solution are potent and specific inhibitors of gamma-secretase. Subtle modifications that disrupt helicity substantially reduce inhibitory potency, suggesting that helical conformation is critical for effective inhibition. These beta-peptides competed with helical peptide-type inhibitor, suggesting that they interact with the substrate binding site of gamma-secretase. The beta-peptide with inhibitory activity at nanomolar concentration should be a useful lead compound for development of gamma-secretase-specific inhibitors and molecular tools to explore substrate recognition by intramembrane proteases.

Novel probes showing specific fluorescence enhancement on binding to a hexahistidine tag.

The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel-nitrilotriacetic acid (Ni2+-NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal-NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal-NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni2+ and Co2+ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni2+ and Co2+ complexes of NTACs in pH 7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)6-Tyr-NH2; Tyr was included to allow convenient concentration measurement) in pH 7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co2+ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide.

Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction.

We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.