Design, Synthesis, and In Vitro Evaluation of Novel Histone H3 Peptide-Based LSD1 Inactivators Incorporating α,α-Disubstituted Amino Acids with γ-Turn-Inducing Structures.

Lysine-specific demethylase 1 (LSD1) mainly removes methyl groups of mono- or di-methylated lysine residues at the fourth position of histone H3 to epigenetically regulate the expression of genes associated with several diseases, such as cancer. Therefore, LSD1 inactivators are expected to be used as therapeutic agents. In this study, to identify novel peptide-based LSD1 inactivators, we focused on the X-ray structure of LSD1 complexed with a H3 peptide-based suicide substrate. It has been proposed that a methylated histone substrate forms three consecutive γ-turn structures in the active pocket of LSD1. Based on this, we designed and synthesized novel histone H3 peptide-based LSD1 inactivators 2a⁻c by incorporating various α,α-disubstituted amino acids with γ-turn-inducing structures. Among synthetic peptides 2a⁻c, peptide 2b incorporating two 1-aminocyclohexanecarboxylic acids at both sides of a lysine residue bearing a trans-2-phenylcyclopropylamine (PCPA) moiety, which is a pharmacophore for LSD1 inactivation, was the most potent and selective LSD1 inactivator. These findings are useful for the further development of histone H3 peptide-based LSD1 inactivators.

Histone H3 peptides incorporating modified lysine residues as lysine-specific demethylase 1 inhibitors.

Lysine-specific demethylase 1 (LSD1) is a flavin-dependent enzyme that removes methyl groups from mono- or dimethylated lysine residues at the fourth position of histone H3. We have previously reported several histone H3 peptides containing an LSD1 inactivator motif at Lys-4. In this study, histone H3 peptides having a trans-2-phenylcyclopropylamine (PCPA), a 2,5-dihydro-1H-pyrrole, and a 1,2,3,6-tetrahydropyridine moiety at Lys-4 were prepared along with related compounds possessing a shorter side chain at the fourth position. Enzymatic assays showed that PCPA peptides containing a longer side chain, which can react with FAD in the active site, are potent LSD1-selective inhibitors.

Potent mechanism-based sirtuin-2-selective inhibition by an in situ-generated occupant of the substrate-binding site, "selectivity pocket" and NAD+-binding site.

Sirtuin 2 (SIRT2), a member of the NAD+-dependent histone deacetylase family, has recently received increasing attention due to its potential involvement in neurodegenerative diseases and the progression of cancer. Potent and selective SIRT2 inhibitors thus represent desirable biological probes. Based on the X-ray crystal structure of SIRT2 in complex with a previously reported weak inhibitor (6), we identified in this study the potent mechanism-based inactivator KPM-2 (36), which is selective toward SIRT2. Compound 36 engages in a nucleophilic attack toward NAD+ at the active site of SIRT2, which affords a stable 36-ADP-ribose conjugate that simultaneously occupies the substrate-binding site, the "selectivity pocket" and the NAD+-binding site. Moreover, 36 exhibits antiproliferative activity in cancer cells and remarkable neurite outgrowth activity. This strategy for the selective inhibition of SIRT2 should allow further probing of the biology of SIRT2, and promote the development of new disease treatment strategies.

Crystal structure of [1,2-bis-(di-phenyl-phosphan-yl)benzene]-hepta-carbonyldi-µ-hydrido-(µ3-2,4,6-tri-methyl-phenyl-phosphin-idene)-triangulo-triruthenium.

The title trinuclear ruthenium cluster, [Ru3(C30H24P2)(C9H11P)(CO)7(µ-H)2], has a triangular Ru3 core that is capped with a mesitylphosphin-idene ligand, µ3-PMes (Mes = mesityl = 2,4,6-tri-methyl-phen-yl). The 1,2-bis-(di-phenyl-phosphan-yl)benzene mol-ecule acts as a bidentate phosphine ligand via two P atoms connecting to a single Ru atom. The title compound crystallizes with two independent mol-ecules in the asymmetric unit.

Design, synthesis, and evaluation of Trolox-conjugated amyloid-ß C-terminal peptides for therapeutic intervention in an in vitro model of Alzheimer's disease.

Two hallmarks of Alzheimer's disease (AD) observed in the brains of patients with the disease include oxidative injury and deposition of protein aggregates comprised of amyloid-ß (Aß) variants. To inhibit these toxic processes, we synthesized antioxidant-conjugated peptides comprised of Trolox and various C-terminal motifs of Aß variants, TxAßx-n (x=34, 36, 38, 40; n=40, 42, 43). Most of these compounds were found to exhibit anti-aggregation activities. Among them, TxAß36-42 significantly inhibited Aß1-42 aggregation, showed potent antioxidant activity, and protected SH-SY5Y cells from Aß1-42-induced cytotoxicity. Thus, this method represents a promising strategy for developing multifunctional AD therapeutic agents.

Evaluation of phenylcyclopropylamine compounds by enzymatic assay of lysine-specific demethylase 2 in the presence of NPAC peptide.

Lysine-specific demethylase 2 (LSD2) demethylates mono- and dimethylated Lys-4 of histone H3 (H3K4me1 and H3K4me2). NPAC protein is known to interact with LSD2 and promote its H3K4 demethylase activity. In this study, we established a demethylation assay system that utilizes recombinant LSD2 in the presence of a synthetic NPAC peptide. Several phenylcyclopropylamine (PCPA)-based inhibitors were examined for their LSD2 inhibitory activity in the LSD2 enzymatic assay with the NPAC peptide. The assay results showed that the PCPA derivatives, including NCD41, selectively inhibited LSD1 in preference to LSD2.

Histone H3 peptide based LSD1-selective inhibitors.

A series of candidates for the histone H3 peptide based LSD1-selective inhibitor were designed and synthesized. Among peptides 1a-c and 2a-c, peptide 1a, which has a phenylcyclopropylamine (PCPA) moiety at Lys-4 of the 21 amino acid residues of histone H3, was the most potent LSD1-selective inhibitor. Truncation studies of peptide 1a revealed the significance of the peptide sequence length. These findings will be useful for the further development of histone H3 peptide based LSD1-selective inhibitors.

Octa-carbonyldi-µ(2)-hydrido-[µ(3)-(1,3,5-trimethyl-phen-yl)phosphinidene](tri-phenyl-phosphane)-triangulo-triruthenium.

In the crystal structure of the title compound, [Ru(3)(C(9)H(11)P)H(2)(C(18)H(15)P)(CO)(8)], the triangular Ru(3) unit is capped with one mesitylphosphin-idene ligand. In the trigonal-pyramidal Ru(3)P core, one Ru(II) atom is coordinated by a triphenyl-phosphane ligand in a terminal fashion. Two hydride ligands bridge over two Ru-Ru bonds. These Ru-Ru bonds [2.9400 (4) and 2.9432 (4) Å] are slightly longer than the nonhydride-bridged Ru-Ru bond [2.8146 (4) Å]. The terminal triphenyl-phosphane ligand coordinates to the Ru(II) atom, which is involved in two hydride bridges.

Development of [Ile4°]HTLV-I protease inhibition assay using novel fluorogenic and chromogenic substrate.

HTLV-I is a debilitating and/or lethal retrovirus that causes HTLV-I-associated myelopathy/tropical spastic paraparesis, adult T-cell leukemia and several inflammatory diseases. HTLV-I protease is an aspartic retropepsin involved in HTLV-I replication and its inhibition could treatHTLV-I infection. A recombinant L40I mutant HTLV-I protease was designed and obtained from Escherichia coli, self-processingand purification by ion-exchange chromatography. The protease was refolded by a one-step dialysis and recovered activity. The cleavage efficiency of the [Ile4°]HTLV-I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His-tagged non-mutated HTLV-I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p-nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV-I protease inhibition potency assay. The HTLV-I protease inhibition assay with the [Ile4°]HTLV-I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost-effective and more time-efficient while being reproducible and less labor-intensive.

Self-assembly pathways of E22Δ-type amyloid ß peptide mutants generated from non-aggregative O-acyl isopeptide precursors.

The recently identified E22Δ-type amyloid ß peptide (Aß) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aß. Aß(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aß oligomers in Alzheimer's disease (or Alzheimer's-type dementia). However, the biochemical and biophysical properties of Aß(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aß(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aß(E22Δ) differed from those of Aß(WT). Aß1-40(E22Δ) underwent a rapid random coil→ß-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aß1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aß1-42(E22Δ) monomer formed ß-sheet-rich oligomers more rapidly than did Aß1-42(WT). Additionally, the Aß1-42(E22Δ) oligomers appear to differ from Aß1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aß(E22Δ) mutants.

Evaluation of superior BACE1 cleavage sequences containing unnatural amino acids.

A recombinant form of BACE1 (ß-site amyloid precursor protein cleaving enzyme-1) corresponding to positions 46-454 of the extracellular domain of the original membrane enzyme was prepared. The recombinant BACE1 (rBACE1) had the kinetic parameters K(m)=5.5µM and k(cat)=1719s(-1). Using several libraries of substrates containing unnatural amino acids, the specificity of rBACE1 was evaluated. LC/MS of digests derived from the libraries clarified that a dodecapeptide containing unnatural amino acids at P(2) to [Formula: see text] was a superior cleavage sequence.

Tetrapeptides, as small-sized peptidic inhibitors; synthesis and their inhibitory activity against BACE1.

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid beta-peptide in Alzheimer's disease and is a major target for current drug design. We previously reported substrate-based peptidomimetics, KMI-compounds as potent BACE1 inhibitors. In this study, we designed and synthesized tetrapeptides as low molecular-sized inhibitors. These exhibited high potency against recombinant BACE1, with the highest IC(50) value of 34.6 nM from KMI-927.

Dehydrocoupling reactions of borane-secondary and -primary amine adducts catalyzed by group-6 carbonyl complexes: formation of aminoboranes and borazines.

Photoirradiation of a solution of BH(3).NHR(2) (1a: R = Me, 1b: R = 1/2C(4)H(8), 1c: R = 1/2C(5)H(10), 1f: R = Et) containing a catalytic amount of a group-6 metal carbonyl complex, [M(CO)(6)] (M = Cr, Mo, W), led to dehydrogenative B-N covalent bond formation to produce aminoborane dimers, [BH(2)NR(2)](2) (2a-c, f), in high yield. During these reactions a borane sigma complex, [M(CO)(5)(eta(1)-BH(3).NHR(2))] (3), was detected by NMR spectroscopy. Similar catalytic dehydrogenation of bulkier amineboranes, BH(3).NH(i)Pr(2) (1d) and BH(3).NHCy(2) (1e, Cy = cyclo-C(6)H(11)), afforded monomeric products BH(2) horizontal lineNR(2) (4d, e). The reaction mechanism of the dehydrocoupling was investigated by DFT calculations. On the basis of the computational study, we propose that the catalytic dehydrogenation reactions proceed via an intramolecular pathway and that the active catalyst is [Cr(CO)(4)]. The reaction follows a stepwise mechanism involving NH and BH activation. Dehydrocoupling of borane-primary amine adducts BH(3).NH(2)R (1g: R = Me, 1h: R = Et, 1i: R = (t)Bu) gave borazine derivatives [BHNR](3) (5g-i).

Synthesis of amyloid beta peptide 1-42 (E22Delta) click peptide: pH-triggered in situ production of its native form.

Amyloid beta peptide (Abeta) 1-42 is known to be involved in the onset of Alzheimer's disease (AD). We developed a click peptide of Abeta1-42 as a useful tool for AD research on the basis of an O-acyl isopeptide method. The click peptide quickly produced intact Abeta1-42 via a pH-dependent O-to-N intramolecular acyl migration (pH-click). Herein, a click peptide (26-O-acyl isoAbeta1-42 (E22Delta)) of a new mutant Abeta1-42 (E22Delta) was synthesized. The mutant click peptide was more water-soluble than Abeta1-42 (E22Delta). Moreover it quantitatively converted to the native peptide under physiological conditions (pH 7.4, 37 degrees C). CD analyses showed a conformational change from a random-coil structure of the click peptide to a beta-sheet structure of the in situ produced Abeta1-42 (E22Delta). This click peptide is a useful precursor of a mutant Abeta1-42 to establish an experiment system for investigating the properties of the mutant.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation.

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.

Switching from the unfolded to the folded state of the helix-loop-helix domain of the Id proteins based on the O-acyl isopeptide method.

The inhibitors of DNA binding and cell differentiation Id1-4 are helix-loop-helix (HLH) proteins that negatively regulate DNA transcription by forming inactive dimers with ubiquitous and tissue-specific bHLH proteins, including E47 and MyoD, respectively. Their highly conserved HLH domains are essential for heterodimerization, but can also self-associate to highly stable, alpha-helix-rich structures at low micromolar peptide concentrations. Here, we show that the introduction of an O-acyl isodipeptide unit involving the putative N-cap serine residue of the C-terminal helix completely abrogates the propensity of the Id HLH analogue for any secondary and tertiary structure, resulting in a random coil, as shown by CD measurements in nonbuffered aqueous solutions. However, the HLH fold reappears as soon as an O-->N intramolecular acyl migration, which occurs spontaneously under physiological conditions, restores the native N-cap serine residue. These results show that changes addressing the N-terminus of the C-terminal helix can dramatically influence the HLH structure, and suggest that local interactions at the junction between the loop and the C-terminal helix might be crucial during the HLH folding process. Furthermore, the present study contributes to the evaluation of the O-acyl isodipeptide unit as a powerful tool to introduce a conformational switch into peptides.

Fmoc-based solid phase chemical synthesis of 71-meric neuregulin 1-beta1, an epidermal growth factor-like domain.

The human neuregulin 1-beta1 (NRG1-beta1, amino acid residues 176-246) was chemically synthesized by Fmoc-based solid phase peptide synthesis (SPPS) followed by folding in a redox buffer. The biological activity of the synthesized NRG1-beta1 was confirmed by ligand-induced tyrosine phosphorylation on Chinese hamster ovary (CHO) cells expressing ErbB-4.

Investigation of the stability of the M-H-B bond in borane sigma complexes [M(CO)5(eta1-BH2R.L)] and [CpMn(CO)2(eta1-BH2R.L)] (M=Cr, W; L=tertiary amine or phosphine): substituent and Lewis base effects.

We investigated the influence of a substituent and a Lewis base on boron upon the thermodynamic stability of metal complexes of borane-Lewis base adducts, [M(CO)5(eta1-BH(2)R.L)] (M=Cr, W) and [CpMn(CO)2(eta1-BH2R.L)], where R=Cl, I, m-C6H4F, Ph, H, Me, Et; L=PMe3, PPh3, NMe3, quinuclidine. In these compounds, the stability of the metal-borane linkage was enhanced by increasing the electron-releasing ability of the substituent on boron. A stronger base L additionally stabilized the complexes. The strength of the borane-metal interaction is thus mainly ascribed to the electron donation from the BH sigma orbital to metal rather than the back-donation into the BH sigma* orbital. This result supports the bonding model for the B-H-M linkage in the borane complexes suggested by MO calculations, where the borane-to-metal electron donation is predominant while the metal back-donation into the BH sigma* orbital is negligible. Such a stability trend of the borane complexes makes a sharp contrast to that of many silane and dihydrogen complexes.