Synthesis and anti-tumor activities in human leukemia-derived cells of polyenylpyrroles with a methyl group at the conjugated polyene terminus.

To develop novel drugs for treating T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) which are highly malignant hematological tumors, a series of analogs having a polyenylpyrrole structure of natural compounds (rumbrin and auxarconjugatin B) were synthesized and investigated their structure-activity relationships (SAR) of in vitro anti-T-ALL and anti-AML activities. We obtained three findings: (1) introduction of a methyl group at the conjugated polyene terminus enhanced anti-T-ALL activity, (2) analogs with a 3-chloropyrrole moiety had even higher selectivity for T-ALL cells, and (3) some analogs were effective against AML-derived cells. Among the studied compounds, 3-chloro-2-(8-ethoxycarbonylnona-1,3,5,7-tetraenyl) pyrrole 4e was the most promising candidate of T-ALL- and AML-treating drug. This study provides useful structural information for designing novel drugs treating T-ALL and AML.

How to Select Firefly Luciferin Analogues for In Vivo Imaging.

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.

Synthesis of polyenylpyrrole derivatives with selective growth inhibitory activity against T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hardly curable disease with a high relapse rate. 20 analogs were synthesized based on the structures of two kinds of fungi-derived polyenylpyrrole products (rumbrin (1) and auxarconjugatin-B (2)) to suppress the growth of T-ALL-derived cell line CCRF-CEM and tested for growth-inhibiting activity. The octatetraenylpyrrole analog gave an IC50 of 0.27 µM in CCRF-CEM cells, while it did not affect Burkitt lymphoma-derived cell line Raji and the cervical cancer cell line HeLa, or the oral cancer cell line HSC-3 (IC50 > 10 µM). This compound will be a promising compound for developing T-ALL-specific drugs.

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases.

Interestingly, only the D-form of firefly luciferin produces light by luciferin-luciferase (L-L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L-L reaction, and the wavelengths of light in the near-infrared (NIR) range (700-900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L-L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure-activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λmax = 705 nm) and mutant luciferase AlaLuc (λmax = 655 nm).

[Hit-to-Lead in Academia: Discovery of a Protein-Protein Interaction Inhibitor of Keap1-Nrf2].

In the process of recent hit-to-lead studies, not only in industry but also in academia, early evaluation of metabolic properties has been one of the key aspects supporting a higher probability of success in drug discovery. In this review, we introduce the development of chemical seeds targeting the Kelch-like ECH-associated protein-1 (Keap1) as an example of an academic hit-to-lead study considering metabolic stability. Keap1 regulates the function of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces various antioxidative or detoxification proteins. An inhibitor of protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 is expected to be a novel target for drug discovery. However, Nrf2 is also activated in several cancers, such as human hepatocellular carcinoma, and causes chemoresistance, which is mediated by phosphorylated p62/Sqstm1 (p-p62), an autophagy-related protein that also undergoes a PPI with Keap1. In this case, an Nrf2 suppressor could be used to attenuate drug resistance. We discovered inhibitors against the Nrf2-Keap1 PPI and p-p62-Keap1 PPI using high-throughput screening and established the synthetic routes for the hit compounds and their derivatives. Furthermore, we assessed the metabolic stability of both of the PPI inhibitors in human liver microsomes and identified the metabolic sites.

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor.

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.

Synthesis of Keap1-phosphorylated p62 and Keap1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity.

The Keap1-Nrf2 system is involved not only in biological defense but also in malignancy progression and chemoresistance. The ubiquitin-binding protein p62/Sqstm1 (p62), which is highly expressed in several cancers, competes with Nrf2 for Keap1 binding, leading to activation of Nrf2-mediated gene expression and survival of cancer cells. We had previously identified an inhibitor for the Keap1-phosphorylated-p62 (p-p62) protein-protein interaction (PPI), the acetonyl naphthalene derivative K67. In this study, we established facile synthetic routes for K67 and derivatives with various side chains on the C-2 position of naphthalene ring. K67 possessed high selectivity in the inhibition of Keap1-p-p62. Other derivatives showed potent Keap1-Nrf2 and Keap1-p-p62 PPI inhibitory activities, though the selectivity between the two activities was lower than K67.

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming.

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Multicolor Bioluminescence Obtained Using Firefly Luciferin.

Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

Crystal structure of 5,5'-di-bromo-3,3'-di-tert-butyl-6,6'-di-methyl-biphenyl-2,2'-diol.

The whole mol-ecule of the title compound, C22H28Br2O2, is generated by twofold rotation symmetry. The dihedral angle of the biphenyl moiety is 85.05 (11)°. The hy-droxy groups show intra-molecular O-H⋯π inter-actions without any other hydrogen-bond acceptors. In the crystal, there are no other significant inter-molecular inter-actions present.

Cathodic reductive coupling of methyl cinnamate on boron-doped diamond electrodes and synthesis of new neolignan-type products.

The electroreduction reaction of methyl cinnamate on a boron-doped diamond (BDD) electrode was investigated. The hydrodimer, dimethyl 3,4-diphenylhexanedioate (racemate/meso = 74:26), was obtained in 85% yield as the major product, along with small amounts of cyclic methyl 5-oxo-2,3-diphenylcyclopentane-1-carboxylate. Two new neolignan-type products were synthesized from the hydrodimer.

A new NF-κB inhibitor based on the amino-epoxyquinol core of DHMEQ.

The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-κB inhibitor DHMEQ (1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-κB, based on NO secretion and MALDI-TOF MS analysis. It was indicated that 6b inhibited the activation by different manner from that of 1.

Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cys188Ser mutant in the liganded form.

Arylmalonate decarboxylase catalyzes the enantioselective decarboxylation of alpha-aryl-alpha-methylmalonate to produce optically pure alpha-arylpropionate. The enzyme is comprised of two alpha/beta domains and contains an active site situated between the two domains. The site is formed by Tyr48, Gly74-Thr75-Ser76, Tyr126, and Cys188-Gly189-Gly190 residues. Since it has been observed that the Gly74Cys/Cys188Ser mutation inverts the enantioselectivity of the enzyme, we determined the crystal structure of the Gly74Cys/Cys188Ser mutant in the liganded form at a resolution of 1.45 A to understand the structural basis for this inversion. The overall structure of the enzyme overlapped well with that of the benzylphosphonate-associated wild-type enzyme, and the mutations had little effect on the structure of the active site. A ligand molecule bound to the active site in an unusual semiplanar conformation resembling the planar enediolate reaction intermediate could be assigned as phenyl acetate. The inversion in enantioselectivity by the paired mutation is explained by the mirror symmetry between Cys74 in the mutant and Cys188 of the wild type with respect to the carbon atom in the ligand to be protonated. Comparison of the wild-type and Gly74Cys mutant crystal structures suggested that ligand binding induces a positional shift of the Cys188-Gly189-Gly190 region toward the Gly74-Thr75 pair which provides two oxyanion holes necessary to stabilize the negatively charged enediolate reaction intermediate. The ligand binding also simultaneously induces the formation of a hydrophobic cluster over the active site cleft. Thus, AMDase is proposed to have "open" and "closed" conformations of the active site that are regulated by ligand binding. These results may provide an effective strategy for the rational design to invert the enantioselectivity of enzymes.

Efficient synthesis of (+/-)-parasitenone, a novel inhibitor of NF-kappaB.

Dehydroxymethylepoxyquinomicin (DHMEQ, 1) is a novel nuclear factor-kappaB (NF-kappaB) inhibitor that inhibits DNA binding of NF-kappaB components including p65. To inspect its biological activity of 1, we synthesized parasitenone (3), possessing the common epoxycyclohexenone moiety of 1. Assessment of the inhibitory activity against NF-kappaB indicated that the epoxycyclohexenone moiety is the most essential element for the NF-kappaB inhibitory activity and the salicylic acid moiety may contribute the binding efficiency and specificity.

Synthesis and biological evaluation on novel analogs of 9-methylstreptimidone, an inhibitor of NF-kappaB.

Synthesis of 9-methylstreptimidone analogs and their inhibitory activities against NF-kappaB (nuclear factor-kappaB) are reported. Among several active derivatives synthesized in this study, 8 with a relatively simple structure, exhibited inhibitory activity against LPS-induced NO production comparable to that of 9-methylstreptimidone.

Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase from Alcaligenes bronchisepticus.

Arylmalonate decarboxylase catalyses the enantioselective decarboxylation of alpha-aryl-alpha-methylmalonates to produce optically pure alpha-arylpropionates. The enzyme was crystallized with ammonium sulfate under alkaline pH conditions with the aim of understanding the mechanism of the enantioselective reaction. X-ray diffraction data collected to a resolution of 3.0 A at cryogenic temperature showed that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 83.13, b = 99.62, c = 139.64 A. This suggested that the asymmetric unit would contain between four and six molecules. Small-angle X-ray scattering revealed that the enzyme exists as a monomer in solution. Thus, the assembly of molecules in the asymmetric unit was likely to have been induced during the crystallization process.

Efficient route to (S)-azetidine-2-carboxylic acid.

A new and efficient route to (S)-azetidine-2-carboxylic acid (>99.9% ee) in five steps and total yield of 48% via malonic ester intermediates was established. As the key step, efficient four-membered ring formation (99%) was achieved from dimethyl (S)-(1'-methyl)benzylaminomalonate by treating with 1,2-dibromoethane (1.5 eq) and cesium carbonate (2 eq) in DMF. Krapcho dealkoxycarbonylation of dimethyl (1'S)-1-(1'-methyl)benzylazetidine-2,2-dicarboxylate, the product of this cyclization procedure, proceeded with preferential formation (2.7:1, 78% total yield) of the desired (2S,1'S)-monoester, with the help of a chiral auxiliary which was introduced on the nitrogen atom. The undesired (2R,1'S)-isomer could be converted to that with proper stereochemistry, by a deprotonation and subsequent re-protonation step. Finally, lipase-catalyzed preferential hydrolysis of the (2S,1'S)-monoester and subsequent deprotection provided enantiomerically pure (S)-azetidine-2-carboxylic acid in a 91% yield from the mixture of (2S,1'S)- and (2R,1'S)-isomers.

Discovery of halogenated eurypamide B analogues as inhibitors of lipid droplet accumulation in macrophages.

Halogenated cyclic isodityrosine-tripeptides were synthesized as analogues of a marine natural product, eurypamide B. Although the original eurypamides showed no inhibitory activity, the new analogues were found to inhibit lipid droplet accumulation in macrophages with a low micromolar IC50 value.

Purification of Shiga-like toxin 1 by pigeon egg white glycoproteins immobilized on Sepharose gels.

The galabiose structure Galalpha1-4Gal is rarely found in natural glycoproteins, but is abundantly present in pigeon egg white proteins as Galalpha(1-4)Galbeta(1-4)GlcNAc termini. Pigeon ovalbumin, ovomucoid, or the whole egg white were immobilized on periodate-oxidized Sepharose CL-6B gels by reductive amination. These gels were found to bind Shiga-like toxin type 1 (SLT-1) specifically and efficiently. SLT-1 was eluted from the gel beads with 0.5 M melibiose, which was more efficient and milder than elution with 4.5 M MgCl(2). SLT-1 was purified to homogeneity from the crude extract of Escherichia coli SLT100 expressing SLT-1 by a single affinity chromatographic step in 83-88% yield. The capacity of the gel was estimated to be ca. 1mg toxin/ml gel. Interestingly, SLT-2 was not bound by these affinity gels containing Galalpha1-4Galbeta1-4GlcNAc termini. Since SLT-2 has been shown to bind to Galalpha1-4Galbeta1-4Glc-terminating compounds, our results suggest that Glc in globotriose moiety is important for binding SLT-2, and replacing the Glc with GlcNAc in this triose renders it ineffective for binding SLT-2.