Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Mechanism of Action of Lycoricidinol, a Plant Growth Inhibitor Isolated from Lycoris radiata Herb.

We studied the action mechanism of lycoricidinol, a plant growthinhibitor isolated from Lycoris radiata Herb. Lycoricidinolinhibited protein synthesis in mung bean hypocotyls, but notRNA synthesis. Protein synthesis in Escherichia coli was notaffected by the inhibitor. Results of in vitro translation experimentswith the wheat germ system and the E. coli system indicatedthat lycoricidinol inhibited only eukaryotic but not prokaryotictranslation. Use of specific inhibitors of initiation and polypeptidechain elongation of polypeptide synthesis revealed that chainelongation was inhibited by lycoricidinol. ¹Permanent address: Department of Biology, Yonsei University,Seoul 120, Korea. (Received September 30, 1983; Accepted December 28, 1983)     

Genetic structure of the Queckchi Indians. Dental microdifferentiation.

Nine oral morphologic characters were investigated. Their frequencies are compared with those published for other populations. The possibility of using such characters to estimate genetic distance between populations is discussed and the conclusion is reached that, although previous studies have suggested this to be a valid approach, further studies testing this subject are needed.     

Limits of artificial selection under unbalanced mating systems

Summary The influence of unbalanced mating systems — factorial mating (FM) and random loss of families after a full diallel crossing (RS) — on the ultimate probability of gene fixation (u()) and the time required to fix or lose a gene (t()) are investigated. The average u() of these systems is smaller than that of random mating, and the range of u() for a given initial parental genotype combination is very large (close to one for most initial genotypic combinations). The average u() of different parental genotypic combinations of a given gene frequency are different. These systems accelerate the t().This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

氦氖激光对绵羊精清中酶活性效应的研究

本文研究结果表明低剂量的氦氖激光可以提高绵羊精清中GOT和LDH酶的活性,并对其机制作了初步的探讨。     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Electron paramagnetic and electron nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase

We have collected electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectra from the hydrogen peroxide compound of yeast cytochrome c peroxidase, termed ES, employing EPR microwave frequencies of 9.6 and 11.6 GHz. We have measured and analyzed the temperature dependence of the spin-lattice relaxation rate (1/T1) of the paramagnetic center of ES over the temperature range 1.9 to 4 K. In addition, an upper bound to exchange coupling between the ferryl heme and EPR-visible centers of ES has been calculated and expressions for the dipolar interaction between a ferryl heme and a free radical have been derived. These results all confirm that the EPR signal of ES is not associated with an aromatic amino acid radical, and in particular not with a tryptophanyl radical. This conclusion has led us to consider an explanation of the EPR signal in terms of a nucleophilically stabilized methionyl radical.