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.     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Pathogenesis of experimental allergic orchitis. III. T lymphocyte requirement in local adoptive transfer by peritoneal exudate cells.

In experimental allergic orchitis (EAO), a lesion characterized by mononuclear invasion of seminiferous tubules can be adoptively transferred within 1 to 4 days by testicular injection of peritoneal exudate cells (PEC) from syngeneic strain 13 guinea pigs (GP) immunized with homologous testicular antigens in complete Freund's adjuvant (CFA). This study examined the role of T lymphocytes, macrophages, and polymorphonuclear neutrophils (PMN) in the adoptive transfer. Guinea pig PEC contained 7% T lymphocytes, rare B lymphocytes, and over 90% of macrophages and PMN. After T lymphocytes were depleted by rabbit erythrocyte (E) rosette and Hypaque-Ficoll gradient centrifugation, cell preparations that contained 73% of original macrophages and 15% original T lymphocytes were obtained, and these cells did not transfer EAO (0 of 18 testes). In contrast, cell preparations enriched in T lymphocytes by nylon wool column or E rosette contained 1.5% of the original macrophages and 59% of the original T lymphocytes transferred EAO to 70% of the testes, starting at 1.5 x 10(6) T lymphocytes per testis. The number of T lymphocytes correlated with the incidence of adoptive transfer; the correlation existed regardless of the number of macrophages or PMN present. Finally, EAO was adoptively transferred to recipients that had total-body irradiation. The results indicate that (a) T lymphocytes are capable of transferring lesions of EAO, (b) in the transfer, the T lymphocytes did not function as helper T cells, since the transfer need not involve participation of host lymphoid cells, and (c) by inference, testis antigen-reactive T lymphocytes exist.     

Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.     

Identification of a prominent 85-kDa cAMP-dependent phosphoprotein associated with late G1 phase in mitogen-stimulated B lymphocytes

In order to elucidate late regulatory events which may be involved in the onset of S phase in B lymphocytes, we studied the effect of anti-Ig on phosphorylation of soluble proteins at late G1 phase. Stimulation of murine splenic B lymphocytes with anti-Ig and other mitogens for 18 h was found to be associated with a major increase in phosphorylation of an 85 kDa/pI approximately 5.3 cytosolic protein, conversely, stimulation of the cells with non-mitogenic stimuli did not induce the phosphorylation of pp85. The increase in phosphorylation of pp85 could not be detected after 30 min, was barely detectable after 6 h, but was very prominent after 18 h of stimulation with anti-Ig. Thus, the increase in phosphorylation of pp85 is not an early signal but is rather correlated with the late G1 phase. pp85 could not be detected in the nuclei of either control or stimulated cells. Stimulation of B cells for 30 min with forskolin induced the phosphorylation of pp85, while phorbol ester did not have any effect. The phosphorylation of pp85 was induced by the catalytic subunit of cAMP protein kinase. Comparison of the phosphopeptide map of pp85 phosphorylated by anti-Ig in intact cells to the phosphopeptide map phosphorylated by forskolin or by the catalytic subunit of cAMP protein kinase, showed a striking similarity indicating that cAMP protein kinase may be involved in phosphorylation of pp85 in mitogen-stimulated cells. An increase in intracellular cAMP levels at late G1 phase was found in B cells stimulated by mitogens. These results implicate an important role for cAMP-dependent phosphorylation events, specifically the phosphorylation of pp85/pI 5.3, at late G1 phase during the cell cycle.     

Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.