Advanced second generation inhibitors of histone deacetylases (HDAC) are currently used in clinical development. This study aimed at comparing the pharmacological properties of selected second generation HDAC inhibitors with the hydroxamate and benzamide head group, namely SAHA, LAQ824/LBH589, CI994, MS275 and MGCD0103. In biochemical assays using recombinant HDAC1, 3, 6 and 8 isoenzymes, SAHA and LAQ824/LBH589 behave as quite unselective HDAC inhibitors. In contrast, the benzamides CI994, MS275 and MGCD0103 are more selective, potent inhibitors of at least HDAC1 and HDAC3. All HDAC inhibitors induce histone H3 hyperacetylation, correlating with inhibition of proliferation, induction of cell differentiation and apoptosis. A broad cytotoxicity is seen across cell lines from different tumor entities with LAQ824/LBH589 being the most potent agents. The apoptosis inducing activity is evident in arrested and proliferating RKO colon cancer cells with inducible, heterologous p21 waf1 expression, indicative for a cell-cycle independent mode-of-action. Differentiation of MDA-MB468 breast cancer cells is induced by benzamide and hydroxamate analogs. The reversibility of drug action was evaluated by pulse treatment of A549 lung cancer cells. Whereas paclitaxel induced irreversible cell cycle alterations already after 6 hr treatment, HDAC inhibitor action was retarded and irreversible after >16 hr treatment. Interestingly, pulse treatment was equally effective as continous treatment. Finally, the efficacy of LAQ824, SAHA and MS275 in A549 nude mice xenografts was comparable to that of paclitaxel at well tolerated doses. We conclude that despite a different HDAC isoenzyme inhibition profile, hydroxamate and benzamide analogs as studied display similar cellular profiles. ' 2007 Wiley-Liss, Inc.Key words: HDAC inhibition; hydroxamate and benzamide head group; isoenzyme selectivity; protein hyperacetylation Posttranslational modification by reversible acetylation of lysine residues in histone proteins and their putative role in RNA synthesis was first described in 1964 by Allfrey et al.1 Since this landmark article, the natural antifungal antibiotic Trichostatin A (TSA) was found to act by inhibition of mammalian histone deacetylases (HDAC).2 Subsequently, the first human HDAC named HD1 (syn. HDAC1), a homolog of yeast transcriptional regulator Rpd3, was isolated.3 Since then, enormous progress was made in understanding reversible protein acetylation in general and histone modifications in particular.4,5 Chromatin condensation and transcriptional activity is regulated by acetylation of N-terminal lysine residues in core histone proteins H3 and H4 by histone acetyltransferases (HATs) and deacetylation by HDACs. HDACs are components of transcriptional silencing complexes as first described for the mRpd3/N-CoR /mSin3 complex.6 Up to now, 11 different HDAC isoenzymes belonging to the class I (HDAC 1, 2, 3, 8), class II (HDAC 4-7, 9, 10) and class IV families (HDAC11) have been described. 7 HDAC class III enzymes, also named Sirtuin...

To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced koff, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30+ target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin’s lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells.

The protein-tyrosine phosphatase SHP-1 is a negative regulator of multiple signal transduction pathways. We observed that SHP-1 effectively antagonized Srcdependent phosphorylations in HEK293 cells. This occurred by dephosphorylation of Src substrates, because Src activity was unaffected in the presence of SHP-1. One reason for efficient dephosphorylation was activation of SHP-1 by Src. Recombinant SHP-1 had elevated activity subsequent to phosphorylation by Src in vitro, and SHP-1 variants with mutated phosphorylation sites in the C terminus, SHP-1 Y538F, and SHP-1 Y538F,Y566F were less active toward Src-generated phosphoproteins in intact cells. A second reason for efficient dephosphorylation is the substrate selectivity of SHP-1. Pull-down experiments with different GST-SHP-1 fusion proteins revealed efficient interaction of Src-generated phosphoproteins with the SHP-1 catalytic domain rather than with the SH2 domains. Phosphopeptides that correspond to good Src substrates were efficiently dephosphorylated by SHP-1 in vitro. Phosphorylated "optimal Src substrate" AEEEIpYGEFEA (where pY is phosphotyrosine) and a phosphopeptide corresponding to a recently identified Src phosphorylation site in p120 catenin, DDLDpY 296 GMMSD, were excellent SHP-1 substrates. Docking of these phosphopeptides into the catalytic domain of SHP-1 by molecular modeling was consistent with the biochemical data and explains the efficient interaction. Acidic residues N-terminal of the phosphotyrosine seem to be of major importance for efficient substrate interaction. Residues C-terminal of the phosphotyrosine probably contribute to the substrate selectivity of SHP-1. We propose that activation of SHP-1 by Src and complementary substrate specificities of SHP-1 and Src may lead to very transient Src signals in the presence of SHP-1. The SH21 domain PTP SHP-1 regulates multiple signal transduction events by dephosphorylation (1-3). These comprise signaling of cytokine receptors such as the erythropoietin receptor (4), and the interleukin-3 receptor (5), and of receptor tyrosine kinases such as c-Kit (6 -8), the colony-stimulating factor-1 receptor (9, 10), and the epithelial kinase Ros (11). SHP-1 modulates also the function of immunoreceptors (12), and cytoplasmic tyrosine kinases such as Lck (13,14). In these and many other cases, SHP-1 regulates signaling in a negative manner. In other pathways, SHP-1 may also exert a positive function. Thus, a role of SHP-1 for differentiation of glia cells (15), and for Ras-dependent activation of mitogen-activated protein kinase (16) have been reported. Also, SHP-1 has the capacity to activate Src kinase by dephosphorylation of the inhibitory phosphotyrosine in the Src C terminus and may thus stimulate Src-dependent phosphorylations in certain cell types (17). SHP-1 can translocate into the nucleus, however, its nuclear substrates are still elusive (18 -21). Among the recently identified substrates of SHP-1 is p120 ctn (22), a cytoplasmic protein that possesses armadillo-like repeats, participates in ...

Nuclear DNA helicase II (NDH II), also designated RNA helicase A, is a multifunctional protein involved in transcription, RNA processing, and transport. Here we report that NDH II binds to F-actin. NDH II was partially purified from HeLa nuclear extracts by ion-exchange chromatography on Bio-Rex 70 and DEAE-Sepharose. Upon gel-filtration chromatography on Sepharose 4B, partially purified NDH II resolved into two distinct peaks. The first NDH II peak, corresponding to the void volume of Sepharose 4B, displayed coelution with an abundant 42-kDa protein that was subsequently identified as actin. Several nuclear proteins such as RNA polymerase II, the U5 small nuclear ribonucleoprotein (RNP)-associated WD40 protein, and heterogeneous nuclear RNPs (hnRNPs) copurified with NDH II. However, only hnRNPs A1 and C were found together with NDH II and actin polymers during gel filtration. NDH II and hnRNP C from the HeLa nuclear extract coeluted with F-actin on Sepharose 4B in an RNase-resistant manner, whereas hnRNP A1 was nearly completely removed from F-actin-associated hnRNP complexes following RNA digestion. The association of NDH II and hnRNP C with F-actin was abolished by gelsolin, an F-actin-depolymerizing protein that fragments actin polymers into oligomers or monomers. Furthermore, NDH II co-immunoprecipitated with F-actin and hnRNP C, respectively. In vitro translated NDH II coeluted with F-actin on Sepharose 4B, whereas no coelution with F-actin was observed for in vitro translated hnRNP A1 or C1. Binding to F-actin requires an intact C terminus of NDH II and most likely a native protein conformation. Electron microscopy indicated a close spatial proximity among NDH II, hnRNP C, and F-actin within the HeLa nucleus. These results suggest an important function of NDH II in mediating the attachment of hnRNP-mRPP RNP complexes to the actin nucleoskeleton for RNA processing, transport, or other actin-related processes.Nuclear DNA helicase II (NDH II) 1 is a nucleic-acid helicase that unwinds double-stranded DNA and RNA in a nucleotidedependent manner (1-3). NDH II is highly conserved among man (4), cow (5), mouse (6), worm (7), and fruit fly (8). NDH II comprises two double-stranded RNA (dsRNA)-binding domains at the N terminus, a helicase catalytic domain in the central part, and a glycine-rich single-stranded nucleic acid-binding domain (RGG box) at the C terminus (9, 10). Sequence analysis revealed that NDH II contains seven helicase core motifs that are conserved among the DEX(D/H) helicase superfamily (4, 5). NDH II displays significant similarities to a group of yeast pre-mRNA splicing factors, including prp2, prp16, and prp22 (4). In general, nucleic-acid helicases may adopt an enzymatic mechanism that transforms energy from nucleotide hydrolysis into the mechanical work for protein translocation and/or disruption of nucleic acid duplices (11). The nucleotide binding and hydrolysis by a helicase are governed by two Walker nucleotide-binding motifs (A and B), which have been originally defined from several ATP...

Inhibitors of histone deacetylases (HDAC) are currently developed for the treatment of cancer. These include compounds with a sulfur containing head group like depsipeptide, alkylthiols, thiocarboxylates, and trithiocarbonates with a carbonyl group in the alpha-position. In the present investigation, we report on the synthesis and comprehensive SAR analysis of HDAC inhibitors bearing a tri- or dithiocarbonate motif. Such trithiocarbonates are readily accessible from either preformed or in situ prepared alpha-halogenated methylaryl ketones. A HDAC isotype selectivity and a substrate competitive mode-of-action is shown for defined analogues. Exploration of the head group showed the necessity of the dithio-alpha-carbonyl motif for potent HDAC inhibition. Highly potent, substrate competitive HDAC6 selective inhibitors were identified (12ac:IC 50 = 65 nM and K i = 110 nM). Trithiocarbonate analogues with an aminoquinoline-substituted pyridinyl-thienoacetyl cap demonstrate a cytotoxicity profile and potency comparable to that of suberoylanilide hydroxamic acid (SAHA) as an approved cancer drug.