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Histone deacetylase inhibitors (HDACis) are one of the last frontiers in pharmaceutical research. Several classes of HDACi have been identified. Although more than 20 HDACi are under preclinical and clinical investigation as single agents and in combination therapies against different cancers, just two of them were approved by the US FDA: Zolinza(®) and Istodax(®), both licensed for the treatment of cutaneous T-cell lymphoma, the latter also of peripheral T-cell lymphoma. Since HDAC enzymes act by forming multiprotein complexes (clusters), containing cofactors, the main problem in designing new HDACi is that the inhibition activity evaluated on isolated enzyme isoforms does not match the in vivo outcomes. In the coming years, the research will be oriented toward a better understanding of the functioning of these protein complexes as well as the development of new screening assays, with the final goal to obtain new drug candidates for the treatment of cancer.

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Regioselective -arylation of acyclic enol ethers by aryl trifluoromethanesulfonates, aryl bromide, aryl iodides, and aroyl chlorides is described. The outcome of the reaction proved to be dependent from the relationship between ligand and counterion in the oxidative addition complex.

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ,90% conversion of cephalosporin C to 7-aminocephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.Keywords: cephalosporin C; 7-amino cephalosporanic acid; protein engineering; directed evolution; site-saturation mutagenesis; enzymes; active sites; structure/function studies; protein sequencing; modification; mass spectrometry; protein structure prediction; kinetics Semisynthetic cephalosporins are the most widely used antibiotics and are primarily synthesized from 7-amino cephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of the natural antibiotic cephalosporin C (CephC). The chemical route includes, however, several expensive steps and requires treatment of toxic wastes. A two-step enzymatic route can also be used that, in two separate reactors, uses D-amino acid oxidase and glutaryl-7-amino cephalosporanic acid (gl-7ACA) acylase activity (see Scheme 1, below): Although this route solves the environmental safety problems, it is also expensive and not entirely satisfactory for industrial production. Therefore, enzymatic conversion of CephC to 7-ACA is of great interest to cephalosporin antibiotics manufacturers (an annual worldwide market of ,400 million US dollars has been estimated) (Pilone and Pollegioni 2002). The greatest hindrance to enzymatic industrial production is that acylase takes glutaryl-7-ACA (gl-7ACA) as a primary substrate, and its specificity (and activity) is too low for CephC. Glutaryl-7-ACA acylases are members of the N-terminal hydrolases (Ntn) class of hydrolytic enzymes. The gene structure of the open reading frame (ORF) of the members of this class consists of a signal peptide, followed by an a-subunit, a spacer sequence (which is not present in the acylase under investigation), and a b-subunit...