Vinyl Sulfones: Inhibitors of SrtA, a Transpeptidase Required for Cell Wall Protein Anchoring and Virulence in <i>Staphylococcus aureus</i>

Several small molecule vinyl sulfones were found to exhibit irreversible time-dependent inhibition of the Staphylococcus aureus sortase SrtA in vitro. A representative of these compounds was shown to impair the ability of S. aureus bacteria to bind fibronectin-coated surfaces through in vivo inhibition of SrtA-mediated linkage of fibronectin to the cell surface. These data highlight the potential use of small molecule vinyl sulfones as chemotherapeutics to prevent adhesion to and colonization of host tissues during S. aureus infection

A Systematic Investigation of the Synthetic Utility of Glycopeptide Glycosyltransferases

Glycosyltransferases involved in the biosynthesis of bacterial secondary metabolites may be useful for the generation of sugar-modified analogues of bioactive natural products. Some glycosyltransferases have relaxed substrate specificity, and it has been assumed that promiscuity is a feature of the class. As part of a program to explore the synthetic utility of these enzymes, we have analyzed the substrate selectivity of glycosyltransferases that attach similar 2-deoxy-l-sugars to glycopeptide aglycons of the vancomycin-type, using purified enzymes and chemically synthesized TDP β-2-deoxy-l-sugar analogues. We show that while some of these glycopeptide glycosyltransferases are promiscuous, others tolerate only minor modifications in the substrates they will handle. For example, the glycosyltransferases GtfC and GtfD, which transfer 4-epi-l-vancosamine and l-vancosamine to C-2 of the glucose unit of vancomycin pseudoaglycon and chloroorienticin B, respectively, show moderately relaxed donor substrate specificities for the glycosylation of their natural aglycons. In contrast, GtfA, a transferase attaching 4-epi-l-vancosamine to a benzylic position, only utilizes donors that are closely related to its natural TDP sugar substrate. Our data also show that the spectrum of donors utilized by a given enzyme can depend on whether the natural acceptor or an analogue is used, and that GtfD is the most versatile enzyme for the synthesis of vancomycin analogues

Alanine Scanning Mutagenesis of the Testosterone Binding Site of Rat 3α-Hydroxysteroid Dehydrogenase Demonstrates Contact Residues Influence the Rate-Determining Step^†

Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3α-hydroxysteroid dehydrogenase (3α-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3α-HSD·NADP+·testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2−3-fold. Fluorescence titrations of the energy transfer band of the E·NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the Kd for testosterone were negligible. Examination of the kcat/Km data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on kcat (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a klim and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave klim and kmax values identical with kcat values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect kcat more than Kd by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, kcat will be limited by the chemical event

Kinetic Analysis of Teicoplanin Glycosyltransferases and Acyltransferase Reveal Ordered Tailoring of Aglycone Scaffold to Reconstitute Mature Teicoplanin

In the maturation of the glycopeptide antibiotic teicoplanin, two glycosyltransferases (tGtfA and tGtfB) and one acyltransferase (tAtf) act on the nascent scaffold to produce the final natural product. In this report, we present detailed kinetic characterization of each of these enzymes, demonstrating the ordered nature of these tailoring transformations. tGtfB acts first on the crosslinked peptide aglycone, adding either an N-acetylglucosamine or 2-aminoglucose moiety to the 4-position of the scaffold. The second glycosyltransferase, tGtfA, preferentially acts on this glycosylated scaffold to condense it with another molecule of N-acetylglucosamine. Although the acyltransferase tAtf is capable of N-acylating the free UDP sugar, UDP-2-acetylglucosamine, its catalytic efficiency is 100000-fold higher when acting on the 4-(2-aminoglucosyl)-teicoplanin scaffold, showing that it prefers to act on the aglycone-tethered sugar. This work expands on our previous studies of ordered tailoring of the vancomycin and chloroeremomycin scaffolds and demonstrates a distinct preference in ordering of the teicoplanin tailoring enzymes

Supplementary Table from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Responses

Supplementary Table from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Response

Supplementary Table 3 from A687V EZH2 Is a Driver of Histone H3 Lysine 27 (H3K27) Hypertrimethylation

Supplementary Table 3: Functional analysis of genes significantly up- and down-regulated in SUP-B8 and NALM-6 cells.</p

Supplementary Table from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Responses

Supplementary Table from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Response

Supplementary Figure 2 from GSK1838705A inhibits the insulin-like growth factor-1 receptor and anaplastic lymphoma kinase and shows antitumor activity in experimental models of human cancers

Supplementary Figure 2. Representative cell cycle histograms from MCF-7 and NCIH929 cells treated with 1 μM of GSK1838705A for 48 h.</p

Supplementary Tables and Figure Legends from GSK1838705A inhibits the insulin-like growth factor-1 receptor and anaplastic lymphoma kinase and shows antitumor activity in experimental models of human cancers

Supplementary Table 1. Kinase selectivity data for GSK1838705A</p

Supplementary Figure 5 from A687V EZH2 Is a Driver of Histone H3 Lysine 27 (H3K27) Hypertrimethylation

Supplementary Figure 5: H3K27me3 enrichment across genes modulated by EZH2 inhibition</p