Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies.With the increasing threat of pathogens, such as Bacillus anthracis, being used as bioweapons, 1 and the rise in the incidence of multi-drug resistant bacteria, 2 the need for new antibiotics that act at novel targets has never been greater. Previous studies within this group 3-5 have revealed that inhibition of one such target, the amidotransferase enzyme nicotinamide adenine dinucleotide (NAD) synthetase (NADs), could hinder both spore outgrowth and vegetative growth, which would provide antibacterial action at two different steps in the bacterial life cycle. 6-10The first class of NADs inhibitors designed by this group consisted of tethered dimers that contain two hydrophobic groups linked by a polymethylene tether, and a positively charged nitrogen on one end. 3-5 These inhibitors were antibacterial, and there was a correlation between the potencies of enzyme inhibition and antibacterial effects. However, the permanent positive charge and detergent-like properties of this class of compounds were unattractive for further drug development. 11,12 More drug-like lead inhibitors were, therefore, sought.Virtual screening of compound databases using the detailed structure of the drug target can serve to greatly enhance success in the lead discovery process. 13-17 Here we use the in silico screening program FlexX 1.20.1(BiosolveIT GmbH ® ) for the virtual screening of commercially available compounds within the catalytic site of NADs to identify new classes *Corresponding author. Tel. +1 205 934 8288; FAX +1 205 934 2543. Email address: wbrou@uab.edu (W. Brouillette). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Supplementary Data Supplementary data, including (a) structures of compounds from Table 1 not shown in Table 2, (b) graphical representations of the binding sites used and poses of selected docked ligands, and (c) a sample HPLC-chromatogram used in the enzyme assay, can be found in the online version. NADs is a large homodimer of approximately 60 kDa that contains two identical binding sites. The crystal structure of the protein from B. subtilis reveals two identical long, linear bind...

Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-␣4-GlcNAc-␤1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino-and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-␤3-GlcNAc-␤1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.The Gram-negative bacterial pathogen Pasteurella multocida Type D is known to cause atrophic rhinitis in swine and pasteurellosis in other domestic animals (1). This microbe produces an extracellular coating of polysaccharide, called a capsule, composed of heparosan (N-acetyl-heparosan or unsulfated, unepimerized heparin) (2) to enhance infection. It is thought that the capsule confers resistance to nonspecific host immunity and perhaps mediates adhesion to certain host cells. In general, most capsules are antigenic, but glycosaminoglycan polysaccharide capsules are a special case because they closely resemble the host molecules and thus may serve as molecular camouflage (3).Two heparosan synthase (PmHS) 2 enzymes have been identified from P. multocida that catalyze the formation of the repeating disaccharide (-4GlcNAc␣1-4GlcUA␤1-) units of the heparosan chain. P. multocida Type D strains encode the 617-residue PmHS1 enzyme in a typical Gram-negative bacterial Type 2 capsule biosynthesis locus (4). Another isozyme, the 651-residue PmHS2, is found in many Type A, D, and F strains (5); the genes are ϳ73% identical. The gene encoding PmHS2 is found in a different region of the chromosome not associated with typical carbohydrate biosynthesis genes and may be expressed during some stage of infection, but its role is not yet known.The dual action PmHS1 and PmHS2 are complex enzymes because the U...

A new lead class of antibacterial drug-like NAD synthetase (NADs) inhibitors was previously identified from a virtual screening study. Here a solution-phase synthetic library of 76 compounds, analogs of the urea-sulfonamide 5838, was synthesized in parallel to explore SAR on the sulfonamide aryl group. All library members were tested for enzyme inhibition against NADs and nicotinic acid mononucleotide adenylyltransferase (NaMNAT), the last two enzymes in the biosynthesis of NAD, and for growth inhibition in a B. anthracis antibacterial assay. Most compounds that inhibited bacterial growth also showed inhibition against one of the enzymes tested. While only modest enhancements in the enzyme inhibition potency against NADs were observed, of significance was the observation that the antibacterial urea-sulfonamides more consistently inhibited NaMNAT.

Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membraneassociated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.