ABSTRACT
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 Bacillus 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.
Subject(s)
Amide Synthases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Bacillus anthracis/drug effects , Bacillus anthracis/enzymology , Sulfonamides/chemistry , Urea/chemistry , Amide Synthases/metabolism , Anthrax/drug therapy , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Humans , Nicotinamide-Nucleotide Adenylyltransferase/antagonists & inhibitors , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Urea/chemical synthesis , Urea/pharmacologyABSTRACT
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 microM (7.6% hit rate), and two were selected for future SAR studies.
Subject(s)
Amide Synthases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Amide Synthases/metabolism , Anti-Bacterial Agents/pharmacology , Computer Simulation , Databases, Factual , Enzyme Inhibitors/pharmacology , SoftwareABSTRACT
Hyaluronan (HA), a linear polysaccharide composed of beta1,3-GlcNAc-beta1,4-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 membrane-associated 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.
Subject(s)
Glucuronosyltransferase/chemistry , Glucuronosyltransferase/metabolism , Pasteurella multocida/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Chromatography, Gel , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Glucosephosphate Dehydrogenase/metabolism , Glucuronosyltransferase/isolation & purification , Glucuronosyltransferase/radiation effects , Hyaluronan Synthases , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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 (-alpha4-GlcNAc-beta1,4-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 (-beta3-GlcNAc-beta1,4-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.
