Rapid Inhibitor Discovery by Exploiting Synthetic Lethality.

Synthetic lethality occurs when inactivation of two genes is lethal but inactivation of either single gene is not. This phenomenon provides an opportunity for efficient compound discovery. Using differential growth screens, one can identify biologically active compounds that selectively inhibit proteins within the synthetic lethal network of any inactivated gene. Here, based purely on synthetic lethalities, we identified two compounds as the only possible inhibitors of Staphylococcus aureus lipoteichoic acid (LTA) biosynthesis from a screen of ∼230,000 compounds. Both compounds proved to inhibit the glycosyltransferase UgtP, which assembles the LTA glycolipid anchor. UgtP is required for ß-lactam resistance in methicillin-resistant S. aureus (MRSA), and the inhibitors restored sensitivity to oxacillin in a highly resistant S. aureus strain. As no other compounds were pursued as possible LTA glycolipid assembly inhibitors, this work demonstrates the extraordinary efficiency of screens that exploit synthetic lethality to discover compounds that target specified pathways. The general approach should be applicable not only to other bacteria but also to eukaryotic cells.

Lipoteichoic acid polymer length is determined by competition between free starter units.

Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (Glc2DAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstituted Staphylococcus aureus LtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred Glc2DAG starter unit, yet the use of Glc2DAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.

The length of lipoteichoic acid polymers controls Staphylococcus aureus cell size and envelope integrity.

The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to both the absence of the encoded protein and to the loss of its products. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress.IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

Antibiotic That Inhibits the ATPase Activity of an ATP-Binding Cassette Transporter by Binding to a Remote Extracellular Site.

Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230 000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan. We purified, for the first time, a WTA transporter and have reconstituted ATPase activity in proteoliposomes. We showed that this new compound series inhibits TarH-catalyzed ATP hydrolysis even though the binding site maps to TarG near the opposite side of the membrane. These are the first ABC transporter inhibitors shown to block ATPase activity by binding to the transmembrane domain. The compounds have potential as therapeutic agents to treat S. aureus infections, and purification of the transmembrane transporter will enable further development.

Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations.

Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

Correction: DNA-catalyzed glycosylation using aryl glycoside donors.

Correction for 'DNA-catalyzed glycosylation using aryl glycoside donors' by Anthony R. Hesser et al., Chem. Commun., 2016, 52, 9259-9262.

DNA-catalyzed glycosylation using aryl glycoside donors.

We report the identification by in vitro selection of Zn(2+)/Mn(2+)-dependent deoxyribozymes that glycosylate the 3'-OH of a DNA oligonucleotide. Both ß and α anomers of aryl glycosides can be used as the glycosyl donors. Individual deoxyribozymes are each specific for a particular donor anomer.

DNA-catalyzed hydrolysis of esters and aromatic amides.

We previously reported that DNA catalysts (deoxyribozymes) can hydrolyze DNA phosphodiester linkages, but DNA-catalyzed amide bond hydrolysis has been elusive. Here we used in vitro selection to identify DNA catalysts that hydrolyze ester linkages as well as DNA catalysts that hydrolyze aromatic amides, for which the leaving group is an aniline moiety. The aromatic amide-hydrolyzing deoxyribozymes were examined using linear free energy relationship analysis. The hydrolysis reaction is unaffected by substituents on the aromatic ring (ρ ≈ 0), suggesting general acid-catalyzed elimination as the likely rate-determining step of the addition-elimination hydrolysis mechanism. These findings establish that DNA has the catalytic ability to achieve hydrolysis of esters and aromatic amides as carbonyl-based substrates, and they suggest a mechanism-based approach to achieve DNA-catalyzed aliphatic amide hydrolysis.