*D. Bulger, Oral Roberts University (ORU).  C. Neylon, Rutherford Appleton Laboratory (RAL), UK.  J. Gaikwad, Oral Roberts...
Overview <ul><li>Sortase Inhibition lessens virulence of Gram + Bacteria </li></ul><ul><li>Inhibition measured by colorime...
Introduction: Sortase A Function <ul><li>Cysteine transpeptidase </li></ul><ul><li>Eight stranded  β -barrel with several ...
Introduction: Sortase A Inhibition <ul><li>Inhibition of Sortase A – inability to display surface proteins: </li></ul><ul>...
Introduction:  Hex  Protein Docking on Sortase A <ul><li>Interactive protein docking and superposition program </li></ul><...
Introduction: Ugi 4 Component Reaction - U4CR <ul><li>Described by Ivor Ugi in 1959 </li></ul><ul><li>Equimolar ratios in ...
Introduction:  H 1  NMR Solubility Determination <ul><li>Determination of Solubility </li></ul><ul><li>Provides approximat...
Aim and Hypothesis <ul><li>AIM: to find synthetic Sortase A inhibitor using protein docking of Ugi Products predicted usin...
Materials and Methods: Assay Design <ul><li>Sortase A and GFP purified from BL21(DE3) transformed with plasmid DNA  </li><...
Results/Discussion:  Protein Purification <ul><li>Transformation of BL21(DE3) with plasmid DNA </li></ul><ul><li>His-trap ...
Results/Discussion:  NMR Solubility and U4CR <ul><li>NMR solubility determination </li></ul><ul><li>(~20% Accuracy) </li><...
Conclusion  <ul><li>Sortase inhibition is useful pharmacologically </li></ul><ul><li>Ugi synthesis produces large variety ...
Future Study <ul><li>Solubility model will be expanded to provide better results </li></ul><ul><li>Ugi Reactions will be o...
Acknowledgements <ul><li>Dr. Robert Stewart, ORU – NMR Lab Technique </li></ul><ul><li>Dr. Hal Reed, ORU – helping with re...
References <ul><li>Arya P, Joseph R, Chou D. Toward High-Throughput Synthesis of Complex Natural Product-Like Compounds in...
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Sortase A Inhibition By Ugi Products (Complex)

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Sortase A Inhibition By Ugi Products (Complex)

  1. 1. *D. Bulger, Oral Roberts University (ORU).  C. Neylon, Rutherford Appleton Laboratory (RAL), UK.  J. Gaikwad, Oral Roberts University. Oral Presentation Section J: Biochemistry D. Bulger, Biology and Chemistry Department, Oral Roberts University, Tulsa, OK 74171 507-475-1516
  2. 2. Overview <ul><li>Sortase Inhibition lessens virulence of Gram + Bacteria </li></ul><ul><li>Inhibition measured by colorimetric assay </li></ul><ul><li>Inhibitors predicted by Hex 5.1 Protein Docking and synthesized using Ugi Reaction </li></ul><ul><li>Solubility Model predicted Ugi Reaction Products that would precipitate quickly from reaction mixture </li></ul><ul><li>H 1 NMR approximated solubilities </li></ul><ul><li>Project implemented open notebook science </li></ul>
  3. 3. Introduction: Sortase A Function <ul><li>Cysteine transpeptidase </li></ul><ul><li>Eight stranded β -barrel with several helices and loops </li></ul><ul><li>Sec secretion pathway </li></ul><ul><li>Photos: (Maresso, 2008) </li></ul><ul><li>http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&term=15117963 </li></ul>
  4. 4. Introduction: Sortase A Inhibition <ul><li>Inhibition of Sortase A – inability to display surface proteins: </li></ul><ul><ul><li>Adhesins </li></ul></ul><ul><ul><li>Immune evasion proteins </li></ul></ul><ul><li>Decreased virulence in various Staphylococcus and Listeria infections (Paterson, 2004) </li></ul><ul><li>Diminished selectivity pressures </li></ul>
  5. 5. Introduction: Hex Protein Docking on Sortase A <ul><li>Interactive protein docking and superposition program </li></ul><ul><li>CombUgi Library 3 used as ligands </li></ul><ul><li>Enzyme docked in active site pocket only </li></ul>
  6. 6. Introduction: Ugi 4 Component Reaction - U4CR <ul><li>Described by Ivor Ugi in 1959 </li></ul><ul><li>Equimolar ratios in solvent (low MW alcohols) </li></ul><ul><li>Fast exothermic reaction (few sec to few min) </li></ul><ul><li>Can convert nearly any combination of Carboxylic acid, Aldehyde, Primary Amine, and Isocyanide </li></ul><ul><li>Merck – HIV protease inhibitor Crixivan ( Furka, 1995) </li></ul>
  7. 7. Introduction: H 1 NMR Solubility Determination <ul><li>Determination of Solubility </li></ul><ul><li>Provides approximation </li></ul><ul><ul><li>About 20% Accuracy </li></ul></ul><ul><li>Ugi reactions occur in 0.5-2.0 M solutions </li></ul><ul><li>Ugi products less soluble than reactants </li></ul>
  8. 8. Aim and Hypothesis <ul><li>AIM: to find synthetic Sortase A inhibitor using protein docking of Ugi Products predicted using solubility model </li></ul><ul><li>Hypothesis: some of the Ugi products that have high Hex protein docking results and precipitate out of solution will inhibit Sortase A as detected through colorimetry </li></ul><ul><li>Null Hypothesis: none of the Ugi products that successfully dock and precipitate with inhibit Sortase A as detected through colorimetry </li></ul>
  9. 9. Materials and Methods: Assay Design <ul><li>Sortase A and GFP purified from BL21(DE3) transformed with plasmid DNA </li></ul><ul><li>Reaction mixture of Sortase A, tris-HCl buffer, tetraglycine, and GFP </li></ul><ul><li>Fluorescence resonance energy transfer (FRET) used </li></ul><ul><li>Absorption can be detected with UV-Vis Spectrophotometer </li></ul><ul><ul><li>Purification tag removal </li></ul></ul><ul><ul><li>Ni resin binding </li></ul></ul><ul><ul><li>Absorbance of free protein at 490 nm </li></ul></ul><ul><li>Ugi synthesis: methanol, carboxylic acids, primary amines, aldehydes, and isocyanides in one dram vials </li></ul><ul><li>Solubility: Jeol 300 MHz H 1 NMR, JSpecView </li></ul>
  10. 10. Results/Discussion: Protein Purification <ul><li>Transformation of BL21(DE3) with plasmid DNA </li></ul><ul><li>His-trap column in Actoprime FPLC </li></ul><ul><li>SDS Gel electrophoresis to confirm </li></ul><ul><li>purity and molecular weight </li></ul><ul><li>Centrifuge and concentration column </li></ul><ul><li>Dialysis </li></ul><ul><li>UV-Vis Conformation of Identity of Protein </li></ul>Fraction 7 Fraction 6 Fraction 5 Fraction 2 Fraction 3 Fraction 4 Fraction 1
  11. 11. Results/Discussion: NMR Solubility and U4CR <ul><li>NMR solubility determination </li></ul><ul><li>(~20% Accuracy) </li></ul><ul><li>Ugi Synthesis </li></ul>Ugi Product 62E
  12. 12. Conclusion <ul><li>Sortase inhibition is useful pharmacologically </li></ul><ul><li>Ugi synthesis produces large variety of organic products </li></ul><ul><li>Number of Ugi products to test limited to those with high Sortase A Hex docking results </li></ul><ul><li>Solubility modeling using NMR predicts the ease of purification </li></ul><ul><li>Colorimetric assay is expected to accurately detect Sortase A inhibition, especially after optimization </li></ul>
  13. 13. Future Study <ul><li>Solubility model will be expanded to provide better results </li></ul><ul><li>Ugi Reactions will be optimized to increase yield </li></ul><ul><li>Better Protein Docking software will be implemented to improve inhibition results </li></ul><ul><li>Optimization of Colorimetric Assay to improve signal quality </li></ul><ul><li>Colorimetric Assay will be used against Ugi Products predicted to inhibit Sortase A </li></ul>
  14. 14. Acknowledgements <ul><li>Dr. Robert Stewart, ORU – NMR Lab Technique </li></ul><ul><li>Dr. Hal Reed, ORU – helping with research funding from ORU Biology Alumni </li></ul><ul><li>ORU Biology Alumni – funding for travel expenses </li></ul><ul><li>Dr. Jean-Claude Bradley and students (Khalid Mirza), Drexel – U4CR Technique and help with Open Notebook Science </li></ul><ul><li>Dr. Andrew Lang, ORU – NMR JSpecViewer and Hex 5.1 </li></ul><ul><li>Dr. Cameron Neylon, RAL – Sortase Assay Development </li></ul><ul><li>Dr. Joel Gaikwad, ORU – Research Advisor </li></ul>
  15. 15. References <ul><li>Arya P, Joseph R, Chou D. Toward High-Throughput Synthesis of Complex Natural Product-Like Compounds in the Genomics and Proteomics Age. Chemistry and Biology Vol.9, 2002. </li></ul><ul><li>Bateman K. Identification of Small Molecule Inhibitors of the Staphylococcus aureus Sortase A Enzyme. Oregon State University Honors Baccalaureate Thesis, 2008. </li></ul><ul><li>Dömling A, Ugi I. Multicomponent Reactions with Isocyanides. Angew. Chem. Int. Ed . 2000, 39 , 3168-3210. </li></ul><ul><li>Furka, A., Drug. Dev. Res. 1995, 36, 1 . </li></ul><ul><li>Paterson G, Mitchell T. The biology of Gram-positive sortase enzymes. TRENDS in Microbiology Vol.12 No.2, 2004. </li></ul><ul><li>Lin, M., Tesconi, M., Tischler, M., Use of H NMR to Facilitate Solubility Measurement for Drug Discovery Compounds, International Journal of Pharmaceutics (2008), doi:10.1016/j.ijpharm.2008.10.038 </li></ul><ul><li>Marraffini L, DeDent A, Schneewind O. Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria. Microbiology and Molecular Biology Reviews Vol.70 No.1, 2006. </li></ul><ul><li>Maresso A, Schneewind O. Sortase as a Target of Anti-Infective Therapy. Pharmacol rev 60:128-141, 2008. </li></ul><ul><li>Maresso A, Schneewind O, et al. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site. The Journal of Biological Chemistry Vol.282, No.32, 2007. </li></ul><ul><li>Musonda M, Chibale K, et al. Application of multi-component reactions to antimalarial drug discovery. Bioorganic & Medicinal Chemistry Letters 14(2004) 3901–3905. </li></ul><ul><li>Pallen M, Lam A, Antonio M, and Dunbar K. An embaressment of sortases – a richness of substrates? TRENDS in Microbiology Vol.9 No.3, 2001. </li></ul><ul><li>Perry A, Ton-That H, Mazmanian S, Schneewind O. Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus . The Journal of Biological Chemistry Vol.277 No.8, 2002. </li></ul><ul><li>Ton-That H, Scheewind O, et al. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif </li></ul><ul><li>Ugi, Werner B, Dömling A. The Chemistry of Isocyanides, their MultiComponent Reactions and their Libraries. Molecules 2003, 8 , 53-66. </li></ul><ul><li>Walsh C. Where will new antibiotics come from? Nature Reviews/Microbiology Vol.1:65-70, 2003. </li></ul><ul><li>Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV. Crystal structures of Staphylococcus aureus sortase A and its substrate complex. J. Biol. Chem. v279, p.31383-31389, 2004. </li></ul><ul><li>  </li></ul><ul><li>ONS Challenge Wiki: </li></ul><ul><li>onschallenge.wikispaces.com </li></ul><ul><li>  </li></ul><ul><li>UsefulChem Wiki: </li></ul><ul><li>usefulchem.wikispaces.com </li></ul><ul><li>  </li></ul><ul><li>David Bulger’s LaBLog: </li></ul><ul><li>biolab.isis.rl.ac.uk/david_bulger </li></ul><ul><li>  </li></ul><ul><li>Hex Useful Chem Wikispace: </li></ul><ul><li>usefulchem.wikispaces.com/D-EXP016 </li></ul><ul><li>  </li></ul><ul><li>UsefulChem Blog: </li></ul><ul><li>usefulchem.blogspot.com </li></ul>
  16. 16. Questions …

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