*D. Bulger, Oral Roberts University (ORU).
C. Neylon, Rutherford Appleton Laboratory (RAL), UK.
                   J. Gaik...
Overview
 Sortase Inhibition lessens virulence of Gram + Bacteria
 Inhibition measured by colorimetric assay
 Inhibitor...
Introduction:
Sortase A Function



 Cysteine transpeptidase
 Eight stranded β-barrel with several helices and
   loops
...
Introduction:
Sortase A Inhibition
 Inhibition of Sortase A – inability to display surface
  proteins:
    Adhesins
   ...
Introduction:
Hex Protein Docking on Sortase A
 Interactive protein
  docking and
  superposition program
 CombUgi Libra...
Introduction:
Ugi 4 Component Reaction - U4CR
 Described by Ivor Ugi in 1959
 Equimolar ratios in solvent (low MW alcoho...
Introduction:
H1 NMR Solubility Determination
 Determination of Solubility
 Provides approximation
    About 20% Accura...
Aim and Hypothesis
 AIM: to find synthetic Sortase A inhibitor using protein
  docking of Ugi Products predicted using so...
Materials and Methods:
Assay Design
 Sortase A and GFP purified from BL21(DE3) transformed with
  plasmid DNA
 Reaction ...
Results/Discussion:
Protein Purification
 Transformation of BL21(DE3) with
  plasmid DNA
 His-trap column in Actoprime F...
Results/Discussion:
NMR Solubility and U4CR
 NMR solubility
  determination
  (~20% Accuracy)
 Ugi Synthesis




       ...
Conclusion
 Sortase inhibition is useful pharmacologically
 Ugi synthesis produces large variety of organic products
 N...
Future Study
 Solubility model will be expanded to provide better results
 Ugi Reactions will be optimized to increase y...
Acknowledgements
 Dr. Robert Stewart, ORU – NMR Lab Technique
 Dr. Hal Reed, ORU – helping with research funding from
  ...
References
Arya P, Joseph R, Chou D. Toward High-Throughput Synthesis of Complex          Ton-That    H, Scheewind O, et a...
Questions …
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Sortase A Inhibition By Ugi Products

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Sortase A Inhibition By Ugi Products

  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  Sortase Inhibition lessens virulence of Gram + Bacteria  Inhibition measured by colorimetric assay  Inhibitors predicted by Hex 5.1 Protein Docking and synthesized using Ugi Reaction  Solubility Model predicted Ugi Reaction Products that would precipitate quickly from reaction mixture  H1 NMR approximated solubilities  Project implemented open notebook science
  3. 3. Introduction: Sortase A Function  Cysteine transpeptidase  Eight stranded β-barrel with several helices and loops  Sec secretion pathway Photos: (Maresso, 2008) http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&t erm=15117963
  4. 4. Introduction: Sortase A Inhibition  Inhibition of Sortase A – inability to display surface proteins:  Adhesins  Immune evasion proteins  Decreased virulence in various Staphylococcus and Listeria infections (Paterson, 2004)  Diminished selectivity pressures
  5. 5. Introduction: Hex Protein Docking on Sortase A  Interactive protein docking and superposition program  CombUgi Library 3 used as ligands  Enzyme docked in active site pocket only
  6. 6. Introduction: Ugi 4 Component Reaction - U4CR  Described by Ivor Ugi in 1959  Equimolar ratios in solvent (low MW alcohols)  Fast exothermic reaction (few sec to few min)  Can convert nearly any combination of Carboxylic acid, Aldehyde, Primary Amine, and Isocyanide  Merck – HIV protease inhibitor Crixivan (Furka, 1995)
  7. 7. Introduction: H1 NMR Solubility Determination  Determination of Solubility  Provides approximation  About 20% Accuracy  Ugi reactions occur in 0.5-2.0 M solutions  Ugi products less soluble than reactants
  8. 8. Aim and Hypothesis  AIM: to find synthetic Sortase A inhibitor using protein docking of Ugi Products predicted using solubility model  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  Null Hypothesis: none of the Ugi products that successfully dock and precipitate with inhibit Sortase A as detected through colorimetry
  9. 9. Materials and Methods: Assay Design  Sortase A and GFP purified from BL21(DE3) transformed with plasmid DNA  Reaction mixture of Sortase A, tris-HCl buffer, tetraglycine, and GFP  Fluorescence resonance energy transfer (FRET) used  Absorption can be detected with UV-Vis Spectrophotometer  Purification tag removal  Ni resin binding  Absorbance of free protein at 490 nm  Ugi synthesis: methanol, carboxylic acids, primary amines, aldehydes, and isocyanides in one dram vials  Solubility: Jeol 300 MHz H1 NMR, JSpecView
  10. 10. Results/Discussion: Protein Purification  Transformation of BL21(DE3) with plasmid DNA  His-trap column in Actoprime FPLC  SDS Gel electrophoresis to confirm purity and molecular weight  Centrifuge and concentration column  Dialysis Fraction 6 Fraction 2 Fraction 3 Fraction 4 Fraction 1 Fraction 5 Fraction 7  UV-Vis Conformation of Identity of Protein
  11. 11. Results/Discussion: NMR Solubility and U4CR  NMR solubility determination (~20% Accuracy)  Ugi Synthesis Ugi Product 62E
  12. 12. Conclusion  Sortase inhibition is useful pharmacologically  Ugi synthesis produces large variety of organic products  Number of Ugi products to test limited to those with high Sortase A Hex docking results  Solubility modeling using NMR predicts the ease of purification  Colorimetric assay is expected to accurately detect Sortase A inhibition, especially after optimization
  13. 13. Future Study  Solubility model will be expanded to provide better results  Ugi Reactions will be optimized to increase yield  Better Protein Docking software will be implemented to improve inhibition results  Optimization of Colorimetric Assay to improve signal quality  Colorimetric Assay will be used against Ugi Products predicted to inhibit Sortase A
  14. 14. Acknowledgements  Dr. Robert Stewart, ORU – NMR Lab Technique  Dr. Hal Reed, ORU – helping with research funding from ORU Biology Alumni  ORU Biology Alumni – funding for travel expenses  Dr. Jean-Claude Bradley and students (Khalid Mirza), Drexel – U4CR Technique and help with Open Notebook Science  Dr. Andrew Lang, ORU – NMR JSpecViewer and Hex 5.1  Dr. Cameron Neylon, RAL – Sortase Assay Development  Dr. Joel Gaikwad, ORU – Research Advisor
  15. 15. References Arya P, Joseph R, Chou D. Toward High-Throughput Synthesis of Complex Ton-That H, Scheewind O, et al. Purification and Natural Product-Like Compounds in the Genomics and Proteomics Age. characterization of sortase, the transpeptidase Chemistry and Biology Vol.9, 2002. that cleaves surface proteins of Staphylococcus Bateman K. Identification of Small Molecule Inhibitors of the Staphylococcus aureus at the LPXTG motif aureus Sortase A Enzyme. Oregon State University Honors Ugi, Werner B, Dömling A. The Chemistry of Isocyanides, Baccalaureate Thesis, 2008. their MultiComponent Reactions and their Dömling A, Ugi I. Multicomponent Reactions with Isocyanides. Angew. Libraries. Molecules 2003, 8, 53-66. Chem. Int. Ed. 2000, 39, 3168-3210. Walsh C. Where will new antibiotics come from? Nature Reviews/Microbiology Vol.1:65-70, 2003. Furka, A., Drug. Dev. Res. 1995, 36, 1. Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV. Paterson G, Mitchell T. The biology of Gram-positive sortase enzymes. Crystal structures of Staphylococcus aureus TRENDS in Microbiology Vol.12 No.2, 2004. sortase A and its substrate complex. J. Biol. Lin, M., Tesconi, M., Tischler, M., Use of H NMR to Facilitate Solubility Chem. v279, p.31383-31389, 2004. Measurement for Drug Discovery Compounds, International Journal of Pharmaceutics (2008), doi:10.1016/j.ijpharm.2008.10.038 ONS Challenge Wiki: Marraffini L, DeDent A, Schneewind O. Sortases and the Art of Anchoring onschallenge.wikispaces.com Proteins to the Envelopes of Gram-Positive Bacteria. Microbiology and UsefulChem Wiki: Molecular Biology Reviews Vol.70 No.1, 2006. usefulchem.wikispaces.com Maresso A, Schneewind O. Sortase as a Target of Anti-Infective Therapy. Pharmacol rev 60:128-141, 2008. David Bulger’s LaBLog: Maresso A, Schneewind O, et al. Activation of Inhibitors by Sortase Triggers biolab.isis.rl.ac.uk/david_bulger Irreversible Modification of the Active Site. The Journal of Biological Chemistry Vol.282, No.32, 2007. Hex Useful Chem Wikispace: Musonda M, Chibale K, et al. Application of multi-component reactions to usefulchem.wikispaces.com/D-EXP016 antimalarial drug discovery. Bioorganic & Medicinal Chemistry Letters 14(2004) 3901–3905. UsefulChem Blog: Pallen M, Lam A, Antonio M, and Dunbar K. An embaressment of sortases – usefulchem.blogspot.com a richness of substrates? TRENDS in Microbiology Vol.9 No.3, 2001. 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.
  16. 16. Questions …

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