*D. Bulger, Oral Roberts University (ORU). C. Neylon, Rutherford Appleton Laboratory (RAL), UK.
J. Gaikwad, Oral Roberts University.
Oral Presentation Section J: BiochemistryD. Bulger,
Biology and Chemistry Department, Oral Roberts University, Tulsa, OK 74171
507-475-1516
Overview
Sortase Inhibition lessens virulence of Gram + BacteriaInhibition measured by colorimetric assayInhibitors predicted by Hex 5.1 Protein Docking and
synthesized using Ugi ReactionSolubility Model predicted Ugi Reaction Products that
would precipitate quickly from reaction mixtureH1 NMR approximated solubilitiesProject implemented open notebook science
Introduction:Sortase A Function
Cysteine transpeptidaseEight stranded β-barrel with several helices and
loopsSec secretion pathwayPhotos: (Maresso, 2008)http://www.ncbi.nlm.nih.gov/sites/entrez?
db=pubmed&term=15117963
Introduction:Sortase A Inhibition
Inhibition of Sortase A – inability to display surface proteins:AdhesinsImmune evasion proteins
Decreased virulence in various Staphylococcus and Listeria infections (Paterson, 2004)
Diminished selectivity pressures
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
Introduction:Ugi 4 Component Reaction - U4CRDescribed by Ivor Ugi in 1959Equimolar 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 IsocyanideMerck – HIV protease inhibitor Crixivan (Furka, 1995)
Introduction: H1 NMR Solubility Determination
Determination of SolubilityProvides approximation
About 20% AccuracyUgi reactions occur in 0.5-2.0 M
solutionsUgi products less soluble than
reactants
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
Materials and Methods:Assay DesignSortase A and GFP purified from BL21(DE3) transformed with
plasmid DNA Reaction mixture of Sortase A, tris-HCl buffer, tetraglycine, and
GFPFluorescence resonance energy transfer (FRET) usedAbsorption can be detected with UV-Vis Spectrophotometer
Purification tag removalNi resin bindingAbsorbance 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
Results/Discussion: Protein PurificationTransformation of BL21(DE3) with
plasmid DNAHis-trap column in Actoprime FPLCSDS Gel electrophoresis to confirm
purity and molecular weightCentrifuge and concentration columnDialysisUV-Vis Conformation of Identity of
Protein Fra
ctio
n 7
Fra
ctio
n 6
Fra
ctio
n 5
Fra
ctio
n 2
Fra
ctio
n 3
Fra
ctio
n 4
Fra
ctio
n 1
Results/Discussion: NMR Solubility and U4CR
NMR solubility determination
(~20% Accuracy)Ugi Synthesis
Ugi Product 62E
Conclusion Sortase inhibition is useful pharmacologicallyUgi synthesis produces large variety of organic productsNumber of Ugi products to test limited to those with high
Sortase A Hex docking resultsSolubility modeling using NMR predicts the ease of
purification Colorimetric assay is expected to accurately detect
Sortase A inhibition, especially after optimization
Future StudySolubility model will be expanded to provide better
resultsUgi Reactions will be optimized to increase yieldBetter Protein Docking software will be implemented to
improve inhibition resultsOptimization of Colorimetric Assay to improve signal
qualityColorimetric Assay will be used against Ugi Products
predicted to inhibit Sortase A
AcknowledgementsDr. Robert Stewart, ORU – NMR Lab TechniqueDr. Hal Reed, ORU – helping with research funding from
ORU Biology AlumniORU Biology Alumni – funding for travel expensesDr. Jean-Claude Bradley and students (Khalid Mirza), Drexel
– U4CR Technique and help with Open Notebook ScienceDr. Andrew Lang, ORU – NMR JSpecViewer and Hex 5.1Dr. Cameron Neylon, RAL – Sortase Assay DevelopmentDr. Joel Gaikwad, ORU – Research Advisor
ReferencesArya 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.
Bateman K. Identification of Small Molecule Inhibitors of the Staphylococcus aureus Sortase A Enzyme. Oregon State University Honors Baccalaureate Thesis, 2008.
Dömling A, Ugi I. Multicomponent Reactions with Isocyanides. Angew. Chem. Int. Ed. 2000, 39, 3168-3210.
Furka, A., Drug. Dev. Res. 1995, 36, 1. Paterson G, Mitchell T. The biology of Gram-positive sortase enzymes.
TRENDS in Microbiology Vol.12 No.2, 2004.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
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.
Maresso A, Schneewind O. Sortase as a Target of Anti-Infective Therapy. Pharmacol rev 60:128-141, 2008.
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.
Musonda M, Chibale K, et al. Application of multi-component reactions to antimalarial drug discovery. Bioorganic & Medicinal Chemistry Letters 14(2004) 3901–3905.
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.
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.
Ton-That H, Scheewind O, et al. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of
Staphylococcus aureus at the LPXTG motifUgi, Werner B, Dömling A. The Chemistry of Isocyanides,
their MultiComponent Reactions and their Libraries. Molecules 2003, 8, 53-66.
Walsh C. Where will new antibiotics come from? Nature Reviews/Microbiology Vol.1:65-70, 2003.
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.
ONS Challenge Wiki: onschallenge.wikispaces.com UsefulChem Wiki:usefulchem.wikispaces.com David Bulger’s LaBLog:biolab.isis.rl.ac.uk/david_bulger Hex Useful Chem Wikispace:usefulchem.wikispaces.com/D-EXP016 UsefulChem Blog:usefulchem.blogspot.com
Questions …