*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 …