*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

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

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&term=15117963

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

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 - 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)

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

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 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

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

UV-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 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

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

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

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 Staphylococcusaureus Sortase A Enzyme. Oregon State University HonorsBaccalaureate 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 SolubilityMeasurement for Drug Discovery Compounds, International Journal ofPharmaceutics (2008), doi:10.1016/j.ijpharm.2008.10.038

Marraffini L, DeDent A, Schneewind O. Sortases and the Art of AnchoringProteins to the Envelopes of Gram-Positive Bacteria. Microbiology andMolecular 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 TriggersIrreversible Modification of the Active Site. The Journal of BiologicalChemistry Vol.282, No.32, 2007.

Musonda M, Chibale K, et al. Application of multi-component reactions toantimalarial drug discovery. Bioorganic & Medicinal Chemistry Letters14(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 SurfaceProteins to the Cell Wall of Staphylococcus aureus. The Journal ofBiological Chemistry Vol.277 No.8, 2002.

Ton-That H, Scheewind O, et al. Purification andcharacterization of sortase, the transpeptidasethat cleaves surface proteins of Staphylococcusaureus at the LPXTG motif

Ugi, Werner B, Dömling A. The Chemistry of Isocyanides,their MultiComponent Reactions and theirLibraries. Molecules 2003, 8, 53-66.

Walsh C. Where will new antibiotics come from? NatureReviews/Microbiology Vol.1:65-70, 2003.

Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV.Crystal structures of Staphylococcus aureussortase A and its substrate complex. J. Biol.Chem. v279, p.31383-31389, 2004.

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