Functional assignments for S' aureus strains

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Functional assignments for S. aureus strains
Lee et al, J. Bact., 2009
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ERGO Annotation, Functions Reactions Overview
Into ERGO
Genome Data
Similarity Computations
Functions
Orf Calling

• Involves calculations of
• Protein families
• Paralogs
• Possible fusions
• Bi-directional best hits
• Pinned orthologs
• Automated annotation
• Automated pathway assertion

• Involves identification of
• Enzymes
• KEGG reactions
• Sort likely reactions
• Transport reactions
• Compounds uptake
• Comparative genomics
• Comparative reactions

IG uses CRITICA, Glimmer IG-proprietary
similarity-based ORF calling tool.
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Metabolic Pathways from Genome
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(No Transcript)
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Essential Enzymes and Pathways
Lee et al, J. Bact., 2009
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• Strain conservation of essential enzymes

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• The FAS II Pathway

• Synthesis of linear and branched fatty acids
• Unique to bacteria
• Each enzyme structurally characterized in
• one or more species
• several known inhibitors

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FabD vs. Human Mitochondrial MCAT
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Computational chemistry-based screening protocol
for finding small molecule inhibitors
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Inhibition of FabD (MCAT)
FDi2
FDi2 FDi14 in FabD pocket (E. coli in red,
human in blue)
FDi10
FDi10
FDi2
FDi14
FDi7
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MCAT Assay and KDH Counterscreen
7 out of 15 selected compounds are active !
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• The FAS II Pathway

• Synthesis of linear and branched fatty acids,
  unique to bacteria
• Each enzyme structurally characterized in one or
  more species
• several known inhibitors
• potential for polypharmacology

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• Ketoacyl ACP Syntheases FabF and FabB

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• Reductases FabI and FabG

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• Reductases FabA and FabZ

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Experimental Verification of Fab Inhibition using
a Gel Elongation Assay
Kodali et al., Nature Protocols DOI
10.1038/nprot.2006.134
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In Vivo Cell Assays
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Cell Toxicity Assays
MTT activity of CL1 fibroblasts in the presence
of 400µg/ml inhibitors or DMSO
Numbers of CL1 cells treated with 400µg/ml
inhibitors or with DMSO
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In Vivo Cell Assays
S. Aureus patient isolate
Human serum added
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Assessing polypharmacologyenzyme assay-based
small molecule-kinase interaction map
Fabian et al, Nat Biotech., 2005, Nolan, Nat Chem
Biol, 2007
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Combining the assessment of S. aureus strains
with bacterial isolate-specific therapy
1,000 S. aureus isolate project Pitt/ PHIDL