Platensimycin - A Selective Fab F Inhibitor
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nsimycin - A selective FabF inhibitor t antibiotic activity Nature|2006|Vol 441|358-361
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Transcript of Platensimycin - A Selective Fab F Inhibitor
Platensimycin - A selective FabF inhibitor with potent antibiotic activity
Nature|2006|Vol 441|358-361
Antibiotic Target Mode of Action
Cell Wall
-LactamsTranspeptidase/
TransglycosylaseBlock crosslinking enzymes
Vancomycin D-Ala-D-Ala TerminiSequestring substrate for
biosynthesis
Protein Synthesis
Tetracyclins Peptidyl Transferases Protein Synthesis
Aminoglycosides Peptidyl Transferases Protein Synthesis
Oxazolidinones Peptidyl Transferases Protein Synthesis
DNA Replication/repair
Fluoroquinolones DNA Gyrase Blocks DNA replication
Modes of action of common antibiotics
O
OH
Cl
Cl
Cl
N BN
S
OH
N
SNH2
N
HNO
NH2
O
NH2
O
O
OO
O
HOS
HO
O
NH
O O
O
OH
OH
HOOC
Triclosan diazoborine ethionamide isoniazid
cerulenin G75 thiolactomycin
PlatensimycinStreptomyces platensis
Fatty Acid biosynthesis (FAB)
Fatty acid synthetic (FAS) pathway
Type I Type II
Discrete soluble proteinsthat catalyze each reactionindividually
Maier, T., et al Science, 2006, 311, 1258 - 1262
Maier, T., et al Science, 2006, 311, 1258 - 1262
Type I FAS pathway
Kodali, S. et al. J. Biol. Chem. 2005;280:1669-1677
Type II fatty acid synthesis pathway in bacteria
Marrakchi, H., etal Biochemical Society Transactions (2002) Volume 30, part 6
Wang, J. etal Nature, 2006, 441, 358-360
In vivo efficacy of platensimycin
Whole cell labeling - Selectivity
DNA (open circles), Cell wall (filled triangles), Protein (open squares) and RNA (open triangles) Phospholipid synthesis (filled circles)
{6-[3H]-Thymidine, (2,3)-[3H]-Alanine, (4,5)-[3H]-Leucine, (5,6)-[3H]-Uracil and 2-[3H]-Glycerol.}
Wang, J. etal Nature, 2006, 441, 358-360
Agar Diffusion - Two plate sensitivity assay
fabF antisense FabF overexpression
ATC- Anhydrotetracyclin - inducer for plasmid over expression of pTet15
Wang, J. etal Nature, 2006, 441, 358-360
Cell free Gel Elongation assay
Purified single-enzyme catalytic assay:
FabF FabH
Wang, J. etal Nature, 2006, 441, 358-360
Binding assays
C12-CoA: Lauroyl-CoA
Purified enzymes: His Tagged
Inhibitor: [3H]-dihydroplatensimycin
NH
O O
O
OH
OH
HOOC
3H
NH
O O
O
OH
OH
HOOC NH
O O
O
OH
OH
HOOC
3H
3H5% Pd/C in i-PrOH
T2 gas
0.2 N NaOH/MeOH
Mutational analysis:K335A: Conformational change in active site.C163Q: Mimics acyl-enzyme intermediate
Inhibitor + Mutant enzyme
Binding IC50 with various inhibitors:Enzyme + lauroyl-CoA + Thiolactomycin (or) Platensimycin(or) Dihydroplatensimycin
Wang, J. etal Nature, 2006, 441, 358-360
Overlay of crystal structures of various enzyme -inhibitor complexes: Yellow: PlatensimycinGreen: thiolactomycin and Cyan: cerulenin. The important residues are shown next to the side chains
Wang, J. etal Nature, 2006, 441, 358-360
A B
Crystal structures showing interaction of Platensimycin with the mutant enzyme C163Q. A) Benzoic acid ring interactions with the residues deep in the malonyl binding site, B) the amide linker and the ketolide moiety interacting with residues near the active site.
Wang, J. etal Nature, 2006, 441, 358-360
Wang, J. etal Nature, 2006, 441, 358-360
Conclusions:
• Platensimycin inhibits bacterial growth through a new mechanism• Mutation of protein in bacteria very unlikely• Serum tolerability• No cross resistance• Selectively inhibit phospholipid synthesis by inhibiting FAB • Very highly specific to Bacteria
• Mammalian cell: >1000 µg/ml• Fungi: >64 µg/ml
