Medicinal Chemistry Journal Club September 2004

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Medicinal Chemistry Journal Club September 2004
NMR structure determination and calcium binding effects of lipopeptide antibiotic Daptomycin
Lee-Jon Ball, Catherine M. Goult, James A.
Donarski, Jason Micklefield and Vasudevan
Ramesh Department of Chemistry, University of
Manchester Institute of Science and Technology, UK

• Konstantinos Ghirtis
• Tuesday September 14th 2004

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Antimicrobial Chemotherapy
Acquired Resistance to Antimicrobial Agents
Produced by the bacterial species that produce
the antibiotic Protect against the action of
that agent Start as a few but after the
introduction of the antibiotic Kill the
sensitive bacteriagtgtgt increase in the resistant
type Shift from the sensitive to the resistance
type.
Wide availability of antimicrobial agents
Irrational use and abuse of these agents Use in
animal husbandries, especially as growth
promoters Wide use in lotions, soaps and other
household items.
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Antimicrobial Chemotherapy
Same basic mechanisms of action 40 years!!

• Cell Wall Biosynthesis (Penicillins-Vancomycin
  Carbepenems-Cephalosporins)
• DNA Synthesis Processing (Sulfonamides
  Fluoroquinolones)
• Protein Biosynthesis Tetracyclines
  Aminoglycosides Aminoglycosides, Macrolides,
  Lincosaminides Streptogrammins

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The Future of Antimicrobial Agents
New Agents Needed
Oxazolidinones First novel agents in thirty
years! Linezolid (Zyvox?) in 2000/others under
development.
Nosocomial Gram () Esp. MRSA, VRE, pneumonia
and multiresistant strains.
Prevent formation of fmet-tRNAmRNA30S complex.
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The Future of Antimicrobial Agents
New Agents Needed
Enter Daptomycin (Cubicin)
New class of antibiotics Acidic Cyclic
lipopeptides
Activity against multiresistant Gram-()
bacteria Staphylococcus aureus, Streptococcus
pyogenes, vancomycin-susceptible strains
Enterococcus faecalis.
Parenteral treatment of major abscesses and
other skin and skin-structure infections.
Current phase III trials for bacteraemic disease
and endocarditis due to staphylococci,
enterococci, etc
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Daptomycin
Structure
Streptomyces roseosporus Cyclic tridecapeptide,
several D- non-proteinogenic AAs N-terminus
acylated n-decanoyl fatty acid side
chain Various straight and branched fatty acid
side chains Major source of toxicity /decanoyl
group exhibits the least C-terminal carboxylate
cyclised side chain OH Thr Decapeptide core.
MeGlu 3 acidic Asp calcium binding and
activity.
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Daptomycin
Mechanism of Action
Act directly on the bacterial cell
membrane Requirement for calcium ions Much less
chance of cross-resistance Known peptide
antimicrobials act on cell membrane may damage
mammalian cells and cause toxicity
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Daptomycin
Mechanism of Action
Lipid tail inserts itself into membrane
Without rupturing Binding of calcium causes
deeper penetration Aggregation create channels
allowing K permeate The membrane is
depolarised, No longer carry out its transport
processes. This kills the bacteria, but they're
not lysed
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Daptomycin
CDA Ca Dependnt Antibiotics
Friulimicin (X NH2, R1 H, R2 CH3)
amphomycin A-1437B (X OH, R1 CH3, R2 H)
from Actinoplanes friuliensis
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Daptomycin
CDA Ca Dependnt Antibiotics
Decapeptide lactone or lactam ring Cyclisation
L-threonine or L-threo-2,3-diaminobutyrate side
chains onto the C-terminal carboxyl group.
Acidic residues (Asp and MeGlu) conserved
Biosynthesised multi-modular nonribosomal
peptide synthetases.gtgtgt So combinatorial
biosynthesis
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Daptomycin
NMR Study
High solubility in water Resonance line widths
large for a small peptide Aggregation tendency
of the lipopeptide Accordingly, the sample was
diluted narrow lines Unique low field shifted
resonance at 5.48 ppm. Side chain H proton of
Thr 4 residue. Evidence for ester linkage of Thr
residue with Ar- Kyn 13
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Daptomycin
Sequence-specific resonance assignment
2D experiments COSY Correlated coupled proton
connectivities, 3JH-H Aromatic side chain spin
systems of (W1),(U13) HSQC Proton-carbon
connectivities, 1JH-C Long aliphatic side chain
spin systems of nonproteinogenic (O6), (E12) AA
residues
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Daptomycin
Sequence-specific resonance assignment
2D experiments TOCSY Intra-residue correlation
exchangeable backbone NHs With non exchangable
side chain Hs
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Daptomycin
Sequence-specific resonance assignment
Except degenerate amide NH _at_ 8.29-8.33 All NHs
assigned Clearly NHs of N- and C-terminal
residues (Trp Kyn) NH proton branching Thr
residue
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Daptomycin
Sequence-specific resonance assignment
2D experiments NOESY Sequential connectivities
due to dipolar correlation (NOE) Amide NHs
with side chain Hs of neighbouring residue
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Daptomycin
Sequence-specific resonance assignment
e.g. Amide proton Kyn at 8.52 ppm NOE cross
peak Me of MeGlu at 0.93 ppm,.
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Daptomycin
Structure of apo-daptomycin
Sequence specific resonance assignment and 142
distance constraints fromm NOESY 30 structures
calculated 20 structures with lowest energy
target function Backbone torsion angles within
the steric repulsion limits.
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Daptomycin
Structure of apo-daptomycin
38 NOE violations Mostly structures
with largest energy function. Best lowest
energy function containing one NOE violation
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Daptomycin
Structure of apo-daptomycin
Extended conformation in solution Turns at Ala8
and Gly10/Ser11. Side chains exposed to
solvent Backbone amide point inside decanoyl
chain is flexible
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Daptomycin
Structure of apo-daptomycin
Distribution of charge
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Daptomycin
Effect of calcium binding
Addition of 0.3 molar loss of fine
structure Further addition of Ca2, increased
broadening Addition of excess no further changes
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Daptomycin
Effect of calcium binding
Raising the temperature from 293 K to 313
K Narrow the lines reduced affinity for
Ca2 Back to 293 K Restored the broad
spectrum Effect of Ca2 binding was
reversible. Pattern of NOEs very similar/no new
NOEs No global conformational change
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Daptomycin
Discussion/Conclusions
Propensity for intermolecular aggregation
Optimisation of the solution conditions to
minimise it Unusual shifted H resonance (5.45
ppm) of Thr 4 Changes NMR resonance line widths
upon Ca2 binding One molar equivalent/ no
further increase to line widths
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Daptomycin
Discussion/Conclusions
Large resonance line widths molecular size of
beyond monomeric Multimeric structure mediated
by an equivalent Ca2 Conformation little
affected by binding Ca2. 3D structure is
relevant to the mechanism of action
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Daptomycin
Discussion/Conclusions
Acidic residues, Asp 3, Asp 7, Asp 9 and MeGlu
12, Not spatially close enough for binding
site Electrostatic in nature, aiding
aggregation Neutralising bridge between
daptomycin molecules Consistent with proposed
mode of action
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THANK YOU