Transcript MedChemJournalClub S..

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
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 bacteria>>> 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.
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
The Future of Antimicrobial Agents
New Agents Needed:
Oxazolidinones First novel agents in thirty years!
Linezolid (Zyvox) in 2000/others under development.
O
O
N
N
Nosocomial Gram (+)
Esp. MRSA, VRE, pneumonia and
multiresistant strains.
O
H
N
CH 3
F
Linezolid (Zyvox®)
O
Prevent formation of fmet-tRNA:mRNA:30S complex.
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
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.
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
NHCH
H
O
Leu (D)
Val
Try
Gly
Leu (D)
Ala
Leu
(D)
Ala Val Val
(D)
Val Try
(D)
Gramicidin A
Leu Try
(D)
CHOH
Lac
(D)
Val Hiv
(D)
Val
(D) Val
Lac
(D) Hiv
Val
Val
Hiv (D)
Lac Val
(D)
Valinomycin
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
Daptomycin
CDA: Ca Dependnt Antibiotics
Friulimicin (X = NH2, R1 = H, R2 = CH3)
amphomycin A-1437B (X = OH, R1 = CH3, R2 = H) fro
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.>>> So combinatorial biosynthesis
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
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), (E*12) AA residues
Daptomycin
Sequence-specific resonance assignment
2D experiments
TOCSY
Intra-residue correlation
exchangeable backbone NHs
With non exchangable side chain Hs
Daptomycin
Sequence-specific
resonance assignment
Except degenerate amide NH
@ 8.29-8.33
All NHs assigned
Clearly NHs of N- and
C-terminal residues (Trp Kyn)
NH proton branching Thr residue
Daptomycin
Sequence-specific resonance assignment
2D experiments
NOESY
Sequential connectivities
due to dipolar correlation (NOE)
Amide NHs with side chain Hs
of neighbouring residue
Daptomycin
Sequence-specific
resonance assignment
e.g.
Amide proton Kyn at 8.52 ppm
NOE cross peak Me of MeGlu
at 0.93 ppm,.
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.
Daptomycin
Structure of apo-daptomycin
38 NOE violations
Mostly structures with
largest energy function.
Best:
lowest energy function
containing one NOE violation
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
Daptomycin
Structure of
apo-daptomycin
Distribution of charge
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
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
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
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
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