Medicinal Chemistry Journal Club Medicinal Chemistry Journal Club September 2004September 2004

Konstantinos GhirtisKonstantinos GhirtisTuesday September 14Tuesday September 14thth 2004 2004

““NMR structure determination and calcium binding NMR structure determination and calcium binding effects of lipopeptide antibiotic Daptomycin”effects of lipopeptide antibiotic Daptomycin”

Lee-Jon Ball, Catherine M. Goult, James A. Donarski, Lee-Jon Ball, Catherine M. Goult, James A. Donarski,

Jason Micklefield and Vasudevan Ramesh*Jason Micklefield and Vasudevan Ramesh* Department of Chemistry, University of Manchester Institute of Science and Technology, UKDepartment of Chemistry, University of Manchester Institute of Science and Technology, UK

Antimicrobial Chemotherapy

Acquired Resistance to Antimicrobial Agents:

Wide availability of antimicrobial agents Irrational use and abuse of these agentsUse in animal husbandries, especially as growth promoters Wide use in lotions, soaps and other household items.

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.

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

Oxazolidinones First novel agents in thirty years! Linezolid (Zyvox) in 2000/others under development.

New Agents Needed:

NO

F

N O

HN

O

CH3

O

Linezolid (Zyvox®)

Nosocomial Gram (+) Esp. MRSA, VRE, pneumonia and multiresistant strains.

Prevent formation of fmet-tRNA:mRNA:30S complex.

The Future of Antimicrobial Agents

Enter Daptomycin (Cubicin)

New Agents Needed:

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

Activity against multiresistant Gram-(+) bacteria: Staphylococcus aureus, Streptococcus pyogenes, vancomycin-susceptible strains Enterococcus faecalis.

New class of antibiotics: Acidic Cyclic lipopeptides

Daptomycin

StructureStreptomyces roseosporus Cyclic tridecapeptide, several D- non-proteinogenic AAs

N-terminus acylated: n-decanoyl fatty acid side chainVarious 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

Act directly on the bacterial cell membraneRequirement for calcium ions Much less chance of cross-resistance

Known peptide antimicrobials act on cell membranemay damage mammalian cells and cause toxicity H O

Val

Gly

Ala Leu Ala Val Val Val Try Leu Try

Leu

Try

Leu

NHCH CHOH

(D) (D) (D) (D)

(D)

(D)

Gramicidin A

Lac ValHiv

Val

Lac

ValHivVal

Lac

Val

Hiv

Val (D)(D)(D)(D)

(D)

(D)

(D)

(D)

(D)

Valinomycin

Mechanism of Action

Daptomycin

Lipid tail inserts itself into membrane

Without rupturing

Binding of calcium causes deeper penetrationAggregation 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

Mechanism of Action

Daptomycin

CDA: Ca Dependnt Antibiotics

Friulimicin (X = NH2, R1 = H, R2 = CH3) amphomycin A-1437B (X = OH, R1 = CH3, R2 = H) from Actinoplanes friuliensis

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

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

NMR Study

Daptomycin

2D experiments

COSYCorrelated coupled proton connectivities, 3JH-HAromatic side chain spin systems of (W1),(U13)

HSQCProton-carbon connectivities, 1JH-C Long aliphatic side chain spin systems of nonproteinogenic (O6), (E*12) AA residues

Sequence-specific resonance assignment

Daptomycin

2D experiments TOCSYIntra-residue correlation exchangeable backbone NHsWith non exchangable side chain Hs

Sequence-specific resonance assignment

Daptomycin

Except degenerate amide NH@ 8.29-8.33

All NHs assigned

Clearly NHs of N- andC-terminal residues (Trp Kyn) NH proton branching Thr residue

Sequence-specific resonance assignment

Daptomycin

2D experiments NOESY

Sequential connectivities due to dipolar correlation (NOE)

Amide NHs with side chain Hs of neighbouring residue

Sequence-specific resonance assignment

Daptomycin

e.g.

Amide proton Kyn at 8.52 ppm NOE cross peak Me of MeGlu at 0.93 ppm,.

Sequence-specific resonance assignment

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.

Structure of apo-daptomycin

Daptomycin

38 NOE violations

Mostly structures withlargest energy function.

Best:lowest energy function containing one NOE violation

Structure of apo-daptomycin

Daptomycin

Extended conformation in solution

Turns at Ala8 and Gly10/Ser11.

Side chains exposed to solvent

Backbone amide point inside

decanoyl chain is flexible

Structure of apo-daptomycin

Daptomycin

Distribution of charge

Structure of apo-daptomycin

Daptomycin

Addition of 0.3 molarloss of fine structure

Further addition of Ca2+, increased broadening

Addition of excess no further changes

Effect of calcium binding

Daptomycin

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

Effect of calcium binding

Daptomycin

Discussion/ConclusionsPropensity 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/ConclusionsLarge 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/ConclusionsAcidic 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