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Medicinal Chemistry Journal Club September 2004. Konstantinos Ghirtis Tuesday September 14 th 2004. 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.
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Medicinal Chemistry Journal Club September 2004 Konstantinos Ghirtis Tuesday September 14th 2004 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
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: OxazolidinonesFirst 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-tRNA:mRNA:30S complex.
The Future of Antimicrobial Agents New Agents Needed: Enter Daptomycin (Cubicin) New class of antibiotics:AcidicCyclic 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
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) 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 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