Nogalamycin

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Nogalamycin

• Nucleic Acids
• Shilpa Maroju
• 1st May 07

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History

• broad spectrum antibiotic
• Streptomyces nogalator
• 1965-isolated by Wiley et al
• 1970-cytotoxicity by Bhuyan et al in mouse tumor
  cell line, invitro
• 1980- Crooke et al-nuclear DNA- intercalation
• significantly less cardiotoxicity
• no clinical application-toxicity
• Menogaril in clinical trials

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ANTHRACYCLINE GLYCOSIDE
Glycone I aminoglucose
Aglycone (anthraquinone) Quinone-hydroquinone
Glycosidic bond
Glycone II nogalose
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Mode of action of Nogalamycin

• Intercalation
• hydrophobic anthraquinone ring
• cause -10 distortion
• Groove binding
• specific H-bonding
• nonspecific-electrostatic
• hydrophobic- vanderwaals
• DNA topoisomerase I inhibition (topoisomerase
  poisons)
• DNA unwinding during replication
• RNA polymerase inhibition
• transcription

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DNA-Topoisomerases/gyrases

• Topoisomerases modulate DNA supercoiling during
  replication
• single/ double strand breaks
• (topoisomerase I/II)
• unwind DNA/release torsional strain
• passing 2nd strand through the nick
• religate the nick

Giorgio,M. Pierantino,M.Emanuela,S.
Gaetano,C. Luca,G. AnthracyclinesMolecular
Advances and Pharmacological Developments in
Antitumour Activity and Cardiotoxicity. Pharmacol
Rev 56185-229, 2004
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Class II Topoisomerase (closed conformation)
GATE
PDB 1bgw-1ei1
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DNA Topoisomerase I cleaves thephosphodiester
bond-nucleophilic attack
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2nd strand passed through the nick DNA covalently
bound to Tyrosine
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Religation-reform phosphodiester bond
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DNA-Topoisomerase poisons (typical)

• Protein-DNA interface
• DNA strand is cut and covalently linked to the
  Tyrosine residue of DNA topoisomerase
• stable enzyme-drug-DNA ternary complex
• terminate TOPI mediated religation of DNA

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Human DNA Topoisomerase I complexed with poison
Camptothecin and covalently complexed to 22 base
pair DNA duplex DNA covalently bound to tyrosine
residue of TOP I (protein) Drug at the
interface Enzyme-Drug-DNA complex
PDB 1T8I
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(No Transcript)
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what is peculiar with Nog ??

• Nonplanar molecule
• It does not bind next to the putative TOP-I
  cleavage site
• enzyme-drug-DNA complex????
• binds distal to cleavage site, but still
  stimulates TOP-I cleavage
• 
  How far is the site?

Barbara et al. Biochemistry, 1997, 36,
13285-13291.
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Further insights

• Shape of molecule
• Specific H-bonding interactions
• X-ray crystal structure
• NMR-solution structure
• Summarize interactions
• Sequence specificity

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Dumbbell shape (U) of Nogalamycin
Yen,C.L. et al, J. Biochemistry 1989, 28,
9918-9926.
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PDB 1N7I
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(No Transcript)
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NOG bound to DNA hexamer d(CGTpSACG)
Yen-Chywan, L. et al, J. Biochemistry 1989, 28,
9913-9918
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solution structure-NMR d(CGTACG)
Robinson,H. et al, Nucleic Acids Research 1990,
18, 4851-4858.
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Summary of possible interactions

• Antraquinone ring
• intercalation -10distortion
• melting and elongation
• Nogalose - minor groove non bulky
• C2-endo
• methoxy substituents - hydrophobic
• Van-der-waals contacts to DNA backbone
  
• expand minor groove
• Amino glucose - major groove - bulky
• C3-endo
• ve charged N-electrostatically interact
• ve phosphates
  

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Specific H-bonding interactions

• 3 H-bonds
• O4G with N7 of G/A- major groove
• O14 with 2-amine of G-minor groove
• O7 with 2-amine of G-minor groove
• DNA is flexible
• Interactions are optimized

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Sequence specificity

• due to dumbbell shape with bulky sugars at C1 and
  C7
• Intercalation needed localized
• strand elongation
• melting- H-bond disruption
• Prefers GC embedded in poly AT

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Why GC embedded in AT bases??

• 2-NH2 of Guanine/absent in Adenine
• Easy (energetically favorable) to break
• 2 H-bonds of ATs than
• 3 H-bonds of GC base pairs

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• Hypothesis
• binds to GC distal to the TOP-I mediated cleavage
  site
• employed DNA footprinting technique

Daniel et al. Biochemistry, 2000, 39, 9928-9934.
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DNA footprinting-endonucleases
protected
or drug
A
T
C
A
T
C
C
CTA
TA
T
A
A
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SEQUENCE INVESTIGATED
hotspot
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(No Transcript)
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Conclusion
Nog binding to -6 GC (upstream) base pair,
stimulated TOP I mediated cleavage at the hotspot
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Thank You

• Dr.Friesen

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References

• Yen,C.L.Howard,H. Jacques,H.B. Andrew,H.J.W.
  Biochemistry, 1989, 28, 9913-9918.
• Sai-Peng,S. Daniel,S.P. Leroy,F.L.
  Biochemistry, 2000, 39, 9928-9934.
• Howard,R. Yen,C.L. Gijs,A.M, Jacques,H.B.
  Andrew,H.J.W. Nucleic Acids Research, 1990, 18,
  4851-4858.
• Giorgio,M. Pierantonio,M. Emanuel,S.
  Gaetano,C. Luca,G. Pharmacol. Rev. 2004, 56,
  185-229.

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• Questions ???

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DNA Foot printing forensic sciences SNPs Coupled
to PCR protein binding sites drug binding
site(s)