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 UV-Vis Spectrometric Quantification of Nitric
Oxide Production by the Anticancer Drug
Suberoylanilide Hydroxamic Acid (SAHA,vorinostat)
Ishita Patel
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Presentation Outline
1 Motivation for Research a. Histone
Deacetylases Inhibitors b. The function of
Nitric Oxide (NO) in the immune system 2
Summary of Research Goals 4 Establishing a
working Protocol 5 Results 6 Discussion
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Histone deacetylase inhibition acetyl groups
shields positively charged amine groups Lysine,
arginine green spheres Hyperacetylated
chromatin is transcriptionally
active Hypoacetylated chromatin is
transcriptionally silent
CANCER altered gene transcription and increased
cell survival
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SAHA structural analogs inhibit the function Of
Zinc-dependent histone deacetylase
HDAC8
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Structural Features of Hybrid Polar Compounds
cap group interacts with the rim of the
catalytic tunnel hydrophobic spacer allows
the molecule to lie into the catalytic
tunnel zinc-binding group complexes the zinc
ion at bottom of catalytic cavity.
(SAHA)
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1 SAHA and HPCs Have Hydroxamate group at the
end 2 Hydroxamates are known to release NO
3 Is NO release by HPCs important?
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Physiological effect of NO
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Research goals

• 1 Experimental set up optimization
• Check reproducibility of NanoDrop Spectrometer
• b) Working with the Griess reagent
• c) Determining Optimal Reactant Concentration
• 2 NO release upon oxidation by metMb/H2O2 for
  each of the HPCs used
• 3 What structural and chemical features of the
  different HPCs make these rates different?

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The Kinetic Assay for NO production
Reaction mixture 1 Hydroxamate 250 µM 2
H2O2 5 mM 3 Met Myoglobin 10 µM Sample
the reaction at different time points Add
Catalase to stop the reactionthen Add both
components of the Griess System
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NO Production Oxidation of Hydroxamates
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The Griess Reaction
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Color Development time for Griess Reaction
500 µL of 100 µM sodium nitrite 250 µL of
Solution A 250 µL of solution B
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Standard Curve for the Griess reagent
200 µL of 20 - 250 µM sodium nitrite 100 µL of
Solution A 100 µL of solution B
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How reproducible are the measurements of the
NanoDrop Spectrometer? The noise level (Error)
does not get larger than 5 and all the rates of
NO production are reproducible as can be seen
from the overlay of trials ABC Is this the
best way to analyze small samples of hard to
obtain chemicals? The NanoDrop gives
reproducible results and the sample volumes were
as low as 200 µL. For samples that are expensive
or in short supply, the NanoDrop is the best
choice.
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Results
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Nitric Oxide Production as a function of time
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NO release rates for the HPCs used
NO production rates have error bars of /- 0.002
µM per min (Standard Deviation of the Mean)
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What structural and chemical features of the
different HPCs make these rates different?
Hydroxy Urea has the fastest NO release
rate SAHA has the slowest NO release rate 1
The trend is that the larger molecules will have
slower oxidation rates. 2 The Hydroxamide has
to make contact with the HEME group inside
Myoglobin, the smaller molecules have easier
access to the HEME group.
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CONCLUSIONS
SAHA has the slowest NO release rate. SAHA is
also the best anticancer drug of the compounds
studied here The conclusion is that NO release
is not the primary function of SAHA. The main
function of SAHA is Histone deacetylase
inhibition The relatively slow NO release rate
means that SAHA is more resistant to oxidation
This would enable SAHA to remain in the body
for longer periods of time, requiring fewer doses
of the drug to be administered to cancer
patients.
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I would like to express my sincere gratitude to
the members of my thesis committee
Prof. Paul Dominguez Prof. Anthony Capetandes
Prof. Uri Samuni Prof. Jorge Ramos