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UV-Vis Spectrometric Quantification of Nitric Oxide Production
by the Anticancer Drug
Suberoylanilide Hydroxamic Acid (SAHA,vorinostat)
Ishita Patel
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
Histone deacetylase inhibition
acetyl groups = shields
positively charged amine groups
Lysine, arginine = green spheres
Hyperacetylated chromatin
is transcriptionally
active
Hypoacetylated chromatin
is transcriptionally
CANCER = altered gene transcription and increased cell survival
silent
SAHA structural analogs inhibit the function
Of Zinc-dependent histone deacetylase
HDAC8
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
(SAHA)
complexes the zinc ion at
bottom of catalytic cavity.
[1] SAHA and HPCs Have Hydroxamate group at the end
[2] Hydroxamates are known to release NO
[3] Is NO release by HPCs important?
Physiological effect of NO
Research goals
[1] Experimental set up optimization
a) 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?
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 reaction…then…
Add both components of the Griess System
NO Production: Oxidation of Hydroxamates
The Griess Reaction
Color Development time for Griess Reaction
500 µL of 100 µM sodium nitrite
250 µL of Solution A
250 µL of solution B
Standard Curve for the Griess reagent
200 µL of 20 - 250 µM sodium nitrite
100 µL of Solution A
100 µL of solution B
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 A+B+C
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.
Results
Nitric Oxide Production as a function of time
NO release rates for the HPCs used
NO production rates have error bars of
+/- 0.002 µM per min (Standard Deviation of the Mean)
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.
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.
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