Download Pasavi-Mechanism Tes..

Mechanism Testing of the Drug
(Modified Megestrol)
Mr.Pasavi Ratchapongsirikul
1
Outline of the Presentation
• Introduction
– Key function of the drug
• OBJECTIVES
– Mechanism testing and the techniques used
• System of the experiments( In vitro
• Methods used in the experiments
• Summary
and In vivo )
2
Mechanism testing of the Drug
• KEY function of the drug
“Able to covalently modify DNA,
forming adducts that bind to the
variant estrogen receptor (vER)”
3
Mechanism testing of the Drug
OBJECTIVES
• To determine binding affinity of the drug for the estrogen
receptors
Competitive Binding Assay
• To examine covalent modification of DNA by the drug
DNA Covalent modification Test
• To examine the drug-modified DNA on the variant
estrogen receptor binding
Electrophoretic Mobility Shift Assay
4
In vitro
•
In vitro (cell free extract)
1. Relative Affinity of the Drug to ERs
2. DNA Covalent modification test
3. The drug modified DNA binding to the variant
estrogen receptor
•
In vitro (cell line)
4. DNA Covalent modification test
In vivo
5. DNA Covalent modification test (in mice)
5
1.Relative Affinity of the Drug to the ERs
(In Vitro, cell free extract)
To determine binding affinity of the drug for the
estrogen receptors
Technique: Competitive Binding Assay
6
Method
• Varied concentrations of the drug or
Estradiol(E2)* are combined with [3H]-E2
• Incubate with wtER or vER
• Remove unbound radioactivity
• Measure the radioactivity of [3H]-E2 bound ER
7
Method
Incubation of:
•
•
•
•
[3H]-E2
Drug (increasing conc)
ER (wtER or vER)
Buffer
Ligand
*Ligand =Drug or [3H]-E2
Ligand
Mixture :Bound ligand* + Free ligand*
Free ligand*
Bound ligand*
8
[3H]-E2
Free ligand
ER
(ER-[3H]-E2)
(ER-Drug)
(Drug),(E2)
(ER-[3H]-E2)
(ER-Drug)
separate “bound
ligand”from “free
ligand”
by
HAP
Measure :
radioactivity
[3H]-E2
extract the [3H]-E2
from the HAP
by ethanol
9
Calculations
• Plot [3H]-E2 bound(%) against drug conc(M)
In the presence of wtER or vER
• Plot [3H]-E2 bound(%) against E2 conc(M)
• estimate: IC50 (50%inhibition of (3H]-E2 binding) of the
drug
“nonlinear curve fitting”
• Calculate: Relative Binding Affinity (RBA)
RBA (%) =
•Data Obtained:
IC 50( E 2)
100
IC 50( Drug )
Receptor
RBA(%) of the Drug
wtER
…?....
vER
…?....
10
2. DNA Covalent modification test
(In Vitro, cell free extract)
• To examine the DNA covalent adduct
formation by the drug
• Method: DNA Covalent Modification Test
11
Methods
• Salmon testes DNA is incubated with 14 (C)
labeled Drug
• Aliquot is removed at various time points
• Hydrolyze DNA covalent compound
(Remove non-covalent DNA adduct)
• Determine the DNA concentration & measure
the radioactivity
12
Measure
Radioactivity
Removed !
Scintillation
Adducts
Relative Abundance
ESI-MS
13
Reaction time (h)
m/z
3. The drug-modified DNA binding to the
variant estrogen receptor
(In Vitro, cell free extract)
• To examine drug-modified
DNA binding to the variant
estrogen receptor
• Method: Electrophoretic
Mobility Shift Assay
14
Method
Electrophoretic Mobility Shift Assay
• Prepare: oligonucleotide
5’-d(AATATTGGCCAATATT)-3’ labeled with
(-32P)ATP at 5’ end
= (32P)DNA
• Incubate:
– Untreated DNA (Control)+ vER (1)
– warhead + DNA + vER (2)
– The drug +DNA + vER (3)
15
Control (1)
Warhead (2)
Drug (3)
DNA
(16 mer)
vER
16
• Gel electrophoresis
• Analyze gel by PhosphoImager
DNA migration
Where;
retarded due to
complex with
ER-LBD
(1)Untreated DNA
(2)Warhead
modified DNA
+vER
(3)Drug modified
DNA+ vER
17
4. DNA modification test
(In vitro, Cell line)
• Incubate HepG2 cell line with the
drug
• Isolate DNA from HepG2 cell
• Hydrolyze DNA covalent
compounds
• Analyze the covalent products by
electrospray ionization mass
spectrometry (ESI-MS)
18
5. DNA modification test
(In vivo, nude mice bearing of hepG2)
• Administer a single dose of the
drug to a xenograph mice
• Isolate DNA from xenograph
• Hydrolyze DNA covalent
compounds
• Analyze the covalent products by
electrospray ionization mass
spectrometry(ESI-MS)
19
Electrospray Ionization Mass Spectrometry
(ESI-MS)
A
•A, The drug reacted with the DNA
•B, The drug reacted with the DNA
in HepG2 cell line
• C, The drug reacted with the
DNA in xenograph mice
Expected:
Relative Abundance
Analysis of the drug –DNA
adducts formed in vitro and in
vivo
B
C
“The Same Compound”
m/z
20
SUMMARY
The experiments are conducted in vitro and
in vivo to test the drug’s mechanisms
• Binding Affinity of the drug to the estrogen receptor is
determined by
“Competitive Binding Assay”
• The drug covalent modification of DNA is examined by
“DNA Covalent modification Test”
• The complex formation between vER and the drugmodified DNA is examined by
“Electrophoretic Mobility Shift Assay”
21