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Mechanism Testing of the Mechanism Testing of the Drug (Modified Megestrol)Drug (Modified Megestrol)

Mr.Pasavi RatchapongsirikulMr.Pasavi Ratchapongsirikul

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Outline of the Presentation

• Introduction– Key function of the drug

• OBJECTIVES– Mechanism testing and the techniques used

• System of the experiments( In vitro and In vivo )

• Methods used in the experiments• Summary

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• KEY function of the drug

“Able to covalently modify DNA, forming adducts that bind to the variant estrogen receptor (vER)”

Mechanism testing of the DrugMechanism testing of the Drug

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OBJECTIVES

• To determine binding affinity of the drug for the estrogen receptors

Competitive Binding AssayCompetitive Binding Assay• To examine covalent modification of DNA by the drug

DNA Covalent modification TestDNA Covalent modification Test

• To examine the drug-modified DNA on the variant estrogen receptor binding

Electrophoretic Mobility Shift Electrophoretic Mobility Shift AssayAssay

Mechanism testing of the Mechanism testing of the DrugDrug

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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 vivo5. DNA Covalent modification test (in mice)

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1.Relative Affinity of the Drug to the 1.Relative Affinity of the Drug to the ERsERs

((In VitroIn Vitro, cell free extract), cell free extract)To determine binding affinity of the drug for the estrogen receptors

Technique: Competitive Binding AssayCompetitive Binding Assay

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MethodMethod

• 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

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Incubation of:• [3H]-E2

• Drug (increasing conc)

• ER (wtER or vER)• Buffer

Method

Mixture :Bound ligand* + Free ligand*

*Ligand =Drug or [3H]-E2

Lig

and

Lig

and

Bound ligand*Free ligand*

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[3H]-E2(ER-[3H]-E2) (ER-Drug) (Drug),(E2)

(ER-[3H]-E2) (ER-Drug)

Free ligand

separate “bound ligand”from “free ligand”

by HAP

extract the [3H]-E2

from the HAP

by ethanol

Measure : radioactivity

[3H]-E2

ER

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• 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)100

)(50

)2(50

DrugIC

EIC

CalculationsCalculations

RBA (%) =

•Data Obtained: Receptor RBA(%) of the Drug

wtER …?....

vER …?....

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2. DNA 2. DNA Covalent modification Covalent modification testtest((In VitroIn Vitro, cell free extract), cell free extract)

• To examine the DNA covalent adduct formation by the drug

• Method: DNA Covalent Modification Test

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Methods

• Salmon testes DNA is incubated with 14 14 (C)(C) labeled Druglabeled Drug

• Aliquot is removed at various time points

• Hydrolyze DNA covalent compound (Remove non-covalent DNA adduct)

• Determine the DNA concentration & measure the radioactivity

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Measure Radioactivity

Removed !

Ad

du

cts

Reaction time (h)m/z

Rel

ativ

e A

bu

nd

ance

ESI-MSScintillation

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3. 3. The drug-modified DNA binding The drug-modified DNA binding to the variant estrogen receptorto the variant estrogen receptor ((In VitroIn Vitro, cell free extract), cell free extract)

• To examine drug-modified DNA binding to the variant estrogen receptor

• Method: Electrophoretic Mobility Shift Assay

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• 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)

MethodMethod

Electrophoretic Mobility Shift AssayElectrophoretic Mobility Shift Assay

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Control (1)

Warhead (2)

Drug (3)

vER(16 mer)

DNA

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• Gel electrophoresis

• Analyze gel by PhosphoImager

DNA migration

retarded due to complex with ER-LBDWhere;

(1)Untreated DNA

(2)Warhead modified DNA +vER

(3)Drug modified DNA+ vER

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4. DNA modification4. DNA modification testtest ((In vitroIn vitro, Cell line), Cell line)

• Incubate HepG2HepG2 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)

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5. DNA modification5. DNA modification testtest ((In vivoIn vivo, nude mice bearing of , nude mice bearing of hepG2)hepG2)• Administer a single dose of the

drug to a xenographa xenograph micemice• Isolate DNA from xenograph• Hydrolyze DNA covalent

compounds• Analyze the covalent products by

electrospray ionization mass spectrometry(ESI-MS)

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Electrospray Ionization Mass Spectrometry Electrospray Ionization Mass Spectrometry (ESI-MS)(ESI-MS)

m/z

A

B

Analysis of the drug –DNA adducts formed in vitro and in vivo

•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:

“The Same Compound”

Rel

ativ

e A

bu

nd

ance

C

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• Binding Affinity of the drug to the estrogen receptor is determined by

““Competitive Binding Assay”Competitive Binding Assay”• The drug covalent modification of DNA is examined by

““DNA Covalent modification Test”DNA Covalent modification Test”

• The complex formation between vER and the drug-modified DNA is examined by

““Electrophoretic Mobility Shift Electrophoretic Mobility Shift Assay”Assay”

SUMMARYSUMMARY The experiments are conducted The experiments are conducted in vitroin vitro and and

in vivoin vivo to test the drug’s mechanisms to test the drug’s mechanisms