1. 1 Mechanism Testing of the Drug (Modified Megestrol) Mr.Pasavi Ratchapongsirikul

2. 2 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

3. 3 KEY function of the drug “Able to covalently modify DNA, forming adducts that bind to the variant estrogen receptor (vER) ” Mechanism testing of the Drug

4. 4 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 Mechanism testing of the Drug

5. 5 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)

6. 6 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 Competitive Binding Assay Technique: Competitive Binding Assay

7. 7 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

8. 8 Incubation of: [ 3 H]-E 2 Drug (increasing conc) ER ( wtER or vER ) Buffer Method Mixture : Bound ligand* + Free ligand* *Ligand =Drug or [3H]-E2 Ligand Bound ligand* Free ligand*

9. 9 [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

10. 10 Plot [3H]-E2 bound(%) against drug conc(M) In the presence of wtER or vER Plot [3H]-E2 bound(%) against E2 conc(M) estimate: IC 50 (50%inhibition of (3H]-E2 binding) of the drug “nonlinear curve fitting” Calculate: Relative Binding Affinity (RBA) Calculations RBA (%) = Data Obtained: ReceptorRBA(%) of the Drug wtER…?.... vER…?....

11. 11 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

12. 12 Methods 14 (C) labeled DrugSalmon 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

13. 13 Measure Radioactivity Removed ! Adducts Reaction time (h) m/z Relative Abundance ESI-MSScintillation

14. 14 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

15. 15 Prepare: oligonucleotide 5’-d(AATATTGGCCAATATT)-3’ labeled with (  - 32 P) ATP at 5’ end = ( 32 P) DNA Incubate: –Untreated DNA (Control)+ vER (1) –warhead + DNA + vER (2) –The drug + DNA + vER (3) Method Electrophoretic Mobility Shift Assay

16. 16 Control (1) Warhead (2) Drug (3) vER (16 mer) DNA

17. 17 Gel electrophoresis Analyze gel by PhosphoImager DNA migrationretarded due tocomplex with ER-LBD Where; (1)Untreated DNA (2)Warhead modified DNA +vER (3)Drug modified DNA+ vER

18. 18 4. DNA modification test (In vitro, Cell line) HepG2Incubate 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)

19. 19 5. DNA modification test (In vivo, nude mice bearing of hepG2) a xenographmiceAdminister 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)

20. 20 Electrospray Ionization Mass Spectrometry (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” Relative Abundance C

21. 21 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 drug- modified DNA is examined by “ Electrophoretic Mobility Shift Assay” SUMMARY The experiments are conducted in vitro and in vivo to test the drug’s mechanisms