1 / 21

Mechanism Testing of the Drug (Modified Megestrol)

Explore the drug's key function and objectives of mechanism testing, including in vitro and in vivo experiments with various techniques such as Competitive Binding Assay and Electrophoretic Mobility Shift Assay. Learn how the drug covalently modifies DNA, affecting estrogen receptors.

emanuelturner
Download Presentation

Mechanism Testing of the Drug (Modified Megestrol)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  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. 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)”

  4. OBJECTIVES Mechanism testing of the Drug • 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

  5. In vitro(cell free extract) Relative Affinity of the Drug to ERs DNA Covalent modification test The drug modified DNA binding to the variant estrogen receptor In vitro(cell line) DNA Covalent modification test In vitro In vivo • DNA Covalent modification test (in mice)

  6. 1.Relative Affinity of the Drug to the ERs (In Vitro, cell free extract) In vitro To determinebinding affinity of the drug for the estrogen receptors Technique: Competitive Binding Assay

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

  9. [3H]-E2 Free ligand Measure : radioactivity ER (ER-[3H]-E2) (ER-Drug)(Drug),(E2) (ER-[3H]-E2) (ER-Drug) [3H]-E2 separate “bound ligand”from “free ligand” by HAP extract the [3H]-E2 from the HAP by ethanol

  10. Calculations • Plot [3H]-E2 bound(%)againstdrug conc(M) In the presence of wtER or vER • Plot [3H]-E2 bound(%)againstE2 conc(M) • estimate: IC50(50%inhibition of (3H]-E2binding) of the drug “nonlinear curve fitting” • Calculate:Relative Binding Affinity (RBA) RBA (%) = • Data Obtained:

  11. 2. DNA Covalent modification test(In Vitro, cell free extract) In vitro • To examine the DNA covalent adduct formation by the drug • Method: DNA Covalent Modification Test

  12. 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

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

  14. In vitro 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. 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)

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

  17. Gel electrophoresis • Analyze gel by PhosphoImager DNA migration retarded due to complexwith ER-LBD • Where; • Untreated DNA • Warhead modified DNA +vER • Drug modified DNA+ vER

  18. 4. DNA modificationtest(In vitro, Cell line) In vitro • IncubateHepG2 cell line with the drug • Isolate DNA from HepG2 cell • Hydrolyze DNA covalent compounds • Analyzethe covalent products by electrospray ionization mass spectrometry (ESI-MS)

  19. In vivo 5. DNA modificationtest(In vivo, nude mice bearing of hepG2) • Administer a single dose of the drug to a xenographmice • Isolate DNA from xenograph • Hydrolyze DNA covalent compounds • Analyzethe covalent products by electrospray ionization mass spectrometry(ESI-MS)

  20. Electrospray Ionization Mass Spectrometry (ESI-MS) A • 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 B Relative Abundance Expected: “The Same Compound” C m/z

  21. 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 drug-modified DNA is examined by “Electrophoretic Mobility Shift Assay”

More Related