Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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