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Phortress 2 nd generation: Screening Cascade

Phortress 2 nd generation: Screening Cascade. MTT: MCF-7, MDA 468. 10 nM, 100 nM, 1 μ M, 10 μ M. MTT: larger concentration range to generate GI 50 values in – Phortress-sensitive cells MCF-7, MDA 468, IGROV-1, TK-10 (CYP 1A1-inducible).

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Phortress 2 nd generation: Screening Cascade

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  1. Phortress 2nd generation: Screening Cascade MTT: MCF-7, MDA 468. 10 nM, 100 nM, 1 μM, 10 μM. MTT: larger concentration range to generate GI50 values in – Phortress-sensitive cells MCF-7, MDA 468, IGROV-1, TK-10 (CYP 1A1-inducible). Address selectivity by testing activity in Phortress – resistant cell lines MDA 231, SKOV-3, CAKI-1 (Neither constitutive nor inducible CYP 1A1). Activity in GW 610 – sensitive (CYP 1A1 non-inducible) colon cell lines – HCC2998, KM12. AhR binding XRE-driven luciferase reporter CYP1A1 induction in (at least) MCF-7, MDA 468, MDA 231; 1 μM, 24 h. DNA damage quantitation: COMET; ELISA - (Cell Biolabs) 8-OXOdG, (Biovision) AP sites Strictly confidential

  2. Phortress 2nd generation: Screening Cascade (ii) MTT: MCF-7, MDA 468, MDA 231 10 nM, 100 nM, 1 μM, 10 μM. MTT: larger concentration range and extended spectrum – Phortress-sensitive cells MCF-7, MDA 468, IGROV-1, TK-10 (CYP 1A1-inducible) and Address selectivity - Phortress – resistant cell lines MDA 231, SKOV-3, CAKI-1 (Neither constitutive nor inducible CYP 1A1). Activity in GW 610 – sensitive (CYP 1A1 non-inducible) colon cell lines – HCC2998, KM12. AhR binding XRE-driven luciferase reporter Western blot: CYP1A1 induction in (at least) MCF-7, MDA 468, MDA 231; 1 μM, 24 h. DNA damage quantitation: ELISA - (Cell Biolabs) 8-OXOdG, (Biovision) AP sites (MET inhibition Western blot; AhR – HIF crosstalk) Strictly confidential

  3. Validating the screening cascade (i) 5F 203 GW 610 IH 352

  4. Rat Aryl hydrocarbon receptor (AhR) binding affinities Binding of [3H] TCDD (1 nM) to CRL:WI rat liver cytosol o/n at 4oC ± 200 nM tetrachloroazoxybenzene ± competitor concentrations. Unbound TCDD is removed by incubation (10 min) with dextran-coated charcoal. Radioactivity in the supernatant quantified. Kd TCDD 0.37 nM Ki (nM) IH 352 36.6 5F 203 2.8 GW 610 6.8

  5. In vitro antitumour activity from NCI screen (i) 5F 203 GW 610 lung lung colon ovarian ovarian renal breast renal breast

  6. NCI screen (i): 2-day screen IH 352; NSC 717147 2-(4-Amino-3-cyanophenyl)-5-fluorobenzothiazole lung colon ovarian renal breast

  7. NCI screen (ii): 2-day screen lung colon ovarian renal breast

  8. Comparative in vitro antitumour activity: MTT assays, 72 h exposure Data generated by Dr Erica Stone

  9. Validating the screening cascade (i) GW 610 IH 352 5F 203

  10. MCF-7 MDA 468 TK-10 5F 203 GW 610 IH 352

  11. Effect of novel benzothiazole analogues on growth of MVF-7 and MDA 468 breast cell lines 1.

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  17. Summary ▪ Novel benzothiazole analogues being synthesised ▪ Cell lines for screening cascade in place

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