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Elucidation of Mechanisms of Action of Novel Antitumour Agents

Elucidation of Mechanisms of Action of Novel Antitumour Agents. Telomere Targeting Agents (TTAs) Aminoflavone and Benzothiazoles – Aryl hydrocarbon ligand antitumour agents. Targeting Telomeres. Telomeres are repeat sequences of DNA at the end of all chromosomes

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Elucidation of Mechanisms of Action of Novel Antitumour Agents

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  1. Elucidation of Mechanisms of Action of Novel Antitumour Agents

  2. Telomere Targeting Agents (TTAs) Aminoflavone and Benzothiazoles – Aryl hydrocarbon ligand antitumour agents

  3. Targeting Telomeres • Telomeres are repeat sequences of DNA at the end of all chromosomes • Cap chromosomes to prevent DNA damage • Telomere erosionduring cell-division leads to senescence and cell death • Telomerase replaces lost telomeric DNA, rendering cells immortal • NOT expressed in most normal cells • Expressed in 85%of cancer cells TTAGGG Strong scientific rationale for targeting telomeres / telomerase

  4. RHPS4

  5. 2500 2000 1500 1000 500 0 0 0 0 10 10 10 20 20 20 30 30 30 1200 2000 1200 PC3 prostate carcinoma TWI 38% H460 lung carcinoma TWI 39% 800 1500 800 1000 400 400 500 0 0 0 0 10 20 30 0 10 20 30 Days after inoculation RHPS4 - single agent activity 2000 HT29 colon carcinoma TWI 41% CG5 breast carcinoma TWI 87% M14 melanoma TWI 50% 1500 1000 500 2/5 cured 0 Tumor weight (mg) RHPS4 15 mg/kg/day x 15 days (days 6-21 after tumor cell implant i.m.) Control: filled symbols Treated: empty symbols

  6. RHPS4 - in vivo synergy with Taxol UXF 1138LX Xenografts (Serial Fragment Xenograft Model) RHPS4 given as single agent to pretreated (Passage 4) UXF1138LX tumours and in combination with a single bolus injection of taxol (20mg/kg i.v) on Day 1. Taxol given as single agent in UXF1138LX xenograft model (s.c) RHPS4 in combination with Taxol resulted in complete remission of most tumours Phatak et al 2007, Br J Cancer 96(8):1223-33

  7. Effect of RHPS4 Combinations on Cell Survival. M14: CPT HCT 116: CPT HT29: CPT HT29: SN-38 Leonetti C et al. Clin Cancer Res 2008;14:7284-7291

  8. Antitumor efficacy of RHPS4 in combination with Irinotecanon HT29 xenografts. Leonetti C et al. Clin Cancer Res 2008;14:7284-7291

  9. MCF-7 Senescence Induction by RHPS4 (15 d) C 1 µM Cookson et al, 2005; MolPharmacol 68; 1551-1558.

  10. Effect of RHPS4 on telomere length MCF-7 cells treated with RHPS4 (17 d) Average TRF length Control 5.2 kb 0.5 µM 4.7 kb 1 µM 4.3 kb Phatak et al 2007, Br J Cancer 96(8):1223-33

  11. Effect of RHPS4 on telomere length Effect of telomere length on response to RHPS4 Phatak et al 2007, Br J Cancer 96(8):1223-33

  12. Tumours with shorter telomeres are more sensitive to RHPS4 Phatak et all2007, Br J Cancer 96(8):1223-33

  13. Effect of RHPS4 on Telomere Length and Chromosome Architecture Phatak et al 2007, Br J Cancer 96(8):1223-33

  14. Effect of RHPS4 on Telomere Length and Chromosome Architecture Atypical Mitoses Phatak et al 2007, Br J Cancer 96(8):1223-33

  15. RHPS4 induces telomere aberrations. Rizzo A et al. Nucl. Acids Res. 2009;37:5353-5364

  16. RHPS4 induces mitotic bridges. Leonetti et al; 2004: Molecular Pharmacol. 66, 1138-1146.

  17. RHPS4 induces DNA double strand breaks. Leonetti et al; 2004: Molecular Pharmacol. 66, 1138-1146.

  18. RHPS4 causes Senescence and Apoptosis Leonetti et al; 2004: Molecular Pharmacol. 66, 1138-1146.

  19. Click on image to magnify. Dual Mechanism of Action of RHPS4 Phatak and Burger, 2007; Br J Pharmacol, 152, 1003-1011

  20. RHPS4 Stabilises G quadruplex DNA - paralyses telomere Disrupts telomere architecture – uncapping – chromosome fusion Blocks telomerase activity – shortens telomeres Potent in vitro activity - Telomerase IC50 0.33µM Potent in vivo activity Observed at doses 15 mg/kg/day Synergistic activity with marketed drugs, inc. paclitaxel, irinotecan Good selectivity for tumour cells vs. normal cells No evidence of myelotoxicity Cardiovascular side-effects preclude further development – medicinal chemistry programme ongoing to design out unwanted effects

  21. Antitumour Activity of (di) Aminoflavone NSC 686288 • Potent, selective in vitro antitumour activity against breast, renal and ovarian cancer cell lines within NCI panel. Kuffel et al; Molecular Pharmacology 2002; 62: 143-153.

  22. Aminoflavone: Role of Aryl hydrocarbon receptor • Nuclear translocation • XRE-driven luciferase activity Loaiza-Perez et al; Mol Can Ther 2004; 3: 715-725..

  23. Aminoflavone: Role of Cytochrome P450 CYP 1A1 Kuffel et al; MolPharmacol 2002; 62: 143-153.

  24. Aminoflavone: Role of Sulfotransferase SULT 1A1 Meng L et al. Cancer Res 2006;66:9656-9664 Pearson correlations (r) between AF activity (−log10 GI50) and gene expression patterns in the NCI-60 cell lines based on data from the Affymetrix HG-U133A chip.

  25. Aminoflavone: Role of Sulfotransferase SULT 1A1 Meng L et al. Cancer Res 2006;66:9656-9664

  26. Aminoflavone: Role of Sulfotransferase SULT 1A1 Meng L et al. Cancer Res 2006;66:9656-9664 AhR AF-N-OH AF-N-O-SO3- AF-NH+ Cell Death DNA DSBs (γH2AX) DNA-/Protein-AF Adducts CYP SULT1A1

  27. 1995 1998 2000 AntitumourBenzothiazoles

  28. 5F 203 Phortress Lung Ovarian Renal Breast * * * NCI GI50 mean graphs Colon cancer Ovarian cancer Renal cancer Breast cancer

  29. MCF-7MDA-MB-435 Control ● ○ 5F 203 (d 0-3) ■ □ In vivoantitumour activity of 5F 203 5mg/kg-1 5F 203 i.p.d 0-3

  30. IGROV-1 Optimum T/C Phortress 25* 48* 12*/12* 47* 67 62 39*+/42*+ 49* 65* Doxorubicin 12* 42* 9* 36* 81 61 79 10* 46* ◯ Control ■ 20 mg/kg d 0,7 ● 20 mg/kg, d 0 □ 4 mg/kg d 0-4 In vivo antitumour activity of Phortress MCF-7 Breast Xenograft 4296 4151 4134 3366 3366/Tam 4586 MCF-7 4898 4000 Fichtneretal; Breast Can Res and Treatment: 87, 97-107, 2004

  31. Control TBDD 10 nM DF 203 1μM 5F 203 1μM Phortress: Role of Aryl hydrocarbon receptor

  32. MCF-7 MDA-MB-435 Control 5F 203 1mM Phortress: Role of Aryl hydrocarbon receptor

  33. L AhR ARNT XRE XRE activation following exposure to 5F 203 Cyp 1a1 transcription

  34. Phortress: Role of cytochrome P450 CYP 1A1 A. MCF-7 B. HCT 116 Western blot analyses following 24 hr exposure of cells to 1 μMfluorinated analogsof DF 203 demonstrated selective induction of CYP1A1 protein in sensitive MCF-7 cells only.

  35. +C -C 4F 5F 6F 7F 5,6F 5Flys CYP1A1 MCF-7 CYP1A1 TK-10 MDA-MB-435 CYP1A1 Phortress: Role of cytochrome P450 CYP 1A1 I Hutchinson et al., J. Med. Chem., 2001, 44, 1446-1455

  36. Induction of DNA adducts by 5F 203 in MCF-7 cells Control 1 μM 10 μM

  37. Generation of Single and Double DNA strand breaks Alkaline (SSB) Neutral (DSB)

  38. Understood mechanism of action of Phortress 5F 203

  39. Phortress: Role of Cytochrome P450s 2W1 and 2S1 • CYP 2W1overexpression has been reported in cancer, including approximately 50% of human colon tumours. It is able to metabolise arachidonic acid and catalyse activation of procarcinogens • CYP 2S1 expressed extrahepatically in epithelial cells of the respiratory and gastrointestinal tract. This P450 metabolises endogenous and exogenous substrates such as retinoic acid and aromatic hydrocarbons. It is also involved in metabolism of topical drugs, mediating response to phytochemotherapyin psoriasis. Induction is regulated by AhR and ARNT. Increased expression in primary colorectal cancer associated with poor prognosis.

  40. Phortress: Role of Cytochrome P450 2S1 • CYP2S1 – Detoxification of AntitumourBenzothiazoles

  41. Phortress: Role of Cytochrome P450 2W1 5F 203 IC50 (µM;48 h) MCF-7 Vector 2W1 si(i) 2W1 si(ii) 0.4 7.5 8.0

  42. Phortress: Role of Cytochrome P450 2W1 5F 203 IC50 (µM;48 h) MCF-7 Vector 2W1 si(i) 2W1 si(ii) 0.4 7.5 8.0 GW 610 • CYP2W1 – Bioactivation of AntitumourBenzothiazoles

  43. Angelika Burger 1965 - 2011 Dedicated Scientist Good friend

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