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Quinols - Discovery Program

Quinols - Discovery Program. Initiated 1998 - novel structures via oxidation of interesting phenols Rigorous antitumour profiling of resulting new chemical entities Lead compounds identified from 2 novel chemical series (AW 464, AJM 290). In Vitro Antitumour Activity.

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Quinols - Discovery Program

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  1. Quinols - Discovery Program • Initiated 1998 - novel structures via oxidation of interesting phenols • Rigorous antitumour profiling of resulting new chemical entities • Lead compounds identified from 2 novel chemical series (AW 464, AJM 290)

  2. In Vitro Antitumour Activity NCI mean LC50 graphs: PMX 464AJM 290 Colon Melanoma Renal Renal Wells et al, J Med Chem 46: 532-541, 2003

  3. PMX 464 and AJM 290– Potent In Vitro Anti-tumour Activity Renal, colon, breast and melanoma cell lines • AW 464 GI50≤ 300 nM • AJM 290 GI50≤ 100 nM HCT 116 and HT29 MTX-resistant cell lines (GI50 > 100 μM cf < 100 nM wt) • AW 464 GI50 < 400 nM • AJM 290 GI50 < 300 nM HCT 116 Cisplatin-resistant cell line (GI5063 - 430µM) • AW 464 GI50< 200 nM • AJM 290 GI50< 50 nM HCT 116Doxorubicin resistant cell line (GI50 70 nM – 5.33 μM) • AW 464 GI50< 250 nM • AJM 290 GI50 < 50 nM

  4. PMX 464 In Vivo Antitumour Activity • Significant growth inhibition in the highly angiogenic renal hypernephroma xenograft RXF 944XL model. A) 15 mg/kg i.p d 1, 8 B) 15 mg/kg i.p d 1, 2 NSC 706704 = AW 464

  5. Comparative efficacy of AJM 290 and PMX 464 against HCT 116 colon xenograft growth. Treatment qd 6-10 * Significant p < .05

  6. Comparative efficacy of AJM 290 and PMX 464 against MDA-MB-435 melanoma xenograft growth. Treatment qd 6-10 * Significant p < .05

  7. PMX 464 -Mechanism(s) of Action: • Dose-dependent inhibition of Thioredoxin signalling • Perturbation of Redox homeostasis • Inhibition of NF-kB activation • Inhibition of HIF signal transduction • Inhibition of angiogenesis • Promotion of apoptosis • Inhibition of tNOX (nM EC50) • Inhibition of PDI • Peroxiredoxin (potential target)

  8. NF-KB, HIF-1a Mechanisms of antitumour activity ASK-1

  9. Binding between PMX 464 and Thioredoxin active site

  10. MS - Human Thioredoxin

  11. Trx + 3 molecules of AW 464 Trx + 4 molecules of AW 464 Trx + 5 molecules of AW 464 MS - PMX 464 Bound to Human Thioredoxin

  12. Trypsin Digestion Specifically cleaves proteins on the carboxy terminal side of Arginine (R) and Lysine (K) residues. PeptidesAmino acid sequence 1-3 MVK 4-8 QIESK 9-21 TAFQEALDAAGDK 22-36 LVVVDFSATWCGPCK 37-48 MIKPFFHSLSEK 49-72 YSNVIFLEVDVDDCQDVASECEVK 73-81 CMPTFQAFFK 82 K 83-65 QVK 86-94 VGEFSGANK 95-96 EK 97-105 LEATINELV

  13. □ 1 M ■ 10  M Trx CDK-1 TR Actin Dose-dependent up-regulation of TR expression and perturbation of TR/Trx signalling

  14. Enzymatic assay of FICIN: cysteine endopeptidase of papain family. Active site contains an essential cysteine sulfhydryl and histidine imidazole unit: cys25and his159 E64: epoxysuccinylleucylamido(4-guanidino)butane – an irreversible inhibitor of cysteine proteases.

  15. Generation of reactive oxygen species (ROS) 1 mM, 3 – 7 h, HCT116 Cell Number - Enhanced potency (10-fold) following depletion of intracellular GSH H2DCFDA fluorescence PMX 464 –Modulation of REDOX regulation

  16. 0 . 3 0 . 2 0 . 1 0 . 0 PMX 464 – Inhibition of HIF signal transduction ● Inhibition of HRE-HIF-1αDNA binding Cos 7/CoCl2 MDA468 hypoxia MDA468 normoxia MDA468 hypoxia AW464 10M MDA468 hypoxia AJM290 10M Cos 7/CoCl2+Competitor Oligo 500ng MDA468 hypoxia +Competitor Oligo 500ng

  17. PMX 464 -Inhibition of HIF signal transduction Inhibition of hypoxia-induced gene transactivation: Dose-dependent down-regulation of BNIP3 mRNA by: A) AW 464 B) AJM 290

  18. μM RCC4 + VHL N Hypoxia AJM290 AW464 C C 1 2.5 10 1 2.5 10 (μM) AJM 290 AW 464 CA-IX BNIP3 PMX 464 -Inhibition of HIF signal transduction • Dose-dependent down regulation of HIF-mediated signal transduction: CA-IX protein expression: MDA 468 ER- mammary carcinoma cells under hypoxia and normoxia • BNIP3 protein expression: MDA 468 cells under hypoxia and normoxia • RCC4 renal carcinoma cells (mutant VHL) • RCC4 cells + VHL transfectant under normoxia and hypoxia

  19. PMX 464 –Anti-angiogenic activity • Dose-dependent inhibition VEGF production AW 464 • Inhibition of human umbilical cord vein endothelial cell proliferation (IC50 500 nM) • Inhibition of endothelial cell differentiation (tubal abortion 500 nM, Matrigel™ model)

  20. PMX 464 -Mechanism(s) of Action: Inhibition of NF-KB • Unstimulated state - IKB tightly bound to NF-KB (p65 nuclear localisation domain) • Release of IKB and activation of NF-KB • IKB phosphorylation by the IKK complex (IKKα and IKKβ) • or reduction of NF-kB (cys 62 p50) catalysed by thioredoxin • p50 and p65 subunits of NF-KB translocate to nucleus, bind DNA and initiate NF-KB-dependent transactivation. IKB IKB p50 p65

  21. IKB p50 p65 p50 p65 P P Ub-Ub-Ub IKK * * P50 p65 * IKB p50 p65 ** Trx IKB PMX 464 -Mechanism(s) of Action: Inhibition of NF-KB • Unstimulated state - IKB tightly bound to NF-KB (p65 nuclear localisation domain) • Release of IKB and activation of NF-KB • IKB phosphorylation by the IKK complex (IKKα and IKKβ) • or reduction of NF-kB (cys 62 p50) catalysed by thioredoxin • p50 and p65 subunits of NF-KB translocate to nucleus, bind DNA and initiate NF-KB-dependent transactivation. In addition: phosphorylation of IKB-α Tyr 42 – role for peroxiredoxin in NF-KB activation

  22. IL-1β alone B PMX 464 + IL-1β C Inhibition of NF-KB activation by PMX 464 • In A549 cells AW 464 (30 μM) inhibits: • IKK Activity (A) • IKB Phosphorylation - cells stimulated with IL-1 alone (B) or pre-incubated with PMX 464 prior to IL-1 stimulation (C) and subsequent degradation

  23. E F Inhibition of NF-KB activation by PMX 464 D • In A549 cells AW 464 (30 μM) inhibits: • Nuclear translocation of p65 (D) and p50 • NF-KB DNA Binding (E) • NF-KB-induced gene transactivation (F)

  24. whole cell lysates IP: Trx  ASK-1  Trx DMSO 1 DMSO 1 DMSO 2 DMSO 2 5μM PMX 290 5μM PMX 290 5μM PMX 464 5μM PMX 464 ●Dissociation of ASK-1 from Trx upon treatment of cells with PMX 464

  25.  p54  p54 phospho-JNK  p46  p46 total JNK actin DMSO 0.5 1 5 10 0 5 30 60 180 180 min DMSO 3h AW 464 (mM) AW 464 (5mM) ●Dissociation of ASK-1 from Trx upon treatment of cells with PMX 464 ●Dose and time-dependent phosphoryation of Jnk

  26. Propidium Iodide CDK-1 Actin Annexin V-FITC C 1 3 6 PMX 464 (μM) PARP Cleaved PARP Caspase 3 DMSO AW 46424 h PMX 464 –Induction of Apoptosis 12-16 h 1 μM PMX 464 G2/M block, preG1 population 1 μM ≥16 h in breast and colon cell lines Dose-dependent CDK-1 down-regulation 3h

  27. PMX 464 -Mechanism(s) of Action: • Dose-dependent inhibition of Thioredoxin signalling • Perturbation of Redox homeostasis • Inhibition of NF-kB activation • Inhibition of HIF signal transduction • Inhibition of angiogenesis • Promotion of apoptosis • Inhibition of tNOX (nM EC50) • Inhibition of PDI • Peroxiredoxin (other potential targets)

  28. Tumour-associated NADH oxidase - tNOX • Cancer-specific, growth related cell surface protein • Protein disulfide-thiol interchange and hydroquinone (NADH) oxidase activities • 34 KDa protein shed into the circulation, forms amyloid filaments • tNOX present - on surface of solid tumours • - on cancer cell lines in culture • - in culture media conditioned by cancer cells • - in sera of cancer patients

  29. 1: 2 + EDAC 2: EDAC 3. 3 + EDAC Coomassie blue stain 2 3 Marker MW (kDa) 1 175 83 62 1 2 47.5 3 4 5 32.5 25 6 16.5 Identification of cellular proteins as molecular targets of quinols Quinol was immobilised onto aminoalkyl agarose beads and the beads were incubated with HCT 116 cell lysates. Bound proteins were separated by SDS-PAGE and the gel was coomassie stained. Gel bands commonly identified in repeated gels were excised, in-gel digested and subjected to MALDI-TOF analysis. Proteins identified in lane 3 of the gel shown were 1: HSP60, 2: β-tubulin, 3: EF-1-γ, 4: β-actin, 5: 40S ribosomal protein, 6: Prx1. Lane 1: negative control, where agarose beads were pretreated with compound 2 and EDAC. Lane 2: negative control, where agarose beads were drug-uncoupled.

  30. quinol EDAC HSP60-V5 - + + + + + WB: V5 HSP60-V5 quinol EDAC Prx1-Flag - + + + + + WB: Flag Prx1-Flag Identification of cellular proteins as molecular targets of quinols Whole cell lysates of HCT 116 cells expressing HSP60-V5 were incubated with beads coupled or uncoupled to quinol. Western blot analysis using anti-V5 antibody only detected presence of HSP60-V5 bound to quinol-coupled beads. Whole cell lysates of HCT 116 cells expressing Prx1-Flag were incubated with beads coupled or uncoupled to quinol. Western blot analysis using anti-Flag antibody only detected presence of Prx1-Flag bound to quinol-coupled beads.

  31. PMX 464: Pharmacokinetics in Rat - IV Vehicle: 10% w/v Captisol No. rats: 2 t½ 0.3h t½ 2.4h Vd 10.2 L/kg Cl 2.9 L/h/kg

  32. PMX 464: Pharmacokinetics in Rat - Oral • Bioavailability: 20% @ 10 mg/kg; 23% @ 50 mg/kg • No significant inhibition of key drug metabolising enzymes • no risk alert with regard to drug-drug interactions • No pharmacological alerts from receptor screening

  33. PMX 464 – IV Acute Toxicology • Rat (male and female; Group size: 6; Vehicle: 20% Captisol) • NOAEL: 40mg/kg • MTD: < 50mg/kg • Mortality: 1 male death (1 day post dosing) • Clinical Obs:Hunched posture, occasional body tremors and piloerection. Reversible 2 – 4 days post dosing including expected increases in body weight gain. • Necropsy: No macroscopic abnormalities • Mouse (male and female; Group size: 6; Vehicle: 20% Captisol) • NOAEL: 75mg/kg • MTD: < 85mg/kg • Mortality: No deaths • Clinical Obs:Hunched posture, occasional body tremors and ptosis. Reversible 1 – 2 days post dosing. • Necropsy: No macroscopic abnormalities

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