Serum Concentration

1. Serum Concentration • HCT116 cells • NCI cultured in 5% FBS • Nottingham cultured in 10% FBS • Cultured in 5% for 1 week prior to experiment • Drug dilutions carried out in appropriate FBS concentration.

2. Serum Concentration

3. Treatment Length • HCT116 cells • Same seeding density • 72 or 48 h MTT assay • Time dependent activity • Previously shown for PMX 464 by A. Seaton

4. Thioredoxin/Thioredoxin Reductase Insulin Reduction Assay – PMX 18034

5. Thioredoxin/Thioredoxin Reductase Insulin Reduction Assay PMX 290, PMX 2058 and MESNA

6. Thioredoxin/Thioredoxin Reductase Insulin Reduction Assay • Carry out further control experiments to try and explain inactivity in full Trx/TR insulin reduction assay • Alternative methods to ensure back-up screening does not miss inhibitors • Insulin reduction assay (EHC method) • Add substrate last rather than NADPH as inhibition of TR was dependent on presence of NADPH • Use rate of reaction rather than final OD • Requires 37 ºC • Direct TR/DTNB assay in parallel • Assess activity as well as expression levels in a panel of cell lines. • colon, breast, melanoma, pancreas

7. Resistant Cells • HCT116 PMX 290 resistant cells • Growing in 0.8 mM (> 4 x GI50) • HCT116 Standard Therapy Resistant Cells • Irinotecan: currently growing in 0.85 mM (~ 1.7 x GI50) • 5-FU: currently recovering in drug free medium having stalled at 35 mM • IGROV1 Cisplatin resisiatant cells grown in 100 mM cisplatin • Not very resistant to cisplatin: IGROV1 GI50 = 3 mM • IGROV1 CISR GI50 = 5 mM • Remain sensitive to PMX 290

8. Phosphatase Inhibition • Phosphatase activity in whole cell lysates of MCF7 and HCT116 cells • 1h treatment at 1 mM • 4-nitrophenyl phosphate as phosphatase substrate • yellow product