STF-118804

The enzymatic activities of NAMPT and NMNAT are measured using in vitro kits and using the Two-Step Method per manufacturer’s instructions. Compounds, NAMPT and/or NMNAT enzymes, and their substrates are mixed and incubated at 30°C for 1 hour. Reagents for the indicator reaction (Wst-1) are then added, and absorbance is read at 450 nm every 5 min at 30°C on a Tecan Infinite M100 multimode plate reader.

Human cell lines (SEM, MV411, Nalm6, 697, Hal01, REH, SUP-B15 and KP-Y-RL) and primary patient leukemic samples

~10 μM

72 hours

Human cell lines or lineage negative cord blood cells were seeded into 96-well plates (6×10⁵ cells per milliliter). Compounds are added in increasing concentrations, and cells are incubated at 37°C/5% CO₂ for 72 hours. To detect viability, CellTiter-Blue reagent is added at 1:10 dilution, and plates are incubated for 4 hours at 37°C/5% CO₂ prior to reading on a Flexstation II 384 or a Synergy H1 reader at an excitation of 555 nm and emission detection of 590 nm. Cell viability is also measured by CellTiter-Fluor. The cell-permeable fluorogenic peptide substrate GF-AFC reagent is added at 1:2 dilution, and plates are incubated for 30 min at 37°C/5% CO₂ prior to reading on a Synergy H1 reader at an excitation of 380 nm and emission detection of 505 nm. Cord blood cells are enumerated on a hemocytometer, and cell viability is assessed with trypan blue exclusion dye. Inhibitory concentration (IC50) is calculated using Prism software. Primary patient samples are plated in 96-well plates and treated with increasing concentrations of STF-118804 for 48 hours at 37°C in 5% CO₂. WST-1 reagent is added to the culture medium (1:10 dilution), and absorbance is measured at 450 nm using a Bio-Rad model 680 microplate reader. All assays are performed in triplicate. IC50 is calculated using CalcuSyn version 2.0 software.

Orthotopic xenograft model of ALL transplanted with MV411 cells

25 mg/kg twice daily

s.c.