Discovery and characterization of novel small-molecule inhibitors targeting nicotinamide phosphoribosyltransferase

Nicotinamide phosphoribosyltransferase (NAMPT) is a promising anticancer target. Using high throughput screening system targeting NAMPT, we obtained a potent NAMPT inhibitor MS0 (China Patent ZL201110447488.9) with excellent in vitro activity (IC50 = 9.87 ± 1.15nM) and anti-proliferative activity against multiple human cancer cell lines including stem-like cancer cells. Structure-activity relationship studies yielded several highly effective analogues. These inhibitors specifically bound NAMPT, rather than downstream NMNAT. We provided the first chemical case using cellular thermal shift assay to explain the difference between in vitro and cellular activity; MS7 showed best in vitro activity (IC50 = 0.93 ± 0.29 nM) but worst cellular activity due to poor target engagement in living cells. Site-directed mutagenesis studies identified important residues for NAMPT catalytic activity and inhibitor binding. The present findings contribute to deep understanding the action mode of NAMPT inhibitors and future development of NAMPT inhibitors as anticancer agents.

The purity of the protein was more than 90% determined by SDS-PAGE and Coomassie Blue (Tiangen, China) staining and further by western blot analysis (Fig. S1).

High throughput screening (HTS)
HTS was performed using our previously reported method 3  The relative enzyme activity (Activity%) regulated by specific compound was calculated according to equation (1): F 0 was the averaged fluorescence of six background controls, representing zero activity from a simulated enzyme reaction with only NAMPT but no NAM and compound; F 100% was the averaged fluorescence of six reference controls, representing 100% activity from intact enzyme reaction without compound perturbation.
Compounds with Activity% less than 40% were considered as inhibitors and subjected to a secondary screen inhibition validation, in which another background control (F C0 ), a simulated enzyme reaction with NAMPT and compound but no NAM, was introduced to eliminate the direct and/or indirect influence from compound. At this stage, the Activity% was calculated according to equation (2): In the screening, the signal-to-noise (S/N) ratio was calculated using the equation: (Mean signal -Mean background ) / SD background 4 . Coefficients of variation (CV) were the ratio of SD to mean. The Z' factor was determined by equation (3)

Determination of IC 50 for NAMPT inhibitors
To determine the IC 50 of inhibitors, 5 μl compound solutions (containing 10% DMSO) with various concentrations were added into 96-well plate. The plate was incubated at 37 °C for 5 min after addition of 16.5 μl reaction buffer containing NAMPT. The enzyme reactions were initiated by 4.5 μl NAM (1.11 μM) following NMN measurement as described above. The IC 50 values were determined by non-linear fitting of the concentration-dependent curves with the four-parameter IC 50 logistic equation.

Cell counting kit-8 (CCK-8) assay
Cell viability was determined by our previous method 9
Briefly, cells were seeded into 96-well plates, cultured overnight, and treated with corresponding compounds for 72 h. Cells were then fixed with 10% trichloroacetic acid and stained with sulforhodamine B (Sigma). Sulforhodamine B in the cells was dissolved in 10 mmol/L Tris-HCl and was measured at 515 nm using a multiwell spectrophotometer (VERSAmax, Molecular Devices, Sunnyvale, CA). The inhibition rate on cell proliferation was calculated for each well as (A515 control cells -A515 treated cells )/A515 control cells ×100% (A515: OD value at 515 nm). The average IC 50 values were determined by Logit method from at least three independent tests.

Cell culture and in vitro experiments
Human atmosphere of 95% air plus 5% CO 2 at 37 ℃, as our previously reported 8,13 .

Isothermal titration calorimetry (ITC)
Thermodynamic parameters of small molecule binding to protein were determined using a MicroCal VP-ITC calorimeter 14  Measurements were performed at 25°C with spacing of 90 s between injections. Background signal (calculated as a mean value) generated by addition of MS0 to buffer was subtracted prior to analysis on Origin 7.5 using software supplied by the manufacturer.

Cellular thermal shift assay (CETSA)
CESTA was performed as previously described 15  were aliquoted into 0.2 mL PCR microtubes, and excess PBS was removed by centrifugation to leave 10 uL or less PBS in each microtube. These cell pellets were heated as previously described and lysed using 2 cycles of freeze-thawing with liquid nitrogen. The soluble fractions were isolated and analyzed by Western blot analysis as described above.

SDS-PAGE and Western blot analysis
Target proteins in CESTA were examined by SDS-PAGE and immunoblotting as previously described 8,13,15,16 . The primary antibodies for Western blotting were specific for the following: NAMPT (Santa Cruz, sc-67020, used at 1:500), HDAC1 (Cell Signaling, 5356, used at