Figure 6.

The small molecule NSM00191 specifically disrupted the interaction between TRADD and TRAF2. TRADD interacted with TRAF2 in yeast cells. Different combinations of plasmids, including pGADT7-TRADD + pGBKT7-TRAF2; pGADT7-TRADD + pGBKT7; and pGADT7 + pGBKT7-TRAF2, were co-transformed into BY4741 cells. Cell growth was determined in media with different nutrient deficiencies, including the lack of Trp or Leu (SC-TL) (top panel), the lack of Trp, Leu and His (SC-HTL) (middle panel), and SC-HTL supplemented with 5 mM 3-amino-1,2,4-trizole (3-AT). Columns in each panel represent the serial decimal dilutions. (B) The protein levels of TRADD and TRAF2 in yeast cells. A Western blot was performed to examine the protein levels of TRADD and TRAF2 in yeast cells used in (A). (C) Schematic representations of screening small molecules that disrupt the TRADD-TRAF2 interaction. The yeast cells harboring pGADT7-TRADD + pGBKT7-TRAF2 were subjected to HTS in the SC-HTL+5 mM 3'-AT medium supplemented with an individual compound in each well. Small molecules that significantly inhibited cell growth (OD₆₀₀<0.2) were selected. The chemical structure of NSM00191 is shown. (D) NSM00191 significantly inhibited SW982 and HFLS-RA cell proliferation. The HFLS, HFLS-RA and SW982 cells were treated with different concentrations (0, 10, 20, 30, 40 and 50 μM) of NSM00191 for 48 hr. Cell viability was determined at 490 nm. (E) NSM00191 specifically disrupted the TRADD-TRAF2 interaction in HFLS-RA and SW982 cells. The HFLS-RA and SW982 cells were first treated with DMSO (lanes 1 and 4), 10 μM NSM00191 (lanes 2 and 5), or 20 μM NSM00191 (lanes 3 and 6), followed by the detection of TNFR1, TRADD, TRAF2 and p65 by Western blot. GAPDH was used as a loading control.