Novel sulfonamides exhibit properties in common with classic gamma-secretase inhibitors. Western blots of cell extracts (a) and media (b, c) after treatment of cultures in duplicate of with gamma-secretase inhibitors indicated above the lanes. Compounds were tested at two doses, approximately 1 X ED50 and approximately 10 X ED50 based on cellular assay results. (a) Full length and c-terminal fragments of APP total cellular lysates recognized by an antibody against the C-terminus of APP (Sigma A8717). The reference gamma-secretase inhibitors L-685,458 and ELN46719 (analog of LY411575, see methods), as well as the novel sulfonamides tested stabilize both α-CTF and β-CTF in a dose-dependent manner, and none of the compounds significantly affect steady-state levels of full-length APP. (b, c) Western blots of the conditioned media samples from this same experiment probed with antibody 8E5 against total secreted APP (b), and 192Sw against the β-secreted APP formed by BACE cleavage of the Swedish mutant APP over-expressed in these HEK293 cells (c). No significant effect on either total sAPPα or sAPPβ was seen by any of the gamma-secretase inhibitors. (d) Quantification of Aβ1-40 in the conditioned media. The results suggest that the sulfonamide only affects gamma-secretase cleavage of APP, and does not appear to have nonspecific effects on overall APP metabolism or cell viability in HEK293 cells.