Figure 3

Binding site analysis by competitive displacement of biotinylated active site isostere. A competitive binding assay employing a biotin conjugated active site isostere to capture gamma secretase from a partially purified cell extract, plus or minus co-incubation with increasing concentrations of reference or test gamma secretase inhibitors, was developed to characterize inhibitor binding site. The eluted enzyme captured on neutravidin beads was detected on western blots with antibodies recognizing either PS-1 amino-terminal fragment (PS1-NTF), or Nicastrin (Nct). The binding assay was carried out using 50 nM affinity probe together with different concentrations of competing compounds. Serial dilutions of affinity captured enzyme were included on the gels to provide a standard curve for densitometric quantitation of test samples following autoradiography of the western blots. (a) The biotinylated isostere is displaced by its non-biotinylated analog L-685,458 in a concentration dependent manner. In subsequent experiments, a 200-fold excess of L-685,458 was employed as a positive control. (b) LY411575 was tested for its ability to displace active site isostere at concentrations ranging up to 1000-fold its IC₅₀ in the Gamma APP enzyme assay. There is no significant displacement of the active site directed compound by at concentrations below 200-fold its IC₅₀ in the enzyme assay. At higher concentrations, a modest dose-dependent effect of LY411575 to displace the active-site-directed compound is observed on both Western blots. Previous observations revealed substrate concentration affects the potency of sulfonamides in cell and enzyme assays (unpublished). Hence, the ability of sulfonamides to displace the active site directed probe was tested in the absence (c and d, left side) or presence (c and d, right side) of 1 Km substrate (MBP-C125sw). Substrate was added to enzyme concurrently with test compound and affinity ligand. In the presence of added APP, the sulfonamides displace the active site probe in a dose-dependent manner. (c) No displacement of active site probe is observed by ELN-318463 in the absence of substrate. In the presence of substrate, ELN318463 displaces approximately 50% of the active site probe at a concentration of approximately 2,000-fold its IC₅₀ in the Gamma APP assay. (d) ELN-475516 does not displace the active site probe at concentrations ranging up to 2,000X its IC₅₀ in the gammaAPP assay. However, in the presence of substrate, ELN475516 displaces 50% of the active site probe at a concentration equivalent to approximately 67-fold its Gamma APP IC₅₀. The results shown in (c) and (d) suggest displacement of active site isostere from gamma-secretase by benzene caprolactam and fused pyrazolo-bicyclic sulfonamide is influenced by the presence of substrate.