Fig. 2.

The primary HTS for DNPEP inhibitors. (A) DNPEP inhibitors identified in a small-scale Asp-AMC hydrolysis screen with predicted high affinity for the DNPEP active site. These compounds were used as search seeds to identify related compounds in the 350,000 UC chemical library with potentially greater efficacy and potency. This search yielded 266 compounds that were added to the 25,000 UC Diversity Set for the HTS. (B) Sorted activity plot of the 26,368 compounds from the 25,000 UC Diversity Set (black dots) and virtual screen (red dots) in the Asp-AMC hydrolysis assay. Each compound was tested at 12.3 µM. The inset table summarizes the primary HTS results. (C) Sorted activity plot of 586 hits, including 367 DNPEP inhibitors and 219 activators identified in the primary HTS, tested in triplicate at 12.3 µM in the Asp-AMC hydrolysis assay. Mean activity score (Mean) and S.D. (residual) are shown as black and red dots, respectively. Results are summarized in the inset table. (D) A sorted EC₅₀ (i.e., IC₅₀ and AC₅₀ for inhibitors and activators) plot of 285 hits, including 282 DNPEP inhibitors and 3 activators, confirmed in the dose-response Asp-AMC hydrolysis screen. Summarized results are presented in the inset table.