Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors

The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid beta 1-25 (A beta(1-25)) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple gamma-secretase inhibitors (GSIs) and (Z-LL)(2) ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51 +/- 79 nM (on SPPL2a) to 5499 +/- 122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465 +/- 93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by gamma-secretase.