Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

Lawrence G. Iben,Barbara J. Robertson,Lynn A. Balanda,C.V.C. Prasad,Sukhanya Jayachandra,Charles F. Albright,Vanessa Hay,Jeremy H. Toyn,John Corradi,Robert Zaczek,Richard E. Olson

doi:10.1074/jbc.m707002200

Lawrence G. Iben, Barbara J. Robertson + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m707002200

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Abstract

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.

Highlights

Summary of IC50 values for inhibition of radioligand binding The means Ϯ S.E. are given for a minimum of three independent experiments
332,000 Ϯ 12,000 a Inhibition of3HL-685,458 binding in SPP-overexpressing HEK293 cell homogenate. b Inhibition of3HL-685,458 binding in non-transfected THP-1 cell homogenate. c Inhibition of3HIN973 binding in non-transfected THP-1 cell homogenate
Calculate dissociation constants; to avoid assumptions about the multiple different potential mechanisms represented by this group of compounds, the results of this experiment are presented as IC50 curves (Fig. 2)

Summary

SPP Binding Pharmacology

A variety of small molecules target both presenilin and SPP, but it has not been determined to what extent the inhibitor binding pharmacology is similar for the two enzymes. To address this question, we utilized an active site-directed ␥-secretase radioligand [17, 18, 37] and took advantage of our finding that it binds with high affinity to both SPP and ␥-secretase. In the course of this study, we found that SPP family protein binding is abundant in cell homogenates, presenting a challenge to the evaluation of ␥-secretase and SPP binding under these conditions Using this approach, we show that ␥-secretase inhibitors exhibit equivalent small molecule binding pharmacology for SPP, implying conserved structure and mechanism in the SPP/presenilin protein family

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION