S 17092-1, a Highly Potent, Specific and Cell Permeant Inhibitor of Human Proline Endopeptidase He ´le `ne Barelli,* Agne `s Petit,* Etienne Hirsch,† Sherwin Wilk,‡ Guillaume De Nanteuil,§ Philippe Morain,§ and Fre ´de ´ric Checler* ,1 *IPMC du CNRS, UPR411, 660 Route des Lucioles, 06560 Valbonne, France; †Hopital Pitie ´-Salpe ˆtrie `re, Paris, France; ‡Mount Sinai School of Medicine, New York, New York; and §Institut de Recherches Servier, Courbevoie, France Received January 22, 1999 Several lines of evidence indicate that proline endo- peptidase (PE) could participate to the symptomatology and/or etiology of Alzheimer’s disease. Thus, proline en- dopeptidase appears to contribute to the degradation of neuropeptides involved in learning and memory and could also control the production of the amyloidogenic peptide A. Therefore the design of potent, selective and permeant inhibitors of human PE should lead to poten- tial probes to assess the genuine contribution of this enzyme in Alzheimer’s pathology. A novel perhydroindol carboxylic derivative, S17092-1 inhibits the hydrolysis of Z-Gly-Pro-7AMC-hydrolysing activity present in human brain nuclei with a high affinity (Ki  1 nM) and behaves as a highly potent (Ki  1.5 nM) inhibitor of partially purified human PE. By contrast, S17092-1 is unable to affect a series of other peptidases including aminopep- tidases B and M, dipeptidylaminopeptidase IV, endo- peptidases 3.4.24.11, 3.4.24.15, 3.4.24.16, calpains and angiotensin-converting enzyme. Furthermore, we show that the embryonic human kidney 293 cell line displays an intracellular PE-like activity that is blocked after preincubating cells with S17092-1, indicating that this inhibitor penetrates in HEK293 cells and could affect intracellular human PE. Altogether, we establish that S17092-1 behaves as a highly potent, specific and cell permeant inhibitor of human proline endopeptidase and can be seen as a probe to examine PE contribution in Alzheimer’s disease. © 1999 Academic Press One of the main cortical stigmate observed in Alz- heimer’s disease (AD) neuropathology is the senile plaque (1), a proteinaceous deposit mainly composed of an insoluble amyloid  peptide (A). A derives from the -amyloid precursor protein (APP) by a combined proteolytic attack triggered by - and -secretases (for review see (2)). The amyloidogenic hypothesis of AD considers the overproduction of A as a central event responsible for subsequent neurodegenerative process. Even if it is not the primary etiological cause, it re- mains that A derives from uncircumventable proteolytic cleavages, therefore suggesting - and/or -secretases as potential therapeutic targets. Besides neuroanatomical lesions, several neuro- chemical alterations have been established in AD brains. They concern the cholinergic neurotransmis- sion (3) but also neuropeptidergic signals (4) which could account for part of the AD symptomatology, and particularly the memory loss. Thus the content of sev- eral neuropeptides reported as memory and learning enhancers are reduced in cortical areas of AD brains (4). Here again, by analogy with the anticholinesterase therapy aimed at protecting endogenous acetylcholine, one can envision to protect neuropeptides from degra- dation with peptidase inhibitors, thereby reducing the neuropeptide-related mnemocognitive deficit. Proline endopeptidase (PE) is a serine endooligopep- tidase involved in the degradation of various neuropep- tides, in vitro and in vivo (for review (5)). This enzyme was also suggested as a putative -secretase, based on the cleavage of synthetic fluorimetric substrates mim- icking the sequence encompassing the site targeted by -secretase (6,7). Accordingly, specific PE inhibitors abolish the formation of A in NG108-15 cells (7) and prevent the amyloid deposition in a mouse model of accelerated senescence (8). Finally, it has been re- ported that the post mortem levels of human PE were affected in AD brains (9,10)). The above data suggest that PE could contribute to both symptomatology and etiology of Alzheimer’s disease, and therefore, could be seen as a potential therapeutic target. In this context, we report here on the characterization of a novel com- pound, S17092-1 that displays high affinity and selec- tivity towards human proline endopeptidase. Further- more, we document the bioavailability of this inhibitor that is cell permeant and blocks endogenous intracel- 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (33) 4 93 95 77 08. E-mail: checler@ipmc.cnrs.fr. Biochemical and Biophysical Research Communications 257, 657– 661 (1999) Article ID bbrc.1999.0366, available online at http://www.idealibrary.com on 657 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.