Articles L-685,458, an Aspartyl Protease Transition State Mimic, Is a Potent Inhibitor of Amyloid -Protein Precursor γ-Secretase Activity Mark S. Shearman,* ,‡ Dirk Beher, ‡ Earl E. Clarke, ‡ Huw D. Lewis, ‡ Tim Harrison, § Peter Hunt, § Alan Nadin, § Adrian L. Smith, § Graeme Stevenson, § and Jose ´ L. Castro § Departments of Molecular Biology and Medicinal Chemistry, Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, England ReceiVed March 9, 2000; ReVised Manuscript ReceiVed May 8, 2000 ABSTRACT: Progressive cerebral amyloid -protein (A) deposition is believed to play a central role in the pathogenesis of Alzheimer’s disease (AD). Elevated levels of A(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid -protein precursor (APP) and the presenilins. Sequential cleavage of APP by the - and γ-secretases generates the N- and C-termini of the A peptide, making both the - and γ-secretase enzymes potential therapeutic targets for AD. The identity of the APP γ-secretase and the mechanism by which the C-termini of A are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving γ-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of APP γ-secretase activity, with a similar potency for inhibition of A(42) and A(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of APP γ-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease. Amyloid -protein (A) 1 is the main constituent of the abundant neuritic plaques that are a characteristic hallmark of Alzheimer’s disease (AD). It has been postulated thats in its many -structured oligomeric and protofibrillar formss A is responsible for, or at least a contributory factor to, the neuronal degeneration that occurs in AD. This hypothesis is supported by the finding that mutations in the amyloid -protein precursor (APP) and presenilin 1 (PS1) and presenilin 2 (PS2) genes, that are causative for familial early- onset AD, alter APP processing to result in elevated formation of the A(42) peptide (1). This peptidesrelative to the predominantly produced A(40) speciessis particu- larly amyloidogenic and appears to form the core of the neuritic plaques. APP is cleaved initially by R- or -secre- tase to generate membrane-bound C-terminal fragments (C83 and C99, respectively). R-Secretase activity appears to be mediated by the disintegrin and metalloprotease (ADAM) family members TACE and ADAM-10 (2). -Secretase (BACE, Asp-2) has recently been cloned and characterized as a novel membrane-bound aspartyl protease (3-6). The C83 and C99 cleavage products serve as substrates for the γ-secretase, with A(40) and A(42) being generated from C99. This cleavage is rather unusual in that it occurs at a site predicted to be within the putative transmembrane domain of APP. The identity of the enzymatic species that generates A(40) and A(42) peptidessfunctionally defined as APP γ-secretasesremains contentious. It has been claimed to be a single pharmacological entity or to exist as multiple species, belonging to either serine, cysteine, or aspartyl protease classes (7-13). Alternatively, following the demonstration of an obligatory role of presenilins in A formation (14, 15), it has been suggested that PS1 itself functions as the γ-secretase (16). We have used directed screening in cell culture-based and in vitro assays of APP processing to discover structurally novel inhibitors of A peptide formation. We report the * To whom correspondence should be addressed. Tel.: 44-1279- 440356; FAX: 44-1279-440712; E-mail: Mark_Shearman@Merck.com. ‡ Department of Molecular Biology. § Department of Medicinal Chemistry. 1 Abbreviations: A, amyloid -protein; AD, Alzheimer’s disease; APP, amyloid -protein precursor; BACE, -site APP cleaving enzyme; CHO, Chinese hamster ovary; DMSO, dimethyl sulfoxide; EuK, europium cryptate; FBS, fetal bovine serum; FRET, fluorescence resonance energy transfer; HEK, human embryonic kidney; HTRF, homogeneous time-resolved fluorescence; PAGE, polyacrylamide gel electrophoresis; PS, presenilin; SELDI-TOF, surface-enhanced laser desorption/ionization time-of-flight. 8698 Biochemistry 2000, 39, 8698-8704 10.1021/bi0005456 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/06/2000