Difluoro ketone peptidomimetics suggest a large S1 pocket for Alzheimer's γ-secretase:: Implications for inhibitor design

The final step in the generation of the amyloid-beta protein (A beta), implicated in the etiology of Alzheimer's disease, is proteolysis within the transmembrane region of the amyloid precursor protein (APP) by gamma-secretase. Although considered an important target for therapeutic design, gamma-secretase has been neither well-characterized nor definitively identified. Previous studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-secretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of difluoro ketone peptide analogues with varying steric bulkiness in the P1 position and tested the ability of these compounds to inhibit A beta production in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chains, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these substituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-molecular-weight compound (<600 Da) that effectively blocked AO production (IC50 similar to 5 mu M) This finding suggests that optimal S1 binding may allow the development of potent inhibitors with ideal pharmaceutical properties. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substituent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues inhibited total A beta and A beta(42) production with the same rank order of potency and increased A beta(42) production at low concentrations, providing further evidence for distinct gamma-secretases that are nevertheless closely similar with respect to active site topology and mechanism.