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

The final step in the generation of the amyloid-beta protein (A beta), impl icated in the etiology of Alzheimer's disease, is proteolysis within the tr ansmembrane region of the amyloid precursor protein (APP) by gamma-secretas e. Although considered an important target for therapeutic design, gamma-se cretase has been neither well-characterized nor definitively identified. Pr evious studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-se cretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of d ifluoro ketone peptide analogues with varying steric bulkiness in the P1 po sition and tested the ability of these compounds to inhibit A beta producti on in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chai ns, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these subs tituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-mol ecular-weight compound (<600 Da) that effectively blocked AO production (IC 50 similar to 5 mu M) This finding suggests that optimal S1 binding may all ow the development of potent inhibitors with ideal pharmaceutical propertie s. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substit uent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues in hibited total A beta and A beta(42) production with the same rank order of potency and increased A beta(42) production at low concentrations, providin g further evidence for distinct gamma-secretases that are nevertheless clos ely similar with respect to active site topology and mechanism.