Subsite specificity of memapsin 2 (beta-secretase): Implications for inhibitor design

Memapsin 2 is the protease known as beta -secretase whose action on beta -a myloid precursor protein leads to the production of the P-amyloid (AP) pept ide. Since the accumulation of A beta in the brain is a key event in the pa thogenesis of Alzheimer's disease, memapsin 2 is an important target for th e design of inhibitory drugs. Here we describe the residue preference for t he subsites of memapsin 2. The relative k(cat)/K-M values of residues in ea ch of the eight subsites were determined by the relative initial cleavage r ates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S-1 subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S2, Asp > Asn > Met; S-3, Ile > Val > Leu; S-4, Glu > Gln > Asp; S-1', Met > Glu > Gln > Ala; S-2', Val > Ile > Ala; S-3', Leu > Trp > Ala; S-4', Asp > Glu > Trp. I n general, S subsites are more specific than the S' subsites. A peptide com prising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) w as found to be hydrolyzed with the highest k(cat)/K-M value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight b inding inhibitors, the consensus preferences were as follows: S2, Asp and G lu; S3, Leu and Ile; S-2', Val; and S-3', Glu and Gln. An inhibitor, OM00-3 , Glu-Leu-Asp-Leu*Ala-Val-Glu-Phe (where the asterisk represents the hydrox yethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K- i of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 re vealed critical improvement of the interactions between inhibitor side chai ns with enzyme over a previous inhibitor, OM99-2.