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Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design
R T Turner1, G Koelsch, L Hong
1Protein Studies Program, Oklahoma Medical Research Foundation, and Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Abstract:
Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates 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: S(2), 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. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was 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 binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene 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 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].
Insights
Memapsin 2, or beta-secretase, is crucial for Alzheimer's disease pathogenesis. Researchers mapped its residue preferences to design potent inhibitors, leading to the discovery of OM00-3, the most effective inhibitor to date.
Area of Science:
- Biochemistry
- Enzymology
- Neuroscience
Background:
- Memapsin 2 (beta-secretase) cleaves beta-amyloid precursor protein, producing beta-amyloid peptides.
- Beta-amyloid accumulation is a key factor in Alzheimer's disease pathogenesis.
- Memapsin 2 is a significant therapeutic target for Alzheimer's disease drug design.
Purpose of the Study:
- To determine the residue preferences for the subsites of memapsin 2.
- To identify optimal residues for designing potent memapsin 2 inhibitors.
- To characterize the binding interactions of novel inhibitors with memapsin 2.
Main Methods:
- Relative k(cat)/K(M) values determined using substrate mixtures and MALDI-TOF mass spectrometry.
- Analysis of binding affinities of memapsin 2 to a combinatorial inhibitor library.
- Molecular modeling of inhibitor-enzyme interactions.
Main Results:
- Each memapsin 2 subsite accommodates multiple residues, with S(1) being the most stringent.
- A peptide with the eight most favored residues showed the highest k(cat)/K(M) value.
- The inhibitor OM00-3, designed from consensus residues, exhibited a K(i) of 3.1 x 10(-10) M, the most potent reported.
Conclusions:
- Detailed residue preferences for memapsin 2 subsites were elucidated.
- These findings facilitate the rational design of highly potent memapsin 2 inhibitors.
- OM00-3 represents a significant advancement in developing therapeutic agents for Alzheimer's disease.