Design of Potent Inhibitors for Human Brain Memapsin 2 (β-Secretase)

Ghosh, Arun K.; Shin, Dong-Chun; Downs, Debbie; Koelsch, Gerald; Lin, Xinli; Ermolieff, Jacques; Tang, Jordan

doi:10.1021/ja000300g

Cited by 236 publications

(205 citation statements)

References 18 publications

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“…This multiplicity of binding sites has been detected previously in studies that examined the binding of radiolabeled A (1-40) to short homologous peptides (39). In addition to the identification of the primary A selfrecognition sequence as A (16)(17)(18)(19)(20), a secondary site was also found within the A (24-34) sequence. We suspect that this region provides an additional weak binding site within the immobilized peptide that accounts for the observed heterogeneity.…”

Section: Resultssupporting

confidence: 63%

Affinity-Based Inhibition of β-Amyloid Toxicity

et al. 2002

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Strategies for interfering with protein aggregation are important for elucidating and controlling the pathologies of amyloid diseases. We have previously identified compounds that block the cellular toxicity of the -amyloid peptide, but the relationship between their ability to inhibit toxicity and their affinity for A is unknown. To elucidate this relationship, we have developed an assay capable of measuring the affinities of small molecules for -amyloid peptide. Our approach employs immobilized -amyloid peptide at low density to minimize the problems that arise from variability in the -amyloid aggregation state. We found that low-molecular weight (MW of 700-1700) ligands for -amyloid can be identified readily by using surface plasmon resonance. The best of these bound effectively (K d ∼ 40 µM) to -amyloid. The affinities measured for peptides in the SPR assay correspond to results from A cell toxicity assays. The most potent ligands for immobilized -amyloid are the most potent inhibitors of the neuronal cell toxicity of -amyloid. Compounds with dissocation constants above ∼100 µΜ did not show significant activity in the cell toxicity assays. Our data support the hypothesis that ligands exhibiting greater affinity for the -amyloid peptide are effective at altering its aggregation and inhibiting cell toxicity.

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Section: Resultssupporting

confidence: 63%

Affinity-Based Inhibition of β-Amyloid Toxicity

et al. 2002

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“…This may suggest that it is not only important to have carbohydrate at these sites, but that specific sugar moieties may be important for a direct interaction with substrate. In addition, the refolded E. coli expressed Asp-2, which is not glycosylated (24), was assayed at a 10-fold higher concentration than our experiments, suggesting that its activity is significantly lower than the mammalian expressed protein. The E. coli expressed material contains asparagine residues at all four consensus glycosylation sites, again supporting the hypothesis that the lack of glycosylation and not the alteration of the asparagine residues is leading to the decrease in activity of Asp-2.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Glycosylation Profiles of Alzheimer's β-Secretase Protein Asp-2 Expressed in a Variety of Cell Lines

Charlwood

Dingwall

Matico

et al. 2001

Journal of Biological Chemistry

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Amyloid 39 -42 ␤ -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39 -42 ␤-peptide is formed from amyloid precursor protein by the sequential action of ␤-and ␥-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have ␤-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.One of the key pathological features of Alzheimer's disease is the formation of brain plaques primarily due to the fibrilization of amyloid 39 -42 ␤-peptides (A␤) 1 (1, 2). The formation of these peptides by the action of proteases on amyloid precursor protein (APP) has been the subject of several studies (3, 4), as the identification of these proteases could lead to the discovery of inhibitors useful in the treatment of Alzheimer's disease. Over the last year there have been a number of reports detailing the discovery, purification, and characterization of a transmembrane aspartic proteinase (Asp-2). This glycoprotein has been shown to cleave the APP at the ␤-secretase site (5-9). High levels of this proteinase were identified in the human brain, and the enzyme activity was found in cells associated with the central nervous system (7) and in cell lines known to produce A␤ via cleavage of APP (5).Asp-2 contains four potential N-linked glycosylation sites at the following asparagine residues: Asn 153 , Asn 172 , Asn 223 , and Asn 354 . The close proximity of some of these glycosylated sites to the three intramolecular disulfide linkages and the catalytic site of Asp-2 has been the subject of more recent interest (10), especially because the type and extent glycosylation of a protein can have a profound effect on its physico-chemical properties (11). For instance, it is well known that N-linked oligosaccharides can influence glycoprotein folding in the endoplasmic reticulum (12) and can protect a protein from protease attack (13).The N-linked oligosaccharide structures on Asp-2 have not yet been characterized in detail, although it has been reported that all four asparagine sites have a heterogeneous mixture of carbohydrate attached (10). Another report has indicated that the glycans in mature Asp-2 are of the complex type, as removal of this carbohydrate is not possible by endoglycosidase H treatment (14).In this study we have released and analyzed the N-linked glycans from Asp-2 expressed as an Fc fusion in ...

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“…Fractions containing BACE were combined, sterile-filtered (0.22 m), and stored at 4°C. The protein was characterized by SDS-PAGE, and a variety of biophysical techniques, including isothermal titration calorimetry, to demonstrate that it was glycosylated, monomeric, catalytically active, and fully competent to bind BACE active site inhibitor OM99-2 (16).…”

Section: Methodsmentioning

confidence: 99%

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

Toulokhonova¹,

Metzler²,

Witmer³

et al. 2003

Journal of Biological Chemistry

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The steady-state kinetic mechanism of ␤-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine-(Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k cat /K m , a large inverse solvent kinetic isotope effect on k cat , and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the ratelimiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at ␦ 13.0 ppm and of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k cat . Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K ii and K is expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of ␤-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.Extracellular amyloid deposits in brain, a characteristic feature of Alzheimer's disease, is a result of proteolytic cleavage of membrane-bound amyloid precursor protein by two enzymes, ␤-secretase and ␥-secretase. The second cleavage activity (␥-secretase) is strongly associated with the presenilin multisubunit complexes (1), whereas ␤-secretase (BACE) 1 has been identified as a novel transmembrane aspartyl protease (2-4).Although aspartyl proteases have been studied for more than 4 decades, new aspects of catalysis and inhibition continue to emerge. A substantial number of these enzymes have been identified as useful targets for chemotherapeutic intervention in human diseases (5-8), yet there has been limited success in identifying clinically relevant inhibitors; hence, it is important to explo...

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Design of Potent Inhibitors for Human Brain Memapsin 2 (β-Secretase)

Cited by 236 publications

References 18 publications

Affinity-Based Inhibition of β-Amyloid Toxicity

Affinity-Based Inhibition of β-Amyloid Toxicity

Characterization of the Glycosylation Profiles of Alzheimer's β-Secretase Protein Asp-2 Expressed in a Variety of Cell Lines

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

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