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

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].     

Structural Features of Human Memapsin 2 （β-Secretase） and Their Biological and Pathological Implications

Alzheimer’s disease (AD), which is characterized bythe progressive destruction of brain functions in olderpeople, was first recognized in the early 20th century.Since then, modern medicine has further increased thenumber of people living to old age. AD h…     

Structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin 2 (beta-secretase)

A series of novel macrocyclic amide-urethanes was designed and synthesized based upon the X-ray crystal structure of our lead inhibitor (1, OM99-2 with eight residues) bound to memapsin 2. Ring size and substituent effects have been investigated. Cycloamide-urethanes containing 14- to 16-membered rings exhibited low nanomolar inhibitory potencies against human brain memapsin 2 (beta-secretase).     

Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor OM00-3

The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (Glu-Leu-Asp-LeuAla-Val-Glu-Phe, K(i) = 0.3 nM, the asterisk denotes the hydroxyethylene transition-state isostere) has been determined at 2.1 A resolution. Uniquely defined in the structure are the locations of S(3)' and S(4)' subsites, which were not identified in the previous structure of memapsin 2 in complex with the inhibitor OM99-2 (Glu-Val-Asn-LeuAla-Ala-Glu-Phe, K(i) = 1 nM). Different binding modes for the P(2) and P(4) side chains are also observed. These new structural elements are useful for the design of new inhibitors. The structural and kinetic data indicate that the replacement of the P(2)' alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P(3)' and P(4)'.     

Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.     

In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors

We have previously reported structure-based design of memapsin 2 (beta-secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a "carrier peptide" penetrated the plasma membrane in cultured cells and inhibited the production of beta-amyloid (Abeta). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimer's mice (Tg2576) resulted in a significant decrease of Abeta level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Abeta reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.     

Potent memapsin 2 (beta-secretase) inhibitors: design, synthesis, protein-ligand X-ray structure, and in vivo evaluation

Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic beta-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K(i) 1.8 nM) and cellular (IC(50)=1 nM in Chinese hamster ovary cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma A beta) in transgenic mice. The X-ray structure of protein-ligand complex of memapsin 2 revealed critical interactions in the memapsin 2 active site.     

Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.     

Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.     

Green tea catechins as a BACE1 (beta-secretase) inhibitor

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity. (-)-Epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate were isolated with IC(50) values of 1.6 x 10(-6), 4.5 x 10(-6), and 1.8 x 10(-6) M, respectively. Seven additional authentic catechins were tested for a fundamental structure-activity relationship. (-)-Catechin gallate, (-)-gallocatechin, and (-)-epigallocatechin significantly inhibited BACE1 activity with IC(50) values of 6.0 x 10(-6), 2.5 x 10(-6), and 2.4 x 10(-6) M, respectively. However, (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin exhibited about ten times less inhibitory activity. The stronger activity seemed to be related to the pyrogallol moiety on C-2 and/or C-3 of catechin skeleton, while the stereochemistry of C-2 and C-3 did not have an effect on the inhibitory activity. The active catechins inhibited BACE1 activity in a non-competitive manner with a substrate in Dixon plots.     

Structural locations and functional roles of new subsites S5, S6, and S7 in memapsin 2 (beta-secretase)

Memapsin 2 (beta-secretase) is the membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP), leading to the production of amyloid-beta (Abeta), a major factor in the pathogenesis of Alzheimer's disease. The active site of memapsin 2 has been shown, with kinetic data and crystal structures, to bind to eight substrate residues (P(4)-P(4)'). We describe here that the addition of three substrate residues from P(7) to P(5) strongly influences the hydrolytic activity by memapsin 2 and these subsites prefer hydrophobic residues, especially tryptophan. A crystal structure of memapsin 2 complexed with a statine-based inhibitor spanning P(10)-P(4)' revealed the binding positions of P(5)-P(7) residues. Kinetic studies revealed that the addition of these substrate residues contributes to the decrease in K(m) and increase in k(cat) values, suggesting that these residues contribute to both substrate recognition and transition-state binding. The crystal structure of a new inhibitor, OM03-4 (K(i) = 0.03 nM), bound to memapsin 2 revealed the interaction of a tryptophan with the S(6) subsite of the protease.     

Apo and inhibitor complex structures of BACE (beta-secretase)

Human BACE, also known as beta-secretase, shows promise as a potential therapeutic target for Alzheimer's disease. We determined the apo structure of BACE to 1.75 A, and a structure of a hydroxyethylamine inhibitor complex derived by soaking. These show significant active-site movements compared to previously described BACE structures. Additionally, the structures reveal two pockets that could be targeted by structure-based drug design.     

Flap position of free memapsin 2 (beta-secretase), a model for flap opening in aspartic protease catalysis

The three-dimensional structure of unbound human memapsin 2 (beta-secretase) protease domain determined at 2.0-A resolution has revealed a new position of the flap region, which appears to be locked in an "open" position. While the structure outside of the flap is essentially the same as the structure of memapsin 2 bound to an inhibitor, the flap positions are 4.5 A different at the tips. The open position of the flap in the current structure is stabilized by two newly formed intraflap hydrogen bonds and anchored by a new hydrogen bond involving the side chain of Tyr 71 (Tyr 75 in pepsin numbering) in a novel orientation. In molecular modeling experiments, the opening of the flap, 6.5 A at the narrowest point, permits entrance of substrates into the cleft. The narrowest point of the opening may function to discriminate among substrates based on sequence and shape. The observed flap opening may also serve as a model for the flap movement in the catalytic mechanism of eukaryotic aspartic proteases and provide insight for the side-chain selection in the design of memapsin 2 inhibitors.     

Synthesis and biological evaluation of phosphino dipeptide isostere inhibitor of human beta-secretase (BACE1)

Phosphino dipeptide (PDP) isosteres are known to be useful analogues of the transition state of metalloprotease substrates. Here we describe the use of this unit for the design of aspartic protease inhibitors. A PDP analogue of OM00-3, a potent BACE1 inhibitor, was synthesized and exhibited high biological activity (IC50 approximately 12 nM).     

GGA proteins mediate the recycling pathway of memapsin 2 (BACE)

Memapsin 2 (BACE, beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta (A beta) and the progression of Alzheimer disease. Both memapsin 2 and APP are transported from the cell surface to endosomes where APP is cleaved by memapsin 2. We described previously that the cytosolic domain of memapsin 2 contains an acid cluster-dileucine motif (ACDL) that binds the VHS (Vps-27, Hrs, and STAM) domain of Golgi-localized gamma-ear-containing ARF-binding (GGA) proteins (He, X., Zhu, G., Koelsch, G., Rodgers, K. K., Zhang, X. C., and Tang, J. (2003) Biochemistry 42, 12174-12180). Here we report that GGA proteins colocalize in the trans-Golgi network and endosomes with memapsin 2 and a memapsin 2 chimera containing a cytosolic domain of a mannose-6-phosphate receptor. Depleting cellular GGA proteins with RNA interference or mutation of serine 498 to stop the phosphorylation of ACDL resulted in the accumulation of memapsin 2 in early endosomes. A similar change of memapsin 2 localization also was observed when a retromer subunit, VPS26, was depleted. These observations suggest that GGA proteins function with the phosphorylated ACDL in the memapsin 2-recycling pathway from endosomes to trans-Golgi on the way back to the cell surface.     

Memapsin 2 (beta-secretase) as a therapeutic target

As beta-secretase, memapsin 2 cleaves amyloid-beta precursor protein, which leads ultimately to the onset of Alzheimer's disease. As such, memapsin 2 is an excellent target of inhibitor drugs for the treatment of this disease. Here we describe the tools for memapsin 2 inhibitor design that have been developed and results from the structure-based inhibitor design. Strategy for the design of memapsin 2 inhibitors with pharmaceutical potential is also discussed.     

Towards rational design of cannabinoid receptor 1 (CB1) antagonists for peripheral selectivity

CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.     

Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases.

The full-length and ectodomain forms of beta-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the beta-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2'0 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM/DAEFR, K(m) = 7 microm, K(cat) = 0.002 s(-1); Swedish mutant, SEVNL/DAEFR, K(m) = 9 microm, K(cat) = 0.02 s(-1)). A new substrate (VVEVDA/AVTP, K(m) = 1 microm, K(cat) = 0.004) was identified by serendipity.     

人β-分泌酶 (BACE1) 在毕赤酵母中分泌表达及纯化

为了获得活性良好的重组人β-分泌酶 (β-secreatase, BACE1),用于研究其与抑制剂的作用模式,构建了携带β-分泌酶proBACE1和BACE1编码序列的重组表达质粒pPIC9K-MetBACE22和pPIC9K-MetBACE46,通过电击法转入毕赤酵母GS115中,分别得到重组子9k-B22和9k-B46。重组菌株在诱导表达培养基中诱导外源基因表达,结果显示9k-B22的上清活性明显高于9k-B46的上清活性。9k-B22表达上清浓缩后经HisTrap亲和柱纯化得到的蛋白具有良好的BACE1活性, SDS-PAGE/高碘酸-希夫试剂染色发现其为糖蛋白,并且其糖基侧链可以被Endo Hf完全切除,得到50 kDa左右的两条蛋白带。肽质量指纹图谱鉴定发现,这两个蛋白分别与proBACE1和BACE1匹配。活性检测发现糖基化BACE1和去糖基化BACE1的活性均低于HEK-293细胞表达的商品BACE1,这说明糖基化及其类型对BACE1的活性非常重要。然而已知的BACE1抑制剂对三者的抑制率无显著差异,这说明糖基化并不影响与抑制剂的相互作用。经过一系列培养条件优化BACE1纯化产量提高到1 mg/L,这为发现并优化BACE1新型抑制剂的相关研究奠定了物质基础。