Sandbox Reserved 817

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.

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BACE1BACE1

spinqualitylabelsall models PDB ID 4ivs Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
Crystal structure of BACE1 with its inhibitor, 4ivs
Ligands:
Activity:	Memapsin 2, with EC number 3.4.23.46
Related:	4ivt
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

Biological functionsBiological functions

BACE1 belong to the beta-secretasesBACE1 belong to the beta-secretases

BACE1 cleaves APP only at the known β-secretase sites of Asp+1 and Glu+11 of Aβ. Moreover, purified recombinant BACE1 directly cleaves APP substrates at these same sites in vitro, demonstrating that the BACE1 molecule intrinsically exhibits protease activity. The sequence specificity of purified BACE1 is the same as β- secretase. For example, it cleaves Swedish mutant APP substrate much more efficiently than wild type, and does not cleave a P1 Met-Val mutant substrate that is resistant to β-secretase cleavage. Like β-secretase, BACE1 has optimal activity at ~pH 4.5, is resistant to inhibition by pepstatin, and is localized within acidic subcellular compartments of the secretory pathway, primarily the Golgi apparatus, TGN and endosomes. Taken as a whole, the properties of BACE1 correlate very well with the previously established characteristics of β-secretase activity in cells and tissues.

Regulation of BACE1Regulation of BACE1

The pattern and level of BACE1 expression is largely consistent with those of β-secretase activity in cells and tissues. The levels of BACE1 mRNA are highest in brain and pancreas and are significantly lower in most other tissues. Moreover, BACE1 mRNA is highly expressed in neurons but little is found in resting glial cells of the brain, as expected for β-secretase. The protein is abundant in both normal human and AD brain. Given the low levels of β-secretase activity in the pancreas, the high pancreatic mRNA expression was initially confusing. However, subsequent reports indicated that BACE1 mRNA transcripts in pancreas largely consist of a splice variant missing the majority of exon 3. This splice variant encodes a BACE1 isoform devoid of β-secretase activity, thus reconciling the paradoxically high BACE1 mRNA levels with the low β-secretase activity found in the pancreas. The functional relevance of this pancreas-specific splice variant remains unclear. BACE1 induces a dramatic increase in β-secretase activity when transfected into stable APP-overexpressing cell lines. The immediate products of β-secretase cleavage, APPsβ and C99, are increased several fold over levels found in un-transfected cells, and Aβ production is also elevated. Interestingly, APPsα levels are reduced upon BACE1 transfection, indicative that α- and β-secretases compete for APP substrate in cells. In contrast to the effects of BACE1 transfection, treatment of APP-overexpressing cells with BACE1 antisense oligonucleotides decreases BACE1 mRNA levels and inhibits β-secretase activity. BACE1 antisense inhibition reduces production of APPsβ, C99, and Aβ in cells; conversely, APPsα and C83 generation is elevated.

StructuresStructures

spinqualitylabelsall models Amino acid sequence of Beta secretase 1 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Structurally, the 501 amino acid sequence of BACE1 belongs to the eukaryotic aspartic proteases of the pepsin family and contains a bilobal structure forming by an N- and a C-terminal domains. Both N- and C-domains are formed by highly twisted β-sheet structures and each domain contributes an aspartic acid to the catalytic module of the enzyme. It is thus proposed that ligands containing a positive charged moiety might be favorable to counteract the negative charged active site. BACE1 has two aspartic protease active site motifs, DTGS () and DSGT (), and mutation of either aspartic acid renders the enzyme inactive. Like other aspartic proteases, BACE1 has an N-terminal signal sequence (residues 1–21) and a pro-peptide domain (residues 22–45) that are removed post-translationally, so the mature enzyme begins at residue Glu46. Importantly, BACE1 has a single transmembrane domain near its C-terminus (residues 455–480) and a palmitoylated cytoplasmic tail. Thus, BACE1 is a type I membrane rotein with a luminal active site, features predicted for β-secretase. The position of the BACE1 active site within the lumen of intracellular compartments provides the correct topological orientation for cleavage of APP at the β-secretase site. As observed with other aspartic proteases, BACE1 has that form three intramolecular disulfide bonds (yellow) and several N-linked glycosylation sites.

InhibitionInhibition

Mecanism of inhibitionMecanism of inhibition

Structural information about the interaction of substrate with the active site of BACE1 would greatly facilitate the rational design of small molecule BACE1 inhibitors. Towards this end, Sauder et al. used molecular modeling to simulate the BACE1 active site bound with wildtype or mutant APP substrates. The basic structure of most aspartic protease active sites is well conserved and the Xray structure of pepsin was used to model BACE1. The molecular modeling identified several residues in BACE1 that potentially contribute to substrate specificity. In particular, Arg296 forms a saltbridge with the P1' Asp+1 residue of the β-secretase cleavage site, thus explaining the unusual preference of BACE1 among aspartic proteases for substrates that are negatively charged at this position. In addition, several hydrophobic residues in BACE1 form a pocket for the hydrophobic P1 residue. The model also showed that the Swedish FAD mutation, LysMet→AsnLeu at P2-P1, interacts more favorably with Arg296 and the hydrophobic pocket of BACE1 than does wild-type substrate, providing an explanation for the enhanced cleavage of this mutation. Conversely, the substitution of Met→Val at P1 blocks the catalytic Asp93 residue, explaining the lack of cleavage of this mutation by BACE1. Shortly after the molecular modeling study, the X-ray structure of the BACE1 protease domain co-crystallized with a transition-state inhibitor was determined to 1.9 angstrom resolution. As expected, the BACE1 catalytic domain is similar in structure to pepsin and other aspartic proteases, despite the relatively low sequence similarity. Interestingly, the BACE1 active site is more open and less hydrophobic than that of other aspartic proteases. Four hydrogen bonds from the catalytic aspartic acid residues (Asp93 and Asp289) and ten additional hydrogen bonds from various residues in the active site are made with the inhibitor, most of which are conserved in other aspartic proteases. The X-ray structure indicates that Arg296 and the hydrophobic pocket of the active site play an important role in substrate binding, confirming the results of the molecular modelling study. In addition, the bound inhibitor has an unusual kinked conformation from P2' to P4'. The BACE1 X-ray structure suggests that small molecules targeting Arg296 and the hydrophobic pocket residues should inhibit β-secretase cleavage. Moreover, mimicking the unique P2'-P4' conformation of the bound inhibitor may increase the selectivity of inhibitors for BACE1 over BACE2 and the other aspartic proteases.

InhibitorsInhibitors

File:Inhibiteur4.jpg

Figure 2 : Docking study on compound 4 bound to BACE1. The coordinates of BACE1 was taken from the crystal structure of 1FKN. The protein is shown in cartoon, while the important residues and ligand 4 are shown in stick model.

In the past, major efforts in designing BACE1 inhibitors were focused on the transition state analogs such as hydroxyethylamines, hydroxyethylene, and tatine-based peptidomimetic inhibitors. Although a large number of potent peptidomimetic inhibitors have been discovered, their relatively large sizes and excessive number of hydrogen-bond donors and acceptors make it difficult for them to penetrate the blood brain barrier. Therefore many researchers in both academia and industry are trying to identify drug-like small molecules as BACE1 inhibitors, which hold great hopes to have good pharmacokinetic (PK) profiles and are suitable for drug development.

The compounds, 1-(2-(1H-indol-1-yl)ethyl)guanidine, showed weak inhibition activity towards BACE1, about 42% inhibition ratio at the ligand concentration of 100 μM in the fluorescence resonance energy transfer (FRET) assay system.

The compound occupied the S1 pocket and the guanidine moiety formed key binding interactions with the two catalytic aspartic acids, Asp32 and Asp228 (Figure 2). As exemplified in some known BACE1 inhibitors in which the guanidine group is usually acylated, we further designed a compound by introducing a carbonyl group into the α-positionof the guanidine moiety.