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

IntroductionIntroduction

BACE1, which stand for β-site of Amyloid precursor protein Cleaving Enzyme 1, is a β secretase also known as memapsin 2. It is an aspartyl protease that initiates the formation of β-amyloid which accumulation has a huge role in the Alzeimer disease. It had been brought to light that not only BACE1 levels rise in the brain of the patients suffering from this disease but that BACE1 knock out mices can’t produce β-amyloid, effectively preventing the formation of amyloid plaques and the development of the Alzheimer disease. This discovery makes the BACE1 a very interesting drug target for Alzheimer disease.The inhibition of this enzyme is a plausible course of action and several inhibitors like the ones that will be presented are considered. However, the total inhibition of BACE1 seems to leads to physiological and behavioural troubles. That would mean that BACE1 could also be involved in several other brain functions. So a regulation of BACE1 activity by partial inhibition seems to be a preferred method. Anyway, a good understanding of the biological function, the structure and the mechanisms of inhibition of BACE1 is highly required.

Localisation and Biological functionLocalisation and Biological function

LocalisationLocalisation

BACE1 is the result of the expression of the BACE1 gene (30 kb) on chromosome 11. It is composed of 9 exons that are linked together during the mRNA maturation. BACE1 is expressed in the majority of human tissues however, the highest levels of activity are found in the neuronal cell. This β secretase, which is a transmembrane protein mainly localised within cholesterol-rich lipid raft, is found in the acidic compartments of the secretory pathway (mostly endosomes and the Trans Golgi Network) with its catalytic site facing the lumen side. This enzyme is regulated both at the translation and transcription steps and its expression is correlated with cell stress signals.

Post translational modificationPost translational modification

BACE1 is synthetised in the endoplasmamic reticulum as a precursor, pro BACE1 and it is inactive. This precursor is then maturated in the Golgi apparatus. It undergoes glycosylation of 4 residus : , Asn(172), Asn(223), and Asn(354), it has a role in activity. 3 cysteine residues of the cytosolic tail are also palmitoylated , it is has a role in the localization of the mature enzyme. The propeptide domain is clived between Arg 45 and Glu 46 by a proprotein convertase (protease). This clivage is known to increase the activity of the enzyme.

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.