A Structure-Based Discovery Platform for BACE2 and the Development of Selective BACE Inhibitors

Abstract

The ability to perform routine structure-guided drug design for selective BACE inhibitors has been limited because of the lack of robust platform for BACE2 expression, purification, and crystallization. To overcome this limitation, we developed a platform that produces 2-3 mg of pure BACE2 protein per liter of E. coli culture, and we used this protein to design macrocyclic compounds that potently and selectively inhibit BACE1 over BACE2. Compound 2 was found to potently inhibit BACE 1 (K_i = 5 nM) with a selectivity of 214-fold over BACE2. The X-ray crystal structures of unbound BACE2 (2.2 Å) and BACE2 bound to compound 3 (3.0 Å and K_i = 7 nM) were determined and compared to the X-ray structures of BACE1 revealing the S1-S3 subsite as a selectivity determinant. This platform should enable a more rapid development of new and selective BACE inhibitors for the treatment of Alzheimer's disease or type II diabetes.

Keywords: Alzheimer’s disease; BACE1; BACE2; selective inhibitors; structure-based drug design; type II diabetes.

Figure 1.

(A) Overlay of BACE2 structures of 3ZKG (marine) and 6UJ0 (magenta, structure determined in this study). The boxed regions and associated insets show the enlargement of the overlay of flap-loop (top) and mobile loop^102-110 (bottom). (B) Overlay of BACE2 flap-loop from different crystal forms. The BACE2 protein structure is shown in gray surface. The catalytic residues are shown in sticks and colored based on the atom types. The flap loops from different crystal forms are shown as ribbons. Structures for unbound BACE2 are shown for 6UJ0 (magenta, determined in this study) and 3ZKG (marine), as well as for inhibitor bound BACE2 (orange, PDB 2EWY). The double arrow indicates the observed flexibility of the flap-loop.

Figure 2.

Ligand interaction plot of inhibitor 3 with (A) BACE1 and (B) BACE2. The plots are adapted from the output of LIGPLOT. The original plots are shown in Figure S6. Inhibitor 3 is shown in green (BACE1) and magenta (BACE2) line structures, and the non-carbon atoms are colored according to the atom types. The polar contacts are indicated by the dash lines with distances shown in angstroms, and the interacting residues are represented as black line. The residues involved in the hydrophobic interactions are labeled and shown as red hashes. Residues involved in BACE1 specific hydrophobic interactions are highlighted with pink circles. (C) Overlay of inhibitor 3 in the active site of BACE1 (green) and BACE2 (magenta). Inhibitor 3 is shown in ball and stick and colored according to the atom types. The 10s loop is labeled and shown as lines. Ser10 (Ser26 in BACE2) and Thr232 (Thr245 in BACE2) are shown in stick and colored based on atom types. The distance from the hydroxyl side chain of Thr232 (Thr245 in BACE2) to the carbonyl oxygen of Ser10 (Ser26 in BACE2) is shown in angstroms. The location of the residues involved in BACE1 specific interactions are highlighted within the pink circle (GQG in 10s loop). See also panel A and Figure S7. (D) Open conformation of 10s loop (shown as a ribbon representation) in unbound BACE1 (PDB: 3TPJ, colored in yellow) and inhibitor 3 bound BACE1 (PDB: 6NV9, colored in green). Ser10, Thr232, inhibitor 3, and an ordered water molecule are shown in ball and stick. The distance between the ordered water and the hydroxyl side chain of Thr232 is shown in angstroms.

Figure 3.

(A) Overlay of inhibitor 3 in the active site of BACE1 (green) and BACE2 (magenta). The set of amino acid residues surrounding the S₁′ binding pocket are shown as stick structures. Loop Thr329–Val332 in BACE1 and Asn341–Val344 are shown in ribbon. (B) Closer view of the S₁′ binding pocket in the active site of BACE1 (top) and BACE2 (bottom). BACE1 and BACE2 are shown in surface presentation and colored in gray. Flap-loop is removed for clarity. Inhibitor 3 is shown in sphere presentation and colored according to the atom types. The S₁′ binding pocket in the active site of BACE1 and BACE2 is highlighted with orange circles.