Fragment Binding to β-Secretase 1 without Catalytic Aspartate Interactions Identified via Orthogonal Screening Approaches

Abstract

An approach to identify β-secretase 1 (BACE1) fragment binders that do not interact with the catalytic aspartate dyad is presented. A ThermoFluor (thermal shift) and a fluorescence resonance energy transfer enzymatic screen on the soluble domain of BACE1, together with a surface plasmon resonance (SPR) screen on the soluble domain of BACE1 and a mutant of one catalytic Asp (D32N), were run in parallel. Fragments that were active in at least two of these assays were further confirmed using one-dimensional NMR (WaterLOGSY) and SPR binding competition studies with peptidic inhibitor OM99-2. Protein-observed NMR (two-dimensional 15N heteronuclear single-quantum coherence spectroscopy) and crystallographic studies with the soluble domain of BACE1 identified a unique and novel binding mode for compound 12, a fragment that still occupies the active site while not making any interactions with catalytic Asps. This novel approach of combining orthogonal fragment screening techniques, for both wild-type and mutant enzymes, as well as binding competition studies could be generalized to other targets to overcome undesired interaction motifs and as a hit-generation approach in highly constrained intellectual property space.

BACE1 inhibitors: Current status and future directions in treating Alzheimer's disease

“…Twenty‐eight of the amino acid residues composing the active site of BACE1 were recognized as ligand‐binding site and all of them within 5 Å from the ligand . The ligand‐binding site is presented as a surface including the subpockets S1, S2, S3, S4, S1′, S2′, S3′, and S4′ . Moreover, optimal BACE1 activity is achieved at acidic pH, as BACE1 is located within acidic intracellular compartments including trans‐Golgi network (TGN) and endosomes where it cleaves its substrates leading to the formation of Aβ protein fragments.…”

“…45 The ligand-binding site is presented as a surface including the subpockets S1, S2, S3, S4, S1′, S2′, S3′, and S4′. 46 Moreover, optimal BACE1 activity is achieved at acidic pH, as BACE1 is located within acidic intracellular compartments including trans-Golgi network (TGN) and endosomes where it cleaves its substrates leading to the formation of Aβ protein fragments. The optimal enzyme activity under acidic conditions suggests that a BACE1 inhibitor having a basic amine residue with a pK a of ca.…”

Applied Biophysical Methods in Fragment-Based Drug Discovery

“…Of these variables, we have some control over the protein immobilization level; however, the NSB problem is more readily attenuated by keeping the compound concentration low. A survey of published SPR FBS conditions 42,[52][53][54] reveals that many users are opting to screen at fragment concentrations of <300 µM in order to combat the problem of NSB. Although it might attenuate NSB, low-concentration SPR fragment screening limits the fragment binding affinity range that is detectable.…”

Prediction of Drug Potencies of BACE1 Inhibitors: A Molecular Dynamics Simulation and MM_GB(PB)SA Scoring

“…The catalytic ectodomain has an aspartic protease fold, with the substrate-binding cleft located between the N-and C-terminal lobes (Figure 1). The crucial catalytic aspartate (Asp) dyad, Asp32 and Asp228, is located at the interface of the two lobes [2]. A hairpin loop "flap" in the N terminal lobe partially covers the cleft in a perpendicular orientation and contains Valine 69 Tyrosine 71 and Threonine 72 (colored blue in Figure 1).…”

“…Amidine containing compounds that form optimal interactions with the Asp32 and Asp228 enhanced the search for BACE1 inhibitors[1] [2] [5]. These Asp-binding amidine and guanidine inhibitors have been studied and the cyclohexyl groups were found to bind the S1 and the lipophilic S1′ pockets (Figure 1).…”