Alzheimer’s Disease: Identification and Development of β‐Secretase (BACE‐1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)

Abstract

The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease β-secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax , KM , and kcat ) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.

Protein‐Templated Fragment Ligations—From Molecular Recognition to Drug Discovery

“…[37][38][39][40][41][42] Finally,asapowerful complement, the detection of fragment ligation products by various bioassays has been developed over recent years. [1,10,[43][44][45][46][47][48] Thed etection of fragment ligation products by LC-MS has become substantially easier over the last two decades because of the rapid development of this method in terms of chromatographic separation and the sensitivity of mass detectors. [13,[25][26][27][28] Not only detection but also quantification and structure elucidation of ligation products is facilitated by using extracted-ion or single-ion chromatography and MS-MS techniques.S ome limitations remain, however.A s chromatographic separation takes some time,L C-MS detection is best suited for irreversible or quasi-irreversible ligation reactions.…”

“…They can be conducted using standard assay equipment such as microtiter plates and automated pipetting devices used for the handling of fragment libraries.I ne nzyme assays,t he sensitivity is further enhanced by the catalytic activity of the protein targets resulting in the rapid turnover of many of the fluorogenic or chromogenic substrate molecules.F luorescence resonance energy transfer (FRET) has also been used for the detection of fragment combinations. [44] To reach the highest sensitivity,t he primary reactive fragment has to be added to the enzyme assay at ac oncentration that results in 10-20 %i nhibition, thereby leaving am easurement window of 80-90 %f or detection of the enhanced inhibition after addition of the secondary fragment.…”

“…[126,127] Imine formation is also ap rocess that equilibrates rapidly and requires nonprotonated amines for reactivity. [10,43,44,46,47,83,84] Thus,t he process depends on the pK a value of the amine nucleophile and the pH value of the reaction buffer.I mines are considerably more stable than hemiacetals,e specially those formed from aromatic amines, and can even be isolated in some cases.…”

“…Dynamic ligation screening has been further extended toward aspartic proteases,t his time by employing af luorescence resonance energy transfer (FRET) assay. [44] Ap eptide aldehyde was used as adirecting probe to identify the activesite binding fragments of the aspartic protease b-secretase (BACE-1). This enzyme is known to be the main culprit in the aggregation of amyloid-b-peptides,w hich is am ajor pathological hallmark of AlzheimersDisease (AD).…”

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

“…In addition to FBDD, DCC14, 15, 16, 17, 18 and dynamic ligation screening (DLS)19, 20, 21, 22 are powerful strategies for identifying/optimizing hit compounds for biological targets. In a dynamic combinatorial library (DCL), the bonds between the building blocks are reversible and are continuously being made and broken.…”