ABSTRACT
Abnormal aggregation of amyloid-ß is a very complex and heterogeneous process. Owing to methodological limitations, the aggregation pathway is still not fully understood. Herein a new approach is presented in which the secondary structure of single amyloid-ß aggregates is investigated with tip-enhanced Raman spectroscopy (TERS) in a liquid environment. Clearly resolved TERS signatures of the amideâ I and amideâ III bands enabled a detailed analysis of the molecular structure of single aggregates at each phase of the primary aggregation of amyloid-ß and also of small species on the surface of fibrils attributed to secondary nucleation. Notably, a ß-sheet rearrangement from antiparallel in protofibrils to parallel in fibrils is observed. This study allows better understanding of Alzheimer's disease etiology and the methodology can be applied in studies of other neurodegenerative disorders.
Subject(s)
Amyloid/chemistry , Hyperspectral Imaging/methods , Spectrum Analysis, Raman , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid/metabolism , Amyloid beta-Peptides/chemistry , Humans , Nanotechnology , Protein Conformation, beta-StrandABSTRACT
The aggregation pathways of neurodegenerative peptides determine the disease etiology, and their better understanding can lead to strategies for early disease treatment. Previous research has allowed modelling of hypothetic aggregation pathways. However, their direct experimental observation has been elusive owing to methodological limitations. Herein, we demonstrate that nanoscale chemical mapping by tip-enhanced Raman spectroscopy of single amyloid fibrils at various stages of aggregation captures the fibril formation process. We identify changes in TERS/Raman marker bands for Aß1-42 , including the amideâ III band (above 1255â cm-1 for turns/random coil and below 1255â cm-1 for ß-sheet conformation). The spatial distribution of ß-sheets in aggregates is determined, allowing verification of a particular fibrillogenesis pathway, starting from aggregation of monomers to meta-stable oligomers, which then rearrange to ordered ß-sheets, already at the oligomeric or protofibrillar stage.
Subject(s)
Amyloid beta-Peptides/chemistry , Nanotechnology , Protein Aggregates , Protein Aggregation, Pathological , Particle Size , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Surface PropertiesABSTRACT
The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
Subject(s)
Drug Discovery , SARS-CoV-2 , Drug Discovery/methods , SARS-CoV-2/metabolism , Catalytic Domain , Magnetic Resonance Spectroscopy , Peptide Hydrolases/metabolism , Protease Inhibitors/metabolism , Antiviral Agents/pharmacology , Molecular Docking SimulationABSTRACT
Structure-based drug discovery (SBDD) largely relies on structural information from X-ray crystallography because traditional NMR structure calculation methods are too time consuming to be aligned with typical drug discovery timelines. The recently developed NMR molecular replacement (NMR2) method dramatically reduces the time needed to generate ligand-protein complex structures using published structures (apo or holo) of the target protein and treating all observed NOEs as ambiguous restraints, bypassing the laborious process of obtaining sequence-specific resonance assignments for the protein target. We apply this method to two therapeutic targets, the bromodomain of TRIM24 and the second bromodomain of BRD4. We show that the NMR2 methodology can guide SBDD by rationalizing the observed SAR. We also demonstrate that new types of restraints and selective methyl labeling have the potential to dramatically reduce "time to structure" and extend the method to targets beyond the reach of traditional NMR structure elucidation.