Activity-based screening assay for mono-ADP-Ribosylhydrolases
| Autor: | Albert Galera-Prat, Heli I. Alanen, Mirko M. Maksimainen, Sarah Wazir, Lari Lehtiö |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Anomer macrodomain Biochemistry Analytical Chemistry 03 medical and health sciences Hydrolysis chemistry.chemical_compound Microtiter plate 0302 clinical medicine ADP-Ribosylation Humans chemistry.chemical_classification Nicotinamide Substrate (chemistry) NAD Fluorescence In vitro Enzyme Activation activity assay 030104 developmental biology Enzyme chemistry 030220 oncology & carcinogenesis ADP-ribosylhydrolases Molecular Medicine Biological Assay screening assay Carboxylic Ester Hydrolases Protein Processing Post-Translational Biotechnology |
| Popis: | ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD⁺, anomer of β-NAD⁺, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD⁺ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|