A novel cell-based sensor detecting the activity of individual basic proprotein convertases.
A549 Cells
Adaptor Proteins, Vesicular Transport
/ genetics
Biosensing Techniques
/ methods
Drug Discovery
/ methods
Enzyme Inhibitors
/ chemistry
Genes, Reporter
HEK293 Cells
HeLa Cells
High-Throughput Screening Assays
/ methods
Humans
Luciferases
/ genetics
Peptidomimetics
/ chemistry
Proprotein Convertases
/ antagonists & inhibitors
Sensitivity and Specificity
furin
high-throughput screening
inhibitor
proprotein convertase
sensor
Journal
The FEBS journal
ISSN: 1742-4658
Titre abrégé: FEBS J
Pays: England
ID NLM: 101229646
Informations de publication
Date de publication:
11 2019
11 2019
Historique:
received:
13
11
2018
revised:
13
05
2019
accepted:
02
07
2019
pubmed:
6
7
2019
medline:
17
6
2020
entrez:
6
7
2019
Statut:
ppublish
Résumé
The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high-throughput screening format. The robust and cost-effective assay format of BPCS makes it suitable to identify novel specific small-molecule inhibitors against basic PCs for therapeutic application. Its cell-based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened 'target range' will enhance the likelihood to identify novel small-molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.
Substances chimiques
Adaptor Proteins, Vesicular Transport
0
Enzyme Inhibitors
0
Peptidomimetics
0
Luciferases
EC 1.13.12.-
Proprotein Convertases
EC 3.4.21.-
sortilin
Z020Y8WIJ4
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
4597-4620Subventions
Organisme : Novartis Stiftung für Medizinisch-Biologische Forschung
ID : 14B062
Pays : International
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
ID : 310030_170108
Pays : International
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
ID : 31003A_153467
Pays : International
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
ID : 31003A_156452
Pays : International
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
ID : 31003A_173178
Pays : International
Organisme : CIHR
ID : 148363
Pays : Canada
Organisme : Canada Research Chairs
ID : 231335
Pays : International
Organisme : Interdisciplinary Research Grant of the Faculty of Biology and Medicine of the University of Lausanne
Pays : International
Informations de copyright
© 2019 Federation of European Biochemical Societies.
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