Characterization of PROTAC-580: A Cereblon-Recruiting
Degrader of Enteroviral 2A Protease
B. Kaščáková1, H. Durmaz3, R. Kumar
Akula3, K. Rox3, H. El Kilani2, R. Hilgenfeld2
and I. Kutá Smatanová1
1Department of
Chemistry, University of South Bohemia, Branišovská 1645/31a, 370 05 Ceske
Budejovice, Czech Republic
2 Institute for
Molecular Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck,
Germany
3 Department of
Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstr. 7,
Braunschweig 38124, Germany
karafb00@jcu.cz
Enteroviruses
(EV) such as EV-A71 and EV-D68 are increasingly recognized as serious public
health threats, particularly among children [1], due to their association with severe
neurological and respiratory illnesses including hand, foot, and mouth disease
(HFMD) [2] and acute flaccid
myelitis (AFM) [3]. Despite their impact, no targeted
antiviral therapies are currently available. To address this gap, we have
developed PROTAC-580, a first-in-class cereblon-recruiting
proteolysis-targeting chimera (PROTAC) designed to degrade the enteroviral 2A
protease (2Apro)—a viral enzyme essential for replication and immune
evasion.
2Apro
mediates polyprotein processing, inhibits host protein synthesis via eIF4G
cleavage, and disrupts type I interferon signaling by degrading IFNAR1 and
cleaving MAVS and MDA5, making it indispensable for viral pathogenesis [4]. Unlike conventional inhibitors that block activity
only transiently, PROTAC-580 should catalytically direct 2Apro to
CRBN-mediated ubiquitination and proteasomal degradation, enabling complete
removal of the target protein, potentially lowering resistance risk, and
expanding the range of druggable viral proteins [5, 6].
Using
integrative structural biology—including X-ray crystallography, molecular
docking, and crosslinking mass spectrometry—we rationally designed PROTAC-580
to achieve high selectivity and sustained degradation of 2Apro. To support these efforts, we established an
optimized recombinant expression system in E. coli, incorporating solubility-enhancing
fusion tags, buffer screening, and refined purification workflows to obtain
milligram-scale quantities of active, monodisperse protein for structural
analysis and degradation assays.
Aligned with the PANVIPREP consortium’s
vision for broad-spectrum antiviral development, PROTAC-580 marks a paradigm
shift from inhibition to targeted protein degradation, offering a promising
therapeutic strategy not only against enteroviruses but also as a platform for
combating other RNA
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This project has received funding
from the European Union’s Horizon Europe research and innovation programme
under Grant Agreement no. 101137229.