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701
A NOVEL HBV CAPSID INHIBITOR COMPOUND SERIES
DEMONSTRATES IMPROVED INHIBITION OF HBV WT AND
T33N CORE PROTEIN VARIANT AND SHOWS A UNIQUE
BINDING MODE TO CORE PROTEIN
Nagraj Mani1, Andrew Cole1, Steven G Kultgen1, Andrzej
Ardzinski1, Junjun Cheng2, Andrea Cuconati1, Bruce David
Dorsey1, Kristi Fan1, Fang Guo1, Ju-Tao Guo2, Zhanying Hu2,
Roseann Kowalski1, Eugen Mesaros1, Cornelis Rijnbrand1,
Holly M Micolochick Steuer1, Kim Stever1 and Michael J
Sofia1, (1)Arbutus Biopharma Inc., Warminster, PA, USA, (2)
Baruch S. Blumberg Institute, Doylestown, PA, USA
Background: The inhibition of HBV pregenomic RNA
(pgRNA) encapsidation by small molecule inhibitors has
been clinically-validated in chronic HBV infected patients.
We present the preclinical antiviral profile and mode of action
studies of a novel chemical series with a unique binding mode
to core protein and improved potencies against both WT and
T33N core protein variant as well as cccDNA formation and
establishment compared to previous generation of capsid
inhibitors Methods: Compounds were evaluated for antiviral
potencies and cytotoxicity in HBV cell culture systems. Mode
of action studies were conducted using HepAD38 or AML12-
HBV10 cells to evaluate compound effects on HBV pgRNA
encapsidation, capsid particle density, size and core protein
phosphorylation status using particle gel assays, sucrose and
CsCl density gradient centrifugations, electron microscopy
and western blot analysis. HBV infection systems were used
to investigate compound effects on capsid uncoating and
cccDNA establishment X-ray crystallography studies were
conducted to determine the core protein binding site A transient
transfection assay was used to determine the effect of T33N
core protein variant on potency Results: A novel chemical
series of HBV capsid inhibitors was optimized for potency and
drug-like properties resulting in compounds with EC50 values
≤10 nM in cell culture models. In HepAD38 cells, compounds
from this series blocked pgRNA encapsidation and produced
HBV capsids devoid of viral nucleic acids without reducing
intracellular pgRNA and core protein levels (class II capsid
inhibitor) X-ray crystallography studies showed a compound
from this series bound to the dimer:dimer interface and
displayed a differential binding mode compared to reported
structures, and displayed a slower gel migration pattern of
capsids formed in HepAD38 cells upon compound treatment
Sucrose and CsCl density gradient centrifugation analyses
and electron microscopic studies suggested that compound
treatment altered the shape/conformation of capsids without
affecting particle density, size or phosphorylation patterns
during pgRNA encapsidation Most advanced compounds
showed EC50 values of sub-300 nM against a T33N variant
of core protein as well as inhibition of capsid uncoating and
cccDNA establishment in the sub-100 nM range Conclusion:
HBV capsid inhibitors from a novel chemotype showed a
unique binding mode to HBV core protein when compared
to previously reported X-ray structures and demonstrated
improved antiviral properties in vitro in comparison to previous
generation of capsid inhibitors.
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发表于 2019-11-4 18:41