Dual Mechanism of Actions of Novel HBV Core Protein Allosteric Modifiers (CpAMs): Inhibiting Viral Replication and Blocking cccDNA Formation
Qi Huang1, Dawei Cai1, Pao-Chen Li1, Alex Mercier1, RenukaKumar1, Emily Connelly1, Yuhua(Sara) Zong1, Ran Yan1, Xiulan Zhou1, Yi Zhou1, Lida Guo1, Ariel Tang1, Geoffrey Chen1, Esteban Carabajal1, Katherine Nabel1, Lichun Li1,
Steve Dunkelbarger1, Sarah Katen1, Jason Deer1, Earl May1, Uri Lopatin1, Adam Zlotnick1,2 and Richard Colonno1
Assembly Biosciences1
Indiana University2
Core Protein Allosteric Modifiers (CpAMs)
Accelerate capsid assembly and induce Cp to form different sizes of aberrant capsids
• Block pgRNA encapsidation and lead to the formation of empty capsids
• First generation of CpAMs is in early clinical development
Zlotnick et al (1999) Biochemistry 38, 14644-52Zlotnick et al (2011) Trends in Microbiology 19, 14-23
• Structurally distinct small molecules allosterically bind to a pocket of Core Protein at the dimer-dimer interface
CpAMs Induce Aberrant Capsid and Cause Cp Aggregation
3
DMSO
TEMCp150/Capsid
ABI-H0731HAPs
• Normal Symmetric Capsid • Variety of irregular sized Capsid
• AD38 IFA• Green:• Cp/Capsid• Blue: • Nucleus
• Mostly diffused cytoplasmic Cp staining
• Cytoplasmic and nuclear aggregates
CpAMs Inhibit HBV Replication by Inducing Empty Capsid Formation
DM
SO
CpA
M
ABI-H
0808
Total Extract(Western Blot) GAPDH
Core (DAKO)
HepAD38 (Tet-, 6 days, 1uM)
ABI-H
0731
CpA
M
ETV
100u
M
GLS
4
ABI-H
0986
Encapsidated RNA(Northern Blot)
No pgRNApackaged
rcDNA
ssDNA
Core DNA(Southern Blot) No viral
replication
Capsid EIA(Western Bot)
AberrantCapsid
NormalCapsid
cccDNA
CpAM
AAA...AAA..
.AAA...
ETVpgRNA
Viral Replication Inhibition
AberrantEmptyCapsid
MatureCapsid
• Capsid Disassembly
CpCp
Viral Replication Inhibition
No viral DNA synthesis
HBV International Meeting 2016
CpAMs Modify Cp Phosphorylation Status
Regulation of specific versus nonspecific RNA packaging of HBV capsidsby the degree of CTD phosphorylation and dephosphorylation
Hu et al (2016) J Virol 90:5830 –5844.
Normal Capsid
CpAMs Mimic Effect?
• Western Blot Evaluation of Cp Phosphorylation
Anti Total Cp ab C-terminal ab*(unphosphorylated 14aa)
GAPDH
C-terminal unphosphorylated Cp
GAPDH
Total Cp
AT-1
30
DMSO
CpAM
GLS
4
ABI-H
986
ABI-H
808
• 2 Day treatment• No effect on Cp expression
• Cp remains hyperphosphorylated
• * Gift from H. Guo
AT-1
30
DMSO
CpAM
GLS
4
ABI-H
986
ABI-H
808
• 1 uM
CpAMs Induce Hyperphosphorylated Cp to Aggregate and Degrade
ABI-H
731
ABI-H
808
GLS
4
• Total Cell Extract• (Sulfuric Acid)
GAPDH
Total
Un-P
HepAD38 (Tet-, 6 days)
Stable total expressionHyper-phosphorylationEnhanced Degradation
• CpAM effect on Cp
• Cytoplasm FL
Un-PCleaved
• NuclearFL
Un-PCleaved
Cleavage/AggregationHyper-phosphorylation
• Cell Pellet• (Sulfuric Acid) Cleaved
FL
Un-P
cccDNA
• Capsid Disassembly
• Hyper-P• Aberrant • Capsid
CpAMs MOA 1
CpAM
AAA...AAA..
.AAA...
ETVpgRNA
Enhanced
P
P
P
PP
PP
PP
P P
P
P
PP
P
PP
PPP
P
P
P
P
PP
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
CpAMs Effectively Inhibit HBV Viral Replication
CpAM
cccDNA
MatureCapsid
• Capsid Disassembly
• Hyper-P• Empty• Capsid
CpAMs MOA 1
CpAM
AAA...AAA..
.AAA...
ETVpgRNA
Viral ReplicationInhibition
P
P
P
PP
PP
PP
P P
P
P
PP
P
PP
PPP
P
P
P
P
PP
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
Viral Replication Inhibition
No viral DNA synthesis
CpAMs block cccDNA Formation in HBV Infected Cells
CpAM
Primary Human Hepatocytes (PHH)
DM
SO
3.3
µM
10 µ
M
ABI-H0731
>1,000x EC50
Entecavir
DM
SO
100
nM
ABI-H0808
cccDNA
DM
SO
0.6
µM
3.0
µM
• Presented at AASLD 2016
Guo et al. J. Virol. 2007Capsid disassembly and rcDNA delivery
CpAM
DM
SO
Inco
min
g ca
psid
3hr Post Infection
Capsid EIA(Western)
Capsid DNA(Particle
Southern)
ABI-H
808
Premature Capsid Disassembly
ETV
HepG2-NTCP HBV Infection
CpAM Dural Mechanisms Of Action: Inhibit Viral Replication and Block cccDNA Formation
Inhibit ViralReplication
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
cccDNA
CpAMETV
AAA...AAA..
.AAA...pgRNA
P
PP
PP
PP
P
P
P
PP
P
PP
P
PP
PPP
P
P
P
P
PP
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
Hyper-PEmptyCapsid
CpAM MOA 1
• Capsid Assembly
Block cccDNAFormation
MatureCapsid
Capsid Disassembly
• Lost
• rcDNAPremature
Capsid Disassembly
CpAM MOA 2CpAM
Inhibit ViralReplication
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
cccDNA
CpAMETV
AAA...AAA..
.AAA...pgRNA
P
PP
PP
PP
P
P
P
PP
P
PP
P
PP
PPP
P
P
P
P
PP
P
PPP
P
PPP
P
PPP
P
PPPP
PPP
P
PPP
Hyper-PEmptyCapsid
CpAM MOA 1
• Capsid Assembly
CpAM
Cytoplasm
nucleus
CpAMs Target Multiple Steps of HBV Lifecycle
Core protein plays multiple roles throughout HBV lifecycle and represents an excellent drug target impacting cccDNA levels
CpAMs represent a new class of direct acting antivirals that are selective for HBV and inhibit de novo cccDNA formation
Assembly Biosciences has established assays to specifically measure cccDNAactivities and its first candidate is currently in clinical development
Acknowledgements
Assembly Biosciences HBV Team
Qi HuangDawei Cai
Pao-Chen LiEmily Connelly
G. Renuka KumarYuhua ZongAlex MercierXiulan Zhou
Katherine NabelYi ZhouRan YanLida Guo
Geoffrey ChenEsteban Carabajal Ariel
TangUri Lopatin
Richard Colonno
Earl MayLichun Li
Sara KatenSteve Dunkelbarger
Jason Deer
Biology BiochemistryChemistry
Leping LiSimon Haydar
Bill TurnerLynn BannenMark Bures
Samson Francis
Adam Zlotnick