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