Translating Molecular Virology into Novel Therapies …regist2.virology-education.com/2011/6HEPPK/docs/07_Glenn.pdfTranslating Molecular Virology into Novel Therapies for Hepatitis

Presented at the 6th Int. Workshop on Clin. Pharmacology of Hepatitis Therapy, 22 – 23 July 2011, Cambridge, USA

Translating Molecular Virology into

Novel Therapies for Hepatitis

Jeffrey S. Glenn, M.D., Ph.D.

Stanford University

6/22/11

Disclosures: Eiger BioPharmaceuticals Inc., Roche, Romark


Hepatitis viruses:

Important world wide causes of liver disease

Unfortunately, current therapies are inadequate for many

Fortunately, molecular virology

new targets

novel therapies


target to lead studies

IND-enabling studies

clinical trial

Examples of such “bench-to-bedside” efforts


Wedemeyer, H. & Manns, M. P. 2010. Nat. Rev. Gastroenterol. Hepatol. 7, 31–40.

Hepatitis delta virus (HDV)

Worst form of human viral hepatitis

~ 15 million world-wide; ~ 70K in U.S.

No effective Rx


HDV HBV

ss circular RNA ds DNA

HBV surface Ag HBV surface Ag

delta antigen HBV core Ag


The HDV life cycle


Double rolling circle model of HDV genome replication


RNA editing generates two types of delta antigen

A G

UAG UGA UGG

195 a.a. 195 + 19 a.a.

delta

antigen

isoform :

196 196 215

small large

required for replication inhibits replication;

required for packaging with HBsAg


prenylation -- site-specific lipid modification of

proteins

Prenylation of large delta antigen is required for HDV assembly

prenylation site = “CXXX box”

mevalonate isoprenoids (prenyl lipids)

-farnesyl (C15)

-geranylgeranyl (C20)

cys arg pro gln



Hypothesis:

Pharmacologic inhibition of large delta antigen

prenylation can prevent HDV

particle formation


large antigen:

Ser 211 large antigen:

(Cys-->Ser)

farnesyl transferase

CXXX CXXX

SXXX SXXX


In vivo treatment of HDV viremia

FTI-277 FTI-2153

(Bordier et al. 2003 JCI))


• target host enzyme

different paradigm: deprive virus access to host function

?more difficult to develop resistance?

• well tolerated

• orally available compounds--ready for clinical trials

HDV = prototype (orphan designation)--> IND approved

multiple viruses --> broaden indication

Attractive features of prenylation inhibition

antiviral therapy:


EBP921

HDV Proof of Concept Trial

• N=16

• Primary Inclusion: Confirmed HDV infection

• Primary Exclusions: HIV, HCV, advanced cirrhosis

• Open Label Rx: 2 dosing regimens

• Duration: 28 days of dosing, option to extend

• Primary Endpoint: : Δ in HDV-RNA from baseline


Turkey CPMC

San Francisco

Henry Ford

Detroit Cornell

New York

University of Ankara

Turkey

US and Ex-US HDV POC Study Sites

NIH


1) HDV--important, fascinating biology, inadequate Rx

2) Prenylation is key to HDV morphogenesis

(pharmacologic target)

3) Farnesyl transferase inhibitors abrogate assembly in

in vitro and in vivo models

4) These types of compounds represent a new

class of potential antiviral agents

(HDV, HAV, HSV, others) --clinical trials

Conclusions


-- important cause of chronic liver disease

(~170 million; 4-5 in US)

#1 cause of HCC, liver transplantation in US

Hepatitis C virus (HCV)

Only 2-3% of HCV patients in the developed world are treated


E1 C E2 NS2 NS4B NS3 NS5A NS5B 3’

core

envelope

protease

helicase polymerase

structural non-structural (NS)

?

Hepatitis C virus (HCV)

(U/UC) 5’ ARFP


5’

IFN ribavirin

?

E1 C E2 NS2 NS4B NS3 NS5A NS5B 3’

protease

helicase polymerase

(U/UC)

IRES

FUTURE:

? ? ?


--27 kD

--nucleotide binding motif; GTPase

--cellular transformation

--intracellular membrane rearrangements

(membranous web)

--amphipathic helix (AH1) assembles NS proteins

NS4B

A E1 C E2 NS2 NS4B NS3 NS5A NS5B 3’ (U/UC)

ARFP

7

p 4 5’


-- NS4B has a second

amphipathic helix (4BAH2)

-- 4BAH2 is highly conserved across HCV isolates

Hypotheses:

-- Role in formation of membranous web

-- Essential for genome replication

-- Represents candidate new anti-HCV target

NS4B

AH2


4BAH2 promotes lipid vesicle aggregation

(Cho et al. 2010, Science Translational Medicine)


An intact amphipathic helix is required for vesicle aggregation



An intact 4BAH2 is required for HCV genome replication



Screen for small molecule inhibitors of 4BAH2 function

fluorescent

vesicles

Vesicles + AH2 + DMSO

Vesicles + AH2 + inhibitor


• identified small molecule inhibitors with nM activity against HCV replication

• inhibitors display synergy with clemizole; IND enabling studies


Conclusions

1) HCV NS4B has a second N-terminal amphipathic helix (4BAH2)

2) Conserved across natural HCV isolates

3) 4BAH2 is essential for HCV RNA replication

4) 4BAH2 promotes lipid vesicle aggregation relevant to formation of membranous web basis for HTS

5) Small molecule inhibitors of 4BAH2 function inhibit HCV

genome replication and can synergize with clemizole

6) 4BAH2 inhibitors represent new potential class of anti-HCV agents; potential for combination with other emerging anti-HCV drugs (a derivative of C4 IND-enabling studies)


Phosphoinositides—classical role in signaling

Also recognized by, and regulate function of, proteins

(e.g. involved in intracellular vesicular membrane trafficking)

4,5-bisphosphate

(PIP2)

phosphatidyl

inositol

Presented at the 6th Int. Workshop on Clin. Pharmacology of Hepatitis Therapy, 22 – 23 July 2011, Cambridge, USA De Matteis et al, Nature Cell Biology (2004)


Tellinghuisen, et al.,Nature (2005) 435:374-9

A E1 C E2 NS2 NS4B NS3 NS5A NS5B 3’ (U/UC)

ARFP

7

p 4 5’

NS5A amphipathic helix (AH):

-- mediates membrane association

-- essential for RNA replication

-- interacts with intracellular membrane trafficking machinery

Hypothesis: PIP2 modulates NS5A function

NS5A AH binds PIP2


The NS5A amphipathic helix specifically binds

lipid vesicles containing PIP-2


PIP-2 binding is mediated by a pair of conserved

positively-charged amino acids.

(A)

Basic Amino Acid PIP2 Pincer (BAAPP) domain




(B)

190 200 210 220 230 240 250

-30000

-20000

-10000

0

10000

20000

30000

40000

Mean R

esid

ue M

ola

r E

llip

ticity (

)

Wavelength (nm)

NS5A AH NS5A AHK20A NS5A AHK26A NS5A AHK20AK26A




0.0 3.0k 6.0k 9.0k 12.0k

-400

-300

-200

-100

0

fr

eque

ncy

(Hz)n=

3/n

=ov

erto

nes

Time (Sec)

PIP2n 4,5 platform AH NH20 NH20,26 NH NH26

0.0 3.0k 6.0k 9.0k 12.0k 15.0k-10

0

10

20

30

40

50

60

70

D

issi

patio

n (x

10-6

)

Time (Sec)

AH NH K20A K26A K20AK26A-500

0

500

1000

1500

2000

2500

3000

3500

area

l mas

s (n

g/cm2

)

target peptides

(C)


NS5A colocalizes with PIP-2 in the context of the HCV

replication complex


PIP-2 mediates HCV RNA genome replication






Potential new anti-HCV strategies:

-- Targeting interaction between PIP2 and NS5A BAAPP domain (e.g. neomycin)

-- Targeting enzymes that regulate PIP2 at HCV replication sites (e.g.

PI PI(4)P PI (4,5)P2

PI 4-kinases PI(4)P 5-kinase

5-phosphatase


Development of small molecule inhibitors of PI-4 kinases

Rationale:

PIP2 binding important for HCV replication

Targeting PIP2 production (e.g. siRNA knockdown of PI4-kinase)

inhibits HCV replication, but well tolerated by host cells.

Have initiated collaboration with Shokat lab:

Non-specific inhibitor removed toxicity increased specificity

potent inhibitors of HCV replication

Ongoing optimization, pharmacokinetics, metabolite analysis

Valuable probes of host cell biology

Applicability to other targets


1) NS5A specifically binds PIP2

2) HCV--first example of PIP2-dependent viral genome

replication

3) HCV NS5A AH--first example of BAAPP domain mediating

PIP2 binding

4) Novel MOA; likely widespread among viruses

5) HTS to identify novel inhibitors of PIP2-BAAPP motif

interaction

6) Specific PI-kinase inhibitors--> anti-HCV and broad-spectrum

antivirals

7) NS proteins (e.g. NS4B, NS5A) represent rich source of novel

targets, basis of future cocktail components

Conclusions


Glenn lab:

Ping Liu

Menashe Elazar

ChoongHo Lee

Nam-Joon Cho

James Lue

Collaborators:

Yale: Andrew Hamilton

Rockefeller: Charlie Rice

Harvard: Greg Verdine

Stanford: Doron Gerber, Steve Quake, Julie Baker, Curt Frank, Suzanne Pfeffer,

Gary Peltz

Hebrew University: Benjamin Aroeti, Tsafi Danieli

Eli Lilly: Tina Myers, Kirk Staschke

UMASS: David Lambright

UCSF: Brandon Tavshanjian, Kevan Shokat

American Liver Foundation, Amgen, AASLD, Burroughs-Welcome Fund, VA Merit Review,

Eli Lilly, Stanford Digestive Disease Center, Goldman Philanthropic Partnerships, Oxnard

Foundation, Chiron, NIH, InterMune, Beckman Foundation, Romark, SPARK, MacFarlane

Foundation, Roche, CTSA, Genentech

Phil Pang

Ed Pham

Michael Gelman

Jerremy Lee

Rick Salazar

Marilyn Masek

Anming Xiong

Mark Winters

Recent alumni:

Meirav Matto

Wonjae Lee

Paul Bryson

Wei Gu

Ella Sklan

Hadas Dvory-Sobol

Shirit Einav

Documents

Translating Molecular Virology into Novel Therapies …regist2.virology-education.com/2011/6HEPPK/docs/07_Glenn.pdfTranslating Molecular Virology into Novel Therapies for Hepatitis