NS5A inhibitor

Raffaele De Francesco (E-mail: [email protected])
Fondazione INGM, Istituto Nazionale di Genetica Molecolare - Milano, Italy
Il ciclo cellulare di HCV ed i target dei
farmaci anti‐virali
(DAA, direct-acting antivirals)
Definition of “Direct-Acting Antivirals
(DAA)” :
Agents that interfere with specific steps in the
virus replication cycle through a direct
interaction with a viral protein or nucleic acid
Goal of HCV antiviral therapy:
Rapid and complete clearance of HCV from the body
Current P.I.-based therapy
(PEG-IFN/RBV/TVR or Boc)
Goals for New DAAs
Severe side effects – high
discontinuation rate
Improved tolerability decreased duration
Several contra-indications
Lower response in cirrhotics
Applicable to all patients
High response in cirrhotics
High pill burden
Fewer pills, ideally once-a-day
PEG-IFN/RBV backbone
Development of IFN-free, RBVfree combinations
60-70% SVR
90+% SVR
Emergence of resistance
Minimal or no resistance
Indicated only for genotype 1
Active across different HCV
genotypes (1 to 6)
Hepatitis C virus (HCV)
Envelope glycoproteins
Membrane
•  Discovered in 1989
•  Small (≈ 60 nm), enveloped virus
(family Flaviviridae, genus hepacivirus)
•  (+)-stranded RNA genome (9.6 kb)
•  Single Large Polyprotein (~3,000 aa)
•  Very high sequence variability
(6 major genotypes, >100 subtypes)
Core Protein
(+) RNA
genome
HCV Genome and Polyprotein Organization
(+) RNA genome
~9.6 kb
ORF
Stop
ATG
Polyprotein
~3,000 aa
Structural proteins
C
E1
E2
Virion components
RNA
Non-structural proteins
p7 NS2
NS3
4A
4B
5A
5B
RNA replication complex
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Most advanced HCV DAAs target
Polyprotein Processing or Viral RNA replication
Binding to
receptors and entry
Polyprotein
Processing
NS3/4A protease
inhibitors
Virus
Maturation
and Exit
Viral RNA
replication
NS5B polymerase
inhibitors,
NS5A inhibitors
Most Important HCV Targets for Antiviral Therapy
Virus Assembly Module
C
E1
E2
RNA Replication Module
p7 NS2
NS3
NS3/NS4A
Serine Protease
4A
4B
5A
NS5A
RNA Replication
Complex
Component
5B
NS5B
RNA-dependent
RNA-Polymerase
The NS3/4A protease as therapeutic target
Virus Assembly Module
C
E1
E2
p7 NS2
RNA Replication Module
NS3
4A
4B
5A
5B
NS3/4A Serine Protease
–  The HCV genome is translated as a single protein precursor
–  The NS3/4A serine protease is responsible for the biogenesis of the
nonstructural proteins (components of the RNA replication
machinery)
–  No NS3/4A protease activity è no viral replication
HCV NS3/NS4A serine protease:
From “undruggable” target to drugs (1996 to 2011)
Ser139
NS4A
•  Long, shallow and exposed active site - little for inhibitors to grasp
• Design of low molecular weight inhibitors is very challenging
NS4A
HCV NS3/4A Protease
Critical issue:
Identification of
active site anchor(s)
Two classes of protease inhibitors with different
active-site anchors
Covalent inhibitors
Non-covalent inhibitors
– 
Substrate-derived
– 
Product-derived
– 
Form a covalent bond with the
enzyme
– 
Better tolerability
– 
Important side effects
O
NH
N
H 3CO
Telaprevir
N
O
O
O
N
Faldaprevir
S
Br
N
H
N
H
N
N
O
H
N
HN
O
H
N
O
O
H
N
N
O
OH
O
O
O
O
S
CH3
OCH3
N
N
Simeprevir
O
Boceprevir
H
N
H
N
O
N
O
O
NH 2
HN
O
O
H
N
H 3C
N
O
O
O
O O
S
N
H
NS3-4A protease inhibitors in clinical development
1) First generation – first wave (covalent, tid, genotype 1 only)
Marketed
Telaprevir (Vertex/J&J)
Boceprevir (Merck)
2) Second-wave (non-covalent, better tolerated, genotype 1,
bid or qd; low barrier to resistance)
FDA-Approved
Simeprevir (Janssen)
BI201335 (Boehringer-Ingelheim)
Phase 3
Vaniprevir (Merck)
Asunaprevir (BMS)
ABT450/r (AbbVie)
Phase 2
Danoprevir/r (Roche)
3) Second-generation (pan-genotype coverage; high barrier to resistance)
Phase 2
Grazoprevir/MK-5172 (Merck)
ACHN-2684 (Achillion)
The NS5B RNA-dependent RNA polymerase as
therapeutic target
• Catalytic center of the viral RNA amplification machine
• Essential for both (-)-stranded and (+)-stranded RNA synthesis
C
E1
E2
Right-hand shape
p7 NS2
NS3
4A
4B
5A
5B
Thumb
Fingers
Palm
The NS5B RNA-dependent RNA polymerase as
therapeutic target
• Catalytic center of the viral RNA amplification machine
• Essential for both (-)-stranded and (+)-stranded RNA synthesis
(+)
5’
RNA Uncoating, translation &
polyprotein processing
3’
(+)
(+)
RNA Replication(+)
(+)
(-)
(+)
(+)
Assembly
Budding
(+)
(+)
(+)
(+)
(-)
The NS5B RNA-dependent RNA polymerase as
therapeutic target
• Catalytic center of the viral RNA amplification machine
• Essential for both (-)-stranded and (+)-stranded RNA synthesis
(+)
5’
RNA Uncoating, translation &
polyprotein processing
3’
(+)
(+)
RNA Replication(+)
(+)
(-)
(+)
(+)
(+)
NO
VIRUS
(+)
(+)
(-)
Inhibitors of Viral Polymerases
C
E1
E2
p7 NS2
NS3
4A
4B
5A
Lesson from other viruses (HIV, HBV, Herpes)
Viral polymerases are target for 23/37 approved antiviral drugs
Two Classes of Inhibitors
1.  ACTIVE SITE INHIBITORS
• 
Nucleoside or nucleotide analogues
2.  ALLOSTERIC INHIBITORS
•  Non-nucleoside inhibitors (NNI)
5B
Several inhibitor-binding sites on the HCV polymerase
NNI site C / PALM 1
NNI site A / THUMB 1
-  Setrobuvir/RG7790; ABT-333; ABT-072
-  Deleobuvir; BMS-791325
A
B
NNI site B / THUMB 2
- Lomibuvir/VX-222;
Filibuvir/PF-00868584
C
D
Active
site
Nucleotide Binding Site
NNI site D / PALM 2
- 
HCV-796; Tegobuvir/GS-9190
- Sofosbuvir; Mericitabine;
VX-135; IDX21437; ACHN-3422
Reviewed in De Francesco & Migliaccio, Nature (2005)
Inhibitors of HCV NS5B RNA-dependent RNA Polymerase
C
E1
E2
p7 NS2
NS3
4A
4B
5A
5B
Two Classes of Inhibitors
1.  NON-NUCLEOSIDE INHIBITORS
• 
Allosteric Inhibitors, inhibit “initiation” of RNA synthesis
• 
Several NNI binding sites on the enzyme surface
• 
Restricted spectrum of action on different HCV
genotypes (target mainly HCV-1b)
2.  NUCLEOSIDE/NUCLEOTIDE ANALOGUES
• 
Active site inhibitors
• 
Chain terminators: terminate RNA synthesis
• 
Equally active on ALL HCV genotypes
Inhibitors of HCV NS5B RNA-dependent RNA Polymerase
C
E1
E2
p7 NS2
NS3
Nucleoside/nucleotide analogs
Approved: Sofosbuvir (Gilead)
Phase 2:
Mericitabine (Roche)
VX-135 (Vertex)
Phase 1:
ACHN-3422 (Achillion)
IDX21437 (Idenix)
Non-Nucleoside Inhibitors (NNI)
Phase 3:
Phase 2:
ABT-333 (AbbVie)
Tegobuvir (Gilead)
Setrobuvir (Roche)
Lomibuvir (Vertex)
BMS-791325 (BMS)
4A
4B
5A
5B
The NS5A protein as therapeutic target
Virus Assembly Module
C
E1
E2
p7 NS2
RNA Replication Module
NS3
- Dimeric protein
- Binds to HCV RNA
- Critical component of membranebound RNA replication complex
4A
4B
5A
HCV
RNA
5B
The NS5A protein as therapeutic target
Virus Assembly Module
C
E1
E2
p7 NS2
Inhibition of NS5A prevents
formation of functional RNA
replication complex
RNA Replication Module
NS3
4A
4B
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5A
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5B
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NS5A inhibitor Daclatasvir (BMS-790052) potently inhibits
HCV genotypes 1 to 5
HCV subtype
Replicon EC50 (pM)
1a
1b
2a
3a
4a
5a
50
M Gao et al. Nature 465, 96-100 (2010)
9
12-63
127
12
33
Inhibitors of HCV NS5A in Clinical Development
C
E1
E2
p7 NS2
NS3
NS5A Inhibitors
Phase 3
Daclatasvir (BMS)
Ledipasvir (Gilead)
ABT267 (AbbVie)
Phase 2
IDX719 (Idenix)
PPI-461 (Presidio)
IDX719 (Idenix)
ACH-2928 (Achillion)
BMS-824393 (BMS)
MK-8742 (Merck)
4A
4B
5A
5B
HCV is Genetically Highly Heterogeneous
Several genotypes, many sub-types, quasispecies
HCV Shows Greater Genetic Diversity than HIV
HCV and HIV variability
Across and Within Genotypes
25
% diversity
20
15
within
10
across
5
across
0
within
HCV genotypes
HIV clades
What have we learned so far about HCV genotype
coverage by different DAA classes?
DAA class
Genotype Coverage
NS3 Inhibitors (PIs)
GT 1
2nd generation NS3 PIs
Pan-genotypic
NS5A inhibitors
Broad Spectrum
NS5B Pol Non-nucleoside Inh.
(NNIs)
Restricted spectrum (mainly 1b)
NS5B Pol nucleoside/nucleotide
analogues
Pan-genotypic
“One definition of a (direct acting) antiviral drug is a drug
that selects for resistance”
DD Richman, Hepatology 32:866-867; 2000
–  Error-prone polymerase, lack of proof-reading mechanism
–  High mutation rate (>1/105 nucleotides/replication cycle)
–  High viral production rate (1012 virions/day)
–  All possible single-nucleotide mutations/double-nucleotide mutations,
and many 3-nucleotide mutations are generated daily
→ therapeutic efficacy of DAA may be limited by emergence of
drug resistant HCV variants
Development of Viral Resistance
Viral Load
Treatment begins
Drug-susceptible quasispecies
Drug-resistant quasispecies
Selection of resistant
quasispecies
Drug selective pressure
Genetic barrier to resistance
Viral fitness of variants
Time (days to weeks)
Not all DAAs are equal
Nucleos(t)ide analogs pose a HIGHER BARRIER TO RESISTANCE
compared to non-nucleoside polymerase or protease inhibitors
McCowen et al, (2008) Antimicrob Agents Chemother
NS3/4A protease
inhibitor (PI)
NS5B non-nucleoside
NS5B nucleoside analog
polymerase inhibitor (NNI) polymerase inhibitor (NI)
Telaprevir
Untreated
1X IC50
10X IC50
15X IC50
HCV-796
1X IC50
10X IC50
Mericitabine/R7128
15X IC50
1X IC50
Blue colonies indicate resistance
•  Resistant colonies readily selected for NNI or PI
•  Treatment with nucleoside analog clears HCV (no resistance)
10X IC50
15X IC50
What have we learned so far about HCV resistance?
Not all DAAs are equal
DAA class
Genotype Coverage
Barrier to Resistance
NS3 Inhibitors (PIs)
GT 1
+
2nd generation NS3
PIs
Pan-genotypic
++
NS5A inhibitors
GT 1-5
+
NS5B Pol Nonnucleoside Inh. (NNIs)
Restricted spectrum
+
Pan-genotypic
+++
NS5B Pol nucleoside/
nucleotide analogues
•  No resistance mutations to nucleoside/nucleotide analogs have
been detected in patients (even under monotherapy regimen!)
SUMMARY: HCV Direct-Acting Antivirals
NS3/4A
Protease
Inhibitors
2nd Generation
NS3/4A PIs
§ Covalent or
Non-covalent
§  Non-covalent
§ Low genetic
barrier to
resistance
§ Genotype 1
§ Higher Barrier
to resistance
§ Pan-genotypic
(1 to 6)
NS5B Polymerase Inhibitors
Nucleos(t)ide
Analogue
NS5A Inhibitors
Nonnucleos(t)ide
§ Active site
§ Bind to several § NS5A has role in
assembly of
§ Incorporated into different
allosteric
replication
RNA chain
enzyme sites
complex
causing chain
termination
§ Low genetic
§ Low genetic
barrier to
barrier to
§ High genetic
resistance
resistance
barrier to
resistance
§ Pan-genotypic
(1 to 6)
§ Very restricted
genotype
coverage
§ Broad genotypic
coverage (1 to 5)
Evolution of HCV standard of care
2010
2012
pegIFN
pegIFN
pegIFN
NS5Bpol
nucleotide
+
+
+
+/-
Ribavirin
Ribavirin
NS5A
inhibitor
NS5B Nuc
+
NS5A
+
+
+/-
and/or
wave
NS3-4A
inhibitor
2nd gen
NS3-4A
inhibitor
Ribavirin
1st
wave
NS3-4A
inhibitor
?
2nd
or
+/-
NS5A
inhibitor
NS5B pol
NNI
or
+/-
NS5B pol
nucleotide
Ribavirin
Once a day pangenotypic
Fixed-dose
Combination
2nd GEN
NS3-4A
inhibitor
Toward 100% SVR with a single pill (and no IFN)
Sofosbuvir (nucleotide analogue)+ Ledipasvir (NS5A inhibitor)
SVR12 Rates From Three Phase 3 Studies Evaluating a Once-Daily Fixed-Dose
Combination of Sofosbuvir and Ledipasvir for Genotype 1 Hepatitis C Patients
Source: http://investors.gilead.com/

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