Transcript Mechanisms of Action

L281816...A PROMISING DRUG LEAD THAT
INHIBITS HCV INFECTIVITY IN A GENOTYPEINDEPENDENT MANNER
Reem Al Olaby, B.Pharm., M.Sc., PhD
Dubai Bio-Expo 2015
2nd to 4th of November
BACKGROUND
Hepatitis C Virus
Hepatitis C Virus
Expert Reviews in Molecular Medicine, 2003
Hepatitis C Virus
281816
Ledipasvir
Public
Health
Interventions
Basic
Science
Research
Drug
Discovery
Drug
Discovery
Project Overview
Aim
Develop a genotype-independent drug that
selectively targets specific residues in the
protein of choice to treat HCV.
Hypothesis
HCV infection and cell to cell transmission could
be inhibited in a genotype-independent manner
when creating a drug that targets HCV E2
glycoprotein.
Project Phases – In Silico
Determine The Top
Virtual Screening Virtual Screening
Hits
Runs Against
Prepare The
Determine The
Target
Protein
Target
Protein
Target Protein
Project Phases – In Vitro
DL Infection
Inhibition Assay
DL confirmatory Binding
Analyzing Results and
Primary
Experimental
Validation
categorizing Drug
Leads
Assay
Key Components of The Project
Autodock Tools 1.5.6 ®
Kong L et al., science, 2013
HCV E2 Crystal Structure
Key Components of The Project
Autodock Tools 1.5.6 ®
HCV E2 Homology Model
Key Components of The Project
Autodock Tools 1.5.6 ®
Kong L et al., science, 2013
HCV E2 Crystal Structure: Model Superposition
Key Components of The Project
Autodock Tools 1.5.6 ®
Ligands – NCI_DSII
Targeting HCV E2
In Silico Homology Modelling
Al Olaby RR et al., PLOS One, 2014
Lawrence Livermore National Laboratory
Autodock Tools 1.5.6 ®
In Silico - Virtual Screening Runs
Ligand ID
• ~1700 Ligands
• First set of virtual
screening hits = 34
ligands
• Experimentally Tested
 Biacore
Al Olaby RR et al., PLOS One, 2014
670283
86467
639174
81462
403379
213700
359472
146554
204232
281816
308835
60785
84100
158413
57103
121861
3076
Free Energy of
Binding
(Kcal/mol)
-7.69
-7.47
-7.81
-6.81
-7.58
-7.89
-7.91
-7.67
-8.54
-8.64
-8.4
-7.48
-6.99
-7.9
-6.36
-8.16
-7.71
Ligand ID
211490
113486
144694
4429
133071
163910
54709
135618
281254
319990
369070
59620
38968
171303
228155
13316
117268
Free Energy of
Binding
(Kcal/mol)
-8.7
-6.26
-7.27
-7.3
-7.5
-7.4
-7.3
-8.7
-6.5
-7.4
-6.3
-7.3
-3.9
-5.8
-8.7
-6.8
-7.6
In Silico - Analysis
Al Olaby RR et al., PLOS One, 2014
Scripps Research Institute
Autodock Tools 1.5.6 ®
Preliminary Binding Assays
(Biacore t200® )
Ligand ID
Binding (RU)
Dissociation 1 (RU)
Dissociation 2 (RU)
670283
86467
639174
81462
403379
213700
359472
146554
204232
281816
308835
60785
84100
158413
57103
121861
117268
3076
211490
113486
144694
4429
133071
54.3
54.9
55.4
57.2
58
62
62
63.4
63.4
64.5
64.8
70.4
71.2
71.2
81.6
88.4
88.5
92.2
102.9
104.7
118.8
155.3
276.3
4
1.9
2.3
9.2
2.8
3.1
2.5
3.1
2.5
3.7
7.1
2.8
4.2
10.3
11.4
26.1
4.1
3.2
6.1
7
6
28.9
1.8
1.4
0.8
0.6
6.5
1.1
0.8
0.8
0.8
0.4
0.9
5.2
0.6
2.2
8.5
2.5
20.4
1.2
1.6
2.1
2.6
2.3
14.2
-2
Al Olaby RR et al., PLOS One, 2014
Protein Expression Center - Caltech
Preliminary Binding Assays (Calibur ® )
Drug Lead 281816
Al Olaby RR et al., PLOS One, 2014
Department of Medicine – Stanford University
HCV Infection Inhibition Assays
(Pangenotypic)
Al Olaby RR et al., PLOS One, 2014
Subtypes
IC50 (µM)
HCVpp 1a
HCVpp 1b
HCVpp 2a
HCVpp 2b
HCVpp 4a
HCVpp 6a
2.95
4.66
2.22
2.93
3.44
3.30
Institute Pasteur De Lille - France
HCV Entry Inhibition
Al Olaby RR et al., PLOS One, 2014
Institute Pasteur De Lille - France
HCV Entry Inhibition
Al Olaby RR et al., PLOS One, 2014
Institute Pasteur De Lille - France
Cell to Cell Transmission
Al Olaby RR et al., PLOS One, 2014
Institute Pasteur De Lille - France
Testing 281816
Analogs
Institute Pasteur De Lille - France
Conclusion
• Ligand 281816:
• Inhibits HCV infectivity (HCVcc)
• Inhibits different stages of HCV entry
• Blocks HCV cell to cell transmission
• 1-3 uM affinity
Conclusion
• 281816 Analogs:
• 10µM 281816 reduced the level of infection to 29.5%.
• Analog A12 had no effect (102%)
• Other analogs had an effect that ranged from:
– Very little (A8, 92.7%)
– Comparable or better than 281816 like A7 and A16 (30% and
24%)
• 3 FDA-approved drugs were tested and were found to
exhibit mild inhibitory effect on HCV infection
(Loxapine, Amoxapine, Olanzapine).
Acknowledgements
• Dr. Hassan Azzazy – Chair of The Research CommitteeChemistry Department- The American University in CairoEgypt
• Dr. Rod Balhorn – SHALs Technologies INC. – Livermore/CA –
USA
Collaborators
#
Professor/ Lab
Contribution
Technology-USA
Dr. Jost Vielmetter
Protein Expression
Center
Surface Plasmon
Resonance Binding
Assays (Biacore t200)
2
Stanford University – USA
Dr. Shoshana Levy
Department of Medicine
3
University of California
Dr. Brett Chromy
Department of Pathology
and Laboratory Medicine
Provision of GST-CD81
protein / Vector
Flow Cytometry Assays
Dual Polarization
Interferometry binding
assays
1
University
California Institute of
Davis
4
Rutgers University -USA
5
6
Scripps Research Institute USA
7
John Hopkins University –
USA
8
Institute Pasteur De Lille France
9
Lawrence Livermore
National Laboratory
Dr. Joseph Marcotrigiano
Department of Chemistry
and Chemical Biology
Provision of
Recombinant E2 protein
Dr. Joel Freundlich
Department of
Pharmacology and
Physiology
Dr. Arthur Olson
Molecular Graphics
Laboratory
Advanced docking and
virtual screening runs
Dr. Photini Sinnis
Department of Molecular
Microbiology and
Immunology
Dr. Jean Dubuisson
Department of Molecular
& Cellular Virology of
Hepatitis C
Dr. Adam Zemla
Sporozoite Invasion
Assays
Dr. Saphon Hok
Preliminary Virtual
Screening Runs
HCV Infection Inhibition
Assays
E2 Homology Model
SHAL7153 synthesis
Questions
Homology Modeling
• Several homology modeling and structure analysis tasks were done using the coordinates of E2c as a
template.
• AS2TS  Final Structural Model
• StralSV algorithm  Identifies protein structures that exhibit structural similarities despite low primary
amino acid sequence similarity A structural search for similar fragments in proteins in the PDB that could
be used to model missing loop regions was performed
• SCWRL  Side-chain prediction was accomplished when residue-residue correspondences did not match.
• Residues that were identical in the template and E2 protein were copied from the template onto the
model.
• Contact-dot algorithm in the MolProbity software package Potential steric clashes were identified in
the unrefined model
• UCSF Chimera  constructed model was finalized
HCVcc
HCVpp
• Retroviral nucleocapsids surrounded by a lipid
envelope that contains authentic HCV
glycoprotein complexes.
• Ideal system to study receptor binding and
entry
• Used intensively to characterize neutralizing
antibodies.
Schematic Representation
Mechanisms of Action
INTERFERON:
• It is a major component of the host innate
response to viral infections.
• Induction of 100s of host genes known as
interferon stimulated genes (ISG), some of
which appear to block viral translation
Mechanisms of Action
RIBAVIRIN:
• Modulation of the immune response.
• Increase in the polymerase mutation rate
when incorporated causing an error
catastrophe.
• Inhibiting the host inosine monophosphate
dehydrogenase which will in turn lower the
GTP level in the cell.
Mechanisms of Action
PROTEASE INHIBITORS:
• They block the enzymatic activity required to
cleave the HCV polyprotein into individual
structural and non-structural individual ones.
Mechanisms of Action
NS5A INHIBITORS:
• Block viral production at an early stage of
assembly so that no viral RNA or nucleocapsid
protein is released
Mechanisms of Action
NS5B INHIBITORS:
• Blocks HCV Replication
Mechanisms of Action
NS4B INHIBITORS (Climizole HCL):
• Altering HCV RNA replication, viral assembly
and release.
Mechanisms of Action
CYCLOPHILIN ANTAGONISTS (Cyclosporin A):
• Inhibit the promoting effect of cyclophilin to
HCV NS5B’s binding to the viral RNA thus
blocking replication.
• Act via modulation of host factors for viral
replication
Mechanisms of Action
miRNA122 INHIBITORS (Miravirsen) – “IND”
• Inhibits the action of miRNA which binds to
HCV RNA and upregulates viral RNA levels.
Thus Miravirsen arrest the upregulation
process.
Mechanisms of Action
HMG CoA reductase inhibitors (statins):
• Have suppressive effect of HCV RNA levels
Lipinski’s Rule of 5
Lipinski's rule states that, in general, an orally active drug has
no more than one violation of the following criteria:
• No more than 5 hydrogen bond donors (nitrogen or oxygen
atoms with one or more hydrogen atoms)
• Not more than 10 hydrogen bond acceptors (nitrogen or
oxygen atoms)
• A molecular mass less than 500 daltons
• An octanol-water partition coefficient[5] log P not greater
than 5
• Polar surface area no greater than 140 Ǻ2
• 10 or fewer rotatable bonds (BAV- Binding Potency) (Rigid
vs. Floppy)
Lipinski’s key reference is Advanced Drug Delivery Reviews, 1997, 23, 3-25.
Ligand 281816
PSA<140
Rotatable bonds <10
M.wt. 357.568 Da
2 H-bond Acceptors
1 H-bond Donor
PSA 8
Rotatable bonds 2
xlogP = 4.14
Good oral bioavailability
JS-81
23 KDa
281816 15.5% M.Wt. of JS-81
JS-81 10-folds > 281816
CD81
• 26 Kda protein
• The gene is expressed in hemopoietic, endothelial and epithelial
cells. It is absent from erythrocytes, platelets and neutrophils.
• Member of tetraspanin family – Tetraspanin Web
• Promote muscle cell fusion and support myotube maintenance.
• Involved in signal transduction
• CD81 gene is localized in the tumor-suppressor gene region 
candidate gene for malignancies
CD81 Structure
Bertaux C , and Dragic T J. Virol. 2006;80:4940-4948
Proposed model for the role of EGFR in HCV entry.
Diao J et al. J. Virol. 2012;86:10935-10949