Shantani Proteome Analytics Pvt. Ltd.€¦ · Peptide-tagged-small molecule coupled...
Transcript of Shantani Proteome Analytics Pvt. Ltd.€¦ · Peptide-tagged-small molecule coupled...
Target Identification: Shantani’s
Chemical Proteomics Tool-Box
ShantaniTM
Leaders in Proteome Analysis
Overview
Innovative Chemical Proteomics organization
Operates from Independent Lab Space @ NCL Innovation Center; Pune, India
Est. 2010 – with Seed Funding from Incubation Park
Dec 2011 – 1st Small Business Innovation Grant to validate the proprietary tech
April 2012 – Private VC firms (India Innovation Fund and Blume Ventures) buys the Stakes
Vision: To be the champion in Chemical Proteomics technologies globally
Mission: Deliver rightful Targets of bioactive compounds for better understanding of
disease biology and development of therapeutics
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www.shantani.com
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Dr. John E. Hale
• Current: Hale Consulting
• Elily Lilly, 22 yrs, Biotech
Research group directing
Biomarkers & Target
Discovery Programs
• Hybritech
Dr. Dongping Zhong
• Current: Prof. of Chemistry &
Biochemistry, The Ohio State
University: research interests
include biomolecular
interactions & ultrafast protein
dynamics
• Robert Smith Prof. of Physics
Dr. Peter T. Kissinger
• Current: Director-Prosolia, IN
& Part Time Prof of
Chemistry-Purdue Uni
• Founder-Bioanalytical
Systems Inc, BASi,
Phlebotics
• Fellow-AAPS
Shrikumar Suryanarayan
• Current: CEO- C6 Synergy
• CEO- Bioscience Cluster, Govt of
India
• Director General-ABLE, India
• President-R&D, Biocon
• Chief Scientific Advisor, Biocon, 24
yrs
Scientific Advisory
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Phenotype / Cell
Based Drug Leads
Target Based
Lead compounds
Drug Repositioning
Target and MoA
Information
Off-targets
Information
New Target &
Elucidation of MoA
Rational Lead Optimization
Tox Profiling & Candidate
Selection Strategies
Scientific Rational for
New Indications
Drug Discovery
Method
Drug
Development Shantani’s Expertise
Shantani’s Expertise and Value Creation
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Target ID Tool-Box
Proprietary Technology
Traditional Chemical
Proteomics
Proprietary in-silico Tools
Fit for Purpose Business Models
Continuous Tech Devpt
Validation Tools
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Shantani’s Tool Box
Value Creation
Operations and Data
Product-ivity Matrix
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a) Location specific
peptide
coupled “bait
molecule”
b) Biological System c) Activity Read-Outs d) Subproteome Selection
X Membrane
Cytoplasm
Nucleus
Prov. Patents 645/MUM/2011, 975/MUM/2012
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1) Sub-Cellular Location Specific target capture technology: Step1
X
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d) Mass-spectrometry e) TARGET
Prov. Patents 645/MUM/2011, 975/MUM/2012
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c) Lysis and affinity
isolation
a) Cytoplasmic
probe
b) Incubation
with cells
Sub-Cellular Location Specific target capture technology: Step2
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Peptide-tagged-small molecule coupled mass-spectrometry based
approaches can identify the relevant targets of small-molecules from cell-lysates.
Reference: J. Proteome Research Vol. 7(8), pp: 3490-97
Year 2008
Year 2009
Year 2012
Peptides of specific properties can be utilized in capturing the small-
molecule targets from cytoplasm and nucleus of the cells
Reference: J. Proteome Research Vol. 8(8), pp: 3951-57
Unique, well-optimized, synthetic peptides can carry the molecule in
subcellular location specific manner (membrane, cytoplasm and / or
nucleus). A functionally relevant concentration of these location specific
peptide-coupled-molecule can capture the target in location specific manner
and assists in narrowing down the false positive identification
Prov. Patents 645/MUM/2011, 975/MUM/2012
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The Methodology Development
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Cytoplasmic Peptide Nuclear Peptide Membrane Peptide
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Location Specificity of the Peptides
The peptides were coupled to FITC, incubated with the cells and later the cells were imaged
using fluorescence microscopy to establish the location specificity of the peptides.
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Immobilize Test
Compounds
On Solid Support / Biotin
Solid
Support
Apply Protein Source
Identify Bound Proteins
using Mass-Spec
Methods
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2) Traditional Chemical Proteomics Workflow
Solid
Support
Extensive Deconvolution exercise using
Mass-spec data, control and competitive
inhibition experiments to identify the specific
target(s)
TARGET
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3) In-Silico Tools
Docking with the Targets Relevant to Phenotype /
Chemotype
Identification of Putative Target > > Data Mining
Around the Targets (Isoforms & Homology
Modelling)
Listing of Targets and
Down-Stream wet-sciences
Validation
Function Relevant Computational Modelling – Relies on Docking the ‘bait-
molecule’ with the Proteins that are relevant to observed phenotype as the starting
point
Random Biopolymer Interaction Modelling – Relies on Docking the ‘bait-molecule’
with > 350 well characterized active sites of different target proteins as the starting
point
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4) Validation Tools
In-silico – Extensive Docking Studies on and around the identified targets
Biophysical Methods – Utilization of Absorption, Fluorescence and ITC based
methods for analysis of ‘bait-molecule-target interaction’
Molecular Biology Methods – siRNA based transient knock down of targets and / or
Over expression of Target Proteins for functional gain or loss
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5) Activities in Development
2014 2015
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4
Timeline
Activities
Different Target-Id
solutions/tools (e.g.
underivatized ‘bait-molecule’)
Optimization of
Network Analysis for
MoA studies
Exte
nd
ing th
e p
oo
l of T
arg
et V
alid
atio
n s
tudie
s
New Targets (Patient samples,
target profile, proteome wide…)
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PoC1: Capture of Membrane Bound Target(s) GPCRs
PoC2: Identifying Sub-cellular Location of Target-Activity
PoC3: Better Deconvolution of Targets
PoC4: Identifying Target(s) and understanding MOAs of novel Bioactive Molecules
Client-1
Client-2
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6) PoC Expts. & Applications
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Molecule: BL11282 Stimulates insulin secretion in unknown fashion.
a) BL11282 does not block ATP-dependent K+ channels
b) BL11282 activity appears to be sensitive to the inhibition of PKA
c) The increase in intracellular calcium upon BL11282 administration
makes only a minor contribution to insulin secretion
d) BL11282 directly influences exocytosis process…..
The Cellular Binding Partners of BL11282 are not know and its action
mechanism is elusive hence the molecule can not be rationally
optimized…
In this PoC we will utilize our chemical-proteomics tools and strategies to
identify and validate primary targets of BL11282
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BL11282
PoC-4: Identification of Targets & understanding of MoA of BL11282
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BL11282 BL-(but) NH2
References: BL11282 - Structure-Activity Relationship (SAR) data
• Bioorganic and Medicinal Chemistry 15: 3284-3265 (2007)
• Bioorganic and Medicinal Chemistry 15: 6782-6795 (2007)
Active site of the molecule
(based on SAR data)
Molecule Derivatization /
Peptide attachment site
Synthesis of small-molecule derivative for the preparation of Target
capturing Probes
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Peptide 1, 2 and 3 are three
different peptides that
targets three different
locations of the cell.
BL11282 was coupled to
these peptides and
functional activity (insulin
secretion ability) of the
constructs was evaluated.
Functional activity of BL11282 derivatives and Peptide coupled Target
capturing Probes
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Immobilization of
Molecule
Incubation
with MIN6 cell
lysate
Washing and
Eluting the Matrix
Proteins in the
Elution Analyzed
by
SDS-PAGE /
Identified by Mass
Spectrometry
Immobilization of molecule on Solid Support for Target Capture and
typical Pull-Down protocol
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Lane 1 = Markers
Lane 2 & 3 = Cell lysates and
Column Flow Through
Lane 4 & 5 = High Salt Column Wash
Lane 6 & 7 = Low Salt Column Wash
Lane 8 = Column Elution in Low
Salt + 1 mM BL11282
Lane 9 = LDS Buffer
1 2 3 4 5 6 7 8 9
Distinct Elution Profile over wash conditions were optimized
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All the runs were within 10% error margin
1 2 3
0.2
0.3
0.4
0.5
Rf
Va
lue
s
(Re
lativ
e M
igra
tion
Dis
tan
ce)
Band Number
Rf Value Variation in Three Distinct Gel-Bands
in Four Different Experiments
Reproducibility over multiple experiments
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Immobilization of
Molecule
Incubation
with MIN6 cell
lysate
Washing and
Eluting the Matrix
Proteins in the
Elution Analyzed
by
SDS-PAGE /
Identified by Mass
Spectrometry
Immobilization of
Molecule
Incubation with
MIN6 cell lysate
+ pre-
incubation with
free molecule
Washing and
Eluting the Matrix
No Immobilization
Incubation
with MIN6 cell
lysate
Washing and
Eluting the Matrix
Control Experiments
Proteins in the
Elution Analyzed
by
SDS-PAGE /
Identified by
Mass
Spectrometry
Immobilization of molecule on Solid Support for Target Capture and
Control Experiments
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Lane 0 = Std
Lane 2 = Control Matrix
Lane 4 = Elution from BL11282 Matrix
Lane 6 = Elution from BL11282 Matrix
(cell-lysate pre-incubated with
BL11282)
Lane 1,3, 5, 7 & 8 = Buffer
*Each of these lanes are proteins pooled from three
different experiments and at least two such runs were
carried out for protein identification.
Note:The proteins were separated only for 7 minutes on
SDS-PAGE gels.
Distinct Elution Profile over Control Experiments
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+ SB202190 - SB202190
Peptide Coupled to SB202190 Control Probe
- SB202190
Probe
Cell Culture
Condition
Number of Identified
Peptides per protein P2 P3 P1
Specificity Ratio = P3 / (P1+P2+P3)
Specific Identification = Protein with specificity ratio > 0.8
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Process of Identifying specific Binding Partners
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Recycling Endocytosis
Or Exocytosis Pathways
Glycolysis /
Gluconeogenesis
Pathways
Fatty Acid Metabolism
Pathways
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Analysis of Specifically Bound Proteins
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Small-molecule affinity based
chromatographic methods will
elute protein complexes
associated with the target protein
rather than The Only Target
Protein.
Proteins that may be directly
interacting with the small-molecule
can be prioritized based on their
binding probability to the targets
Continued Analysis using ‘in-silico’ direct Protein-Ligand Docking
Chemical
Proteomics may
identify the Target
and associated
protein complex
Proteins directly interacting with
the small molecule-based on
binding probability to targets
Calculating possible free
energy: Protein-Ligand
docking
a)
b)
c)
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Target Predicted Free Energy
(kcal/mol)
Predicted (Kd)
uM
1 -8.71 0.41
2 -8.61 0.49
3 -8.23 0.93
4 -8.03 1.30
5 -7.94 1.50
6 -7.54 2.96
7 -7.52 3.07
8 -7.43 3.60
9 -7.44 3.77
10 -7.4 3.79
Prioritizing Identified Protein Targets based on probability that it may
interact with the ‘bait-molecule’
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BL11282 Inhibits Identified Target 1 and 2 – Biophysical Validation
Inhibition of Target – 1 by BL11282 (Spectrophotometric Assay Monitoring Product of
the Enzyme Reaction)
Inhibition of Target – 2 by BL11282 (ELISA based Cell Based Assay)
40%
50%
60%
70%
80%
90%
100%
110%
0 100 200 500
% E
nzym
e A
cti
vit
y
Concentration of BL11282 (nM)
-10%
10%
30%
50%
70%
90%
110%
NaturalSubstrate +
BL11282(1:0)
NaturalSubstrate +
BL11282(1:1)
NaturalSubstrate +
BL11282(1:2)
NaturalSubstrate +
BL11282(1:7)
NaturalSubstrate +
BL11282(1:10)
% E
nzym
e A
cti
vit
y I
nh
ibit
ion
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siRNA and Pharmacological Inhibition Based Functional Validation of Target(s)
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Both the targets if inhibited together increases insulin secretion !!!
• Cell Type – MIN6 (Mouse Insulinoma)
• Target knock down by siRNA was
optimized and established separately.
• Data are plotted after subtracting
respective control.
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Study of molecule SAR allowed preparation of
functionally active target capturing probe
Appropriate chemical proteomics tools were
used to identify cellular binding partners of
BL11282
Identified binding partners were thoroughly
analyzed using in-silico tools and their putative
relation with phenotype to deconvolute the
targets
Prioritized targets were validated using
biophysical and molecular biology tools
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Problem Analysis
Picking right Chemical
Proteomics Tool
Identifying Rightful Targets
Validating the targets
Conclusions from BL11282-Chemical Proteomics
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Client-1: Target ID of Quercetin-Derivative
a) Proprietary Derivative
(R- is client specific) b) Preparation of bait-molecule Specific Matrix
No visible difference in Elution Profile > Multiple Runs > Protein Pool
> Mass-Spec Based Target ID and Deconvolution
Validation
c) Silver Stained Gel
Elution Control Matrix Elution bait-molecule
specific Matrix
d) Western Blot Analysis using Antibody against the
Deconvoluted Target
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Client-2: Deconvoluting 180 degree opposite targets
Proteins with
pink circle were
identified in pull-
down
In-silico studies provided explanation that identified proteins
with predicted higher kd are involved in apoptosis and with
lower kd are linked to proliferation.
Clients’ proprietary molecule
At low-concn, molecule induces cell proliferation + @ 20X
concnmolecule induces cell dealth through apoptosis
SAR for cell proliferation –well established
Shantani’s Chemical Proteomics Tools
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L-0: Brainstorm-Consult-
Suggest Best Fit Platform
+ Model (scientific &
business team)
Off-Target Toxicity
PDD or TDD
Target -Id ? Go/No Go
Decisions
MoA
Un-known
Drug Repositio
ning
7) Collaborate with Whom & How: Business Models
Shantani
Project Basis
FTE
RS/B2S
L0/L1 L2/L3 RS B2S
*Note: L0,1,2,3 on next slide; RS-Risk Sharing; B2S-Build2Suit
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Target ID
Methods
Points of
Considerations
Typical # of
Deconvoluted
Targets
Target
Validation and
MoA Analysis
Time
Req.
Level 0 Consulting, Cost, Tech Subject to Tech Subject to Tech ~1 week
Level 1 Fast & Inexpensive ~25-30 None 5-7 weeks
Level 2 More Specific Target
Information ~12-18 ‘in-silico’ analysis 7-10 weeks
Level 3
Shantani’s
Advanced
Technology
In-depth target & MOA
information allowing
‘Go / No Go’ decisions
~4-8
For almost all
Identified Targets +
‘in-silico’ studies +
wet-lab validation
12-25
weeks
Different Levels of Engagement with Shantani + Productivity Matrix
OR
New Methods/Technologies: Polymer based-underivatized molecule (client focused)
Off-the-Shelf: Proteomics related other MS based solutions can be provided based on the
requirements.
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Operational processes & HR
Internationally experienced management & technical teams
Documented protocols to protect clients’ intellectual property
Secure IT-enabled data management processes & eLNB for fast data retrieval
Laboratory Infrastructure
2000 square feet of lab space (5 shared instrument facility rooms)
Lab blocks guarded by double layers of security
Well practiced Environment Health & Safety (EHS) policies
Routine calibration of instruments
Scientific rigor and customer focus attitude are our biggest assets
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Operations
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Step Activity Timelines
0 Interest from clients 24-48 hrs to respond
1 Signing of CDA (if needed) and sharing
project information
Largely client and project
dependent
2 Shantani send quotation detailing
milestones, timelines of deliverables
Within 7 days after project
description
3 Client revises / accepts the quotation and
Shantani initiate the project
Client and project dependent
4 Shantani provides milestone based report • Typical Target ID - 6-8 weeks
• Target ID, Validation/ MOA
project length 4-6 months
5 Shantani and Client agrees on the final
outcome, project considered close
Data support stays for another 12
months
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Working with Clients: Overview
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Publications & AppNotes
Saxena C., Affinity-Based Chemoproteomics with Small Molecule-Peptide Conjugates ,
Methods Mol Biol (2012) 803 pp. 39-54.
Saxena et al, Capture of Drug Targets from Live Cells Using a Multipurpose Immuno-
Chemo-Proteomics Tool , J. Proteome Res. (2009) 8 pp. 3951-3957.
Saxena C., Small-molecule affinity chromatography coupled mass spectrometry for
drug target deconvolution, Expert Opinion on Drug Discovery (2009) 4 pp. 701- 714.
Saxena et al, An Immuno-Chemo-Proteomics Method for Drug Target Deconvolution ,
J. Proteome Res. (2008) 7 pp. 3490-3497.
Technology Patent: Provisional Patents 645/MUM/2011, 975/MUM/2012
Application Notes: http://www.shantani.com/Notes.htm
More Questions: http://www.shantani.com/FAQ.htm
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Hope we can work together to improve the drug
discovery process
Thank you.
Shantani Proteome Analytics Pvt. Ltd.
100 National Chemical Laboratories Innovation Park
Dr. Homi Bhabha Road, Pune, MH, India, 411 008
Email: [email protected] (Aditya Ghosh-Global Business Dvpt)
Phone: M: +91-7774037073, T: +91-20-64103918
http://www.shantani.com