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: aghosh@shantani.com (Aditya Ghosh-Global Business Dvpt)

Phone: M: +91-7774037073, T: +91-20-64103918

http://www.shantani.com