Rmc phenotypic screening

Building an in-silico phenotypic screening approach with Reaxys

Olivier BARBERAN

Senior Product Manager

Reaxys Medicinal Chemistry

February 2016

Target-directed and Phenotypic screening: parallel pathways to the

same goal

Phenotypic screening identifies compounds

that produce a biological response in a cell

or animal model

Target screening identifies

compounds that produce a biological

response on an isolated target

Phenotypic screening is the most successful approach

in Drug Discovery Strategy

• Majority of small-molecule first-in-class NMEs

that were discovered between 1999 and 2008

were first discovered using phenotypic assays

• 28 of the first-in-class NMEs came from

phenotypic screening approaches, compared

with 17 from target-based approaches.

• This is despite the current focus of small-

molecule drug discovery on target-based

approaches.

• A possible contributing factor to this trend could

have been a lag time between the introduction

of new technologies and strategies, and their

impact in terms of the number of approved first-

in-class NMEs derived from these approaches.

Bibliographic reference : How were new medecines discovered? D. Swinney

and J. Anthony (Nature Reviews), July 2011

A phenotype-led approach is still very success successful

Bibliographic reference : How were new medecines discovered? D. Swinney

and J. Anthony (Nature Reviews), July 2011

Phenotype - led vs. Target - led

However, a drawback is the need to use time-consuming genetic, chemical and/or

biophysical methods to identify the targets of compounds that are active (Target

deconvolution)

Phenotypic screening can find molecules that have more optimized drug-like

properties (such as cell penetration) than target-based screens.

Why using an in silico Phenotypic screening strategy in drug

discovery

Development of an In Silico Phenotypic screening approach in order to :

2) Building an automated tool in order to establish a target FingerPrint for

disease specific cell lines

1) identify pharmacological targets involved in cell based assay and

understand the molecular mechanisms of action of drugs (MMOA).

7

Scenario Melanoma cells predominantly occur in skin. Melanoma is less common than

other skin cancer. However, it is much more dangerous if it is not found in the

early stages. It causes the majority (75%) of deaths related to skin cancer.

Which are the targets involved in proliferation of melanoma cells lines ? And

which substances are acting on ?

Melanoma Cells

Search In RMC

Which Targets are involved in melanoma cells proliferation ?

Searching Melanoma cells line in Reaxys Medicinal chemistry using cell

taxonomy

Retrieving compounds active on Melanoma cells lines

66666 Substances tested on melanoma cell lines

Including actives and inactives substances Activity < 1µM

17600 Substances inhibiting melanoma cell lines

proliferation (IC50<1µM)

Compounds tested

melanoma cell lines

Melanoma cell lines

On which target active compounds on melanoma cells line are active

on?

Move from

cell lines

dimension

to target

dimension

BRAF, BRAF (V600E),

Ceramide glucosyltransferase

etc….

Targets

Substances

Cell lines

Targets

Affinity

On which target anti proliferative compounds (melanoma cell lines)

are active on?

BRAF (V600E),BRAF,

Ceramide

glucosyltransferase,

Melanocortin 1

Receptor, etc…

IC50<1µM

IC50<10 nM

Melanocortin 1

Receptor, ERK2,

BRAF, Ceramide

glucosyltransferase,

, BRAF (V600E), ,

Melanocortin 4

receptor,

CB1 receptors,

In silico Phenotypic screening Automated Process using

API and Pipeline pilot node

Target Fingerprint on A375 Melanoma cells : Overview

996

molecules

Cell line A375

IC50 <= 1µM

996 molecules +In

vitro Biological

results on targets

In vitro Biological

results on targets

Define activity class of

compounds

active / inactive on target

Target active

ratio calculation

Biological Threshold @ 1µM

Actives

Inactives

For each activity point!

PipelinePilot process to define activity class

Define activity class of compounds

For each compound and its given target:

Compound_Active_Ratio = (#Actives) / (#Actives + #Inactives)

Active if Active_Ratio ≥ 0.8

Inactive if Active_Ratio ≤ 0.2

Target Fingerprint on A375 Melanoma cells : Overview

996

molecules

Cell line A375

IC50 <= 1µM

996 molecules +In

vitro Biological

results on targets

In vitro Biological

results on targets

Define activity class of

compounds

active / inactive on target

Target active

ratio calculation

Target active ratio calculation

For each Target, count the number of Active and Inactive molecules

Target_Active_Ratio = (#Actives - #Inactives) / (#Actives + #Inactives)

Target FingerPrint for A375 cell line

#Molecules >= 20

Target FingerPrint of A375 cell lines

#Molecules >= 20

Average Activity

Rate in RMC

All compounds are

active on the target

Activity rate =1

All compounds are

not active on the

target

Activity rate = -1

Target Fingerprint of A 375 Melanoma cells based on Similar Compounds

996

molecules

Cell line A375

IC50 <= 1µM

3525 molecules

+In vitro Biological

results on targets

In vitro Biological

results on targets

Define activity class of

compounds

active / inactive on target

Target active

ratio calculation

3525

molecules

Chemical Similarity

Search 85%

Target Fingerprint of A375 based on similar Compounds

(85% similarity)

Target Fingerprint of A375 based on similar Compounds

(85% similarity)

#Molecules >= 20

Average Activity

Rate in RMC

A3R and A375 melanoma cells relationship in literature

• On A375 cells, A3R stimulation activated PI3K which induced Akt

phosphorylation resulting in reduced levels of phosphorylated

ERK1/2.

• A3R agonist (Cl-IB-MECA) inhibits cell proliferation

• Agonist effect abolished by A3 receptor antagonist

• No effect of A1, A2a and A2b receptors antagonists

• Merighi et al., 2005 (J. Biol. Chem.)

Conclusion

Using Reaxys API and Pipeline pilot an automated process was set up to

establish a target FingerPrint for disease specific cell lines.

Reaxys Medicinal chemistry provides High Quality data to identify

pharmacological targets involved in phenotypic screening and understand the

molecular mechanisms of action of drugs (MMOA).