Natural Product-inspired Macrocyclic Toolbox for

“Undruggable Targets”: Our Wnt Journey

Prabhat AryaFounder and Chief Scientific Officer, SignMod

Chief Scientific OfficerSignMod-Transcell Oncologics, Joint Venture

www.signmod.org

prabhat.arya@signmod.org

June 25, 2019 1

June 25, 2019

Underestimated the complexity of human biology!

2

…Our current understanding of molecular pathways is insufficient as a platform for effective pharmaceutical discovery…

…Several biotechnology companies have focused on the known elements of a few key pathways to target them with new medicines. But for the genome to be translated into medicines with any reliability and regularity, far more work needs to be done. Defining the role of pathways in complex diseases will undoubtedly take many years…

Mark FishmanPresident

NIBR, 2002-2016

Defining the target (could be

enzymes or isolated

protein(s)

Structural information

on the target – finding the pocket

Structural guided chem

/med chem

Lipinski’s Rule of 5

Classical Approaches

• Complex, multiple protein-

protein interactions

• Dynamic and temporal

processes!

• Regulation (normal) and de-

regulation (disease)

tough journey From genes to pathways:

Science 2003, 300, 445-452

The sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation....

However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior.

June 25, 2019 5

• Map large surface area• Shallow surface • Combination of several weak

interactions• Extended hydrophobic

interactions• Possible hot spots

2014

6

O

Me Me

H OH

O

O

Me

OMe

OH

OH

Me

Me

Me

MeO O

O N

O

H

FK506

O

O

O

OHH2N

O

OO O

H

H

O OO

H

Eribulin

7

• present complex 3D architectures • dense display of stereo-defined groups • challenging task in placing them on the drug

discovery path!

• excellent track record as small molecule modulators of protein-protein interactions

• can serve as a good source of inspiration for developing

novel scaffolds in the drug discovery arena!

Finding Molecules as Effective Modulators of Pathways!

BioactiveNatural

Products

Exploring MacrocyclicChemical Space!

• Cyclic compounds• Large surface area• Pre-organization• Less freely rotating bonds

• Enhanced cell permeation

Building A Chemical Toolbox for Challenging “Undruggable” Targets

Natural ProductInspired

Sub-structures

Natural Product Fragments as Sub-structures

Our working model

Key features in our design:

• 3D architectures • sufficient complexity• stereochemical and skeletal diversity • synthesis in a reasonable time-scale • easy to follow-up medicinal chemistry studies

From Arya Research Team:

Chem. & Biol.163 (2005)

Curr. Opin. Chem. Biol. 247 (2005)

Chem. Rev. 1999 (2009)June 25, 2019 8

June 25, 2019 9

Angew Chem 2001

Chem & Eng News 2004

June 25, 2019 10

Natural Product-Inspired, Functionalized 14- and 17-Membered Rings

Macrocyclic Toolbox

1

2

3

Indoline / Tetrahydroquinoline (Alkaloids) and Benzofuran (Flavonoids)-Inspired

Macrocyclic Toolbox

Glyco-based Macrocyclic Toolbox

Examples of Our Early Work

11

Org. Lett. (2013) Eur. J. Org. Chem. (2013)

Org. Lett. (2013)

Org. Lett (2008)Eur. J. Org. Chem. (2014)

Angew Chem (2005)Eur. J. Org. Chem. (2013)

12

Example 1: Rapamycin fragment-based Macrocyclic Toolbox

Rapamycin

from amino

alcohol der

Org. Lett. 480-483 (2015)

Recent Examples

13

• Isolated from Red Sea sponge

Negombata magnifica

• Potent G-actin Inhibitors

• Latrunculol A is more potent

compared to other family members

O

O

HN

S

H

HO H

O

O

H

O

O

HN

S

H

HO H

O

O

H

OH

O

O

OH

HN

S

H

HO H

O

O

H

Latrunculin B Latrunculin A Latrunculol A

Example 2: Latrunculin-derived Macrocyclic Toolbox

O

HN

O

O

OO

R1

O

MeO

PMBN

S

O

H

H HO

HN

O

O

OO

R1

O

MeO

PMBN

S

O

H

H H

13 15

17

13 15

17

15-Membered ring

Diversity

site

Org. Lett. 472-475 (2015)

O

O

O

OHH2N

O

OO O

H

H

O OO

H

2932 30

31

34

Eribulin

Practical and scalable synthesis

Precise

fragment-based

Diastereomeric

fragment-based

A

B

14Org. Lett. 468-471 (2015)

Example 3: Macrocyclic Toolbox based on Eribulin Sub-structures

Our ApproachOur Approach

Jimmidi, Krishna Reddy, Arya Org. Lett. 2015, 17, 468-471 15

single diastereomer

single diastereomer

Synthesis of Diastereomeric Fragment

16

June 25, 2019 17

Synthesis Special Issue (2017)

Different Macrocyclic Architectures

18

Leading to A New Family of Macrocyclic Architectures

Isomeric A

Precise

Isomeric B

O

OP1

HOOH

O

EtOCCH

H108

3

7

Submitted for publication

Growing Faith in Emerging Chemical Biology Model

Modern Organic Synthesis

novel chemical toolbox toexplore the scope of biologically relevant chemical space// natural product-inspired compounds

placing them onto the drug discovery path!

clinically-relevant questions

Cell Signaling Biology(biological questions/novel assays)

cellular/zebrafish studiesin vivo studies

Phenotypic Unbiased Screens

Discovery of functional small molecules

understanding the mode of action – biophysics, genomic,CRISPR-Cas9 tools

June 25, 2019 19

• Tumor heterogeneity

• Every patient is different!

• Cancer cells vs cancer stem

cells/tumor initiating cells

• Selective killing – understanding of

signaling pathways involved

• Tumor metastasis – Understanding of

signaling pathways involved

Challenges in Cancer Challenges in Cancer

Nat Rev Drug Discov 2014June 25, 2019 20

Cancer Stem Cell-derived Signaling Pathways

Cell Stem Cell 2019

our interest

our interest

our interest21

Cancer Patient-derived Banking Platform

• >900 Indian patient samples banking with primary cancer cells, tumorspheres (enriched with cancer stem cells).

• Patient-derived tumor xenograft (1st

generation, 10 samples as proof of concept).

• Novel procedure for obtaining tumorspheres that are rich in tumor initiating cells/cancer stem cells

June 25, 2019 22

Indian Patient-derived Transcriptomic Study

Tumor Biopsy Primary Cancer Cells Tumorspheres

• Oncogenic Signatures

(Broad Inst)

• Cancer Hall marks

(Broad Inst)

• Apoptosis

K-RAS

c-Myc

Wnt

Unpublished 23

Transcriptomic Data – Breast Cancer

TS PCC Control

TS: Tumorspheres

PCC: Primary Cancer

Cells

Down

UpKRAS

target genes

Myc target genesTS PCC Control

unpublished24

Breast Cancer

TS PCC Control

TS: Tumorspheres

PCC: Primary Cancer CellsDown

Up

Wnt target genes

TS PCC Control Anti apoptotic genes

Unpublished

25

Wnt Signaling and Stemness

• “Undruggable” domain

• Multiple cell surface interactions

• Multiple protein-protein interactions (Cytoplasm)

• Multiple DNA-protein-protein interactions (Transcription/Nucleus)

• Play a key role to maintain stemness / epithelial to mesenchymal transition / critical for cancer stem cells / metastasis

Nat Rev Drug Disc 2014

Transcription as the target

June 25, 2019 26

30,000 cells per well //

Incubate the plates

overnight in CO2 incubator

Add agonist (Wnt 3a), antagonist (DKK1), control (DMSO)

Add 100 μL luciferase substrate mixture

Incubate for 10 min and then read the chemi-luminescence signal

Primary Screening – WnT Inhibitors

IWR-1

XAV

ICG001

Controls

June 25, 2019 27

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

DMSO WNT3A IWR1 61 62 63 64 65 66 67 68

Luci

fera

se a

ctiv

ity

Con. 10 uM

Wnt Luciferase Screening Data

@10 µM

SM-2015-61/62/63/64/65SM-2015-66/67/68/69/70

open 17-memmacrocycles

Family 1 Hits

Five

Analogs

Single olefin geometry

The effect of small molecule on cytoplasmic protein complexes?

The effect of small molecule on transcriptional machinery – related to multiple protein-protein // DNA-protein-protein interactions?

Tools and Methods:

RT-PCRWestern blotsRNA sequencing Quantitative proteomicsFinally, the target pull-down plan

Our Chemical Biology Journey!

On the Functional Biology Front:

- The effect on tumorspheres from established cell lines?

- The effect on tumorspheres from patients-derived cells?

- The effect on organoids synthesized from patient organs/tissues?

- The effect on stemness / EMT / cancer stem cells?

RT-PCR Data – Transcription Genes

IWR-1 (control)

O

O

NH

O

OBnO

OO

O

O

O

H

H

NH

O

OBnO

OO

O

SM-61 SM-66 SM-110 SM-115

@ 10 uM

0

0.5

1

1.5

2

2.5

DMSO 61 66 IWR-1

Fold

Change

TCF4

0

0.5

1

1.5

2

2.5

3

DMSO 61 66 IWR-1

Fold

Change

LEF

0

0.2

0.4

0.6

0.8

1

1.2

1.4

DMSO 110 115 IWR-1

Fold

Change

TCF4

0

0.5

1

1.5

2

2.5

3

DMSO 110 115 IWR-1

Fold

Change

LEF

30

RT-PCR Data (contd.) IWR-1Selected genes related to…

Migration / Invasion

66: macrocycle

61: precursor

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Fo

ld c

ha

nge

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Fo

ld c

ha

nge

0

0.2

0.4

0.6

0.8

1

1.2

Fo

ld c

ha

nge

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6F

old

ch

an

ge

Metastasis Proliferation

00.20.40.60.8

11.21.41.6

Fo

ld c

ha

nge

With Family 1 hits 31

Western Blots

IWR-1

Moving on to proteins

O

O

NH

O

OBnO

OO

O

O

O

H

H

NH

O

OBnO

OO

O

SM-61 SM-66 SM-110 SM-115 32

Western Blots (contd)

GAPDH

TCF1

β-catenin

GAPDH

TCF1

β-catenin

GAPDH

Cyclin D1

c-Myc

p300

GAPDH

Cyclin D1

c-Myc

p300

June 25, 2019 33

34

Axin 2: 66 Showed Axin 2 stabilization compared to 61. 110 Showed Axin 2 stabilization compared to 115.

LEF 1: 61, 66, 110 and 115, all showed decreased expression levels of LEF1 but 110 was more potent.

CBP: 66 and 110 decreased the expression of CBP

Axin 2

LEF1

CBP

GAPDH

Western Blots (contd)

Source: http://www.jnsci.org/content/36

p300

CBP: (CREB, cAMP response element-binding protein)-binding protein

O

O

NH

O

OBnO

OO

O

O

O

H

H

NH

O

OBnO

OO

O

RNA Seq Study

SM-2015-61 vs SM-2015-66small molecule treated transcriptome study

SM-2015-66

SM-2015-61

2017

anti

pro

Bhatia’s lab, McMaster

What’s next

ICG001

35

RNA Seq Study

SM-2015-66

SM-2015-61Cell Migration Gene Set

IWR-1 (control)

down

up

Family 1 Hits

O

O

NH

O

OBnO

OO

O

O

O

H

H

NH

O

OBnO

OO

O

36

Metastasis Breast Cancer Gene Set

IWR-1 (control)

down

up

RNA Seq Study (contd)

SM-2015-66

SM-2015-61

Family 1 Hits

O

O

NH

O

OBnO

OO

O

O

O

H

H

NH

O

OBnO

OO

O

June 25, 2019 37

Epithelial to Mesenchymal Transition

down

up

RNA Seq Study (contd)Stemness

Transcription

down

up

Translation

Gene sets / pathways are taken from the public data bases:KEGG: https://www.genome.jp/kegg/genes.htmlGSEA: http://software.broadinstitute.org/gsea/msigdb/REACTOME: https://reactome.org/

open macrocycle

39

Cell Migration Gene SetEpithelial to Mesenchymal

Transition Gene Set

down

up

O

O

NH

O

MeOBnO

O

O

O

O

NH

O

MeOH

H

BnO

O

O

SM-110

SM-115

Family 2 Hits

Effect on Tumorspheres (G2*) from HCT 116

DMSO IWR1 SM-61SM-66

(Macrocycle)

48

Hr

72

Hr

96

Hr

12

0H

r1

68

Hr

G2* = 2nd generation

100 µm

SM-2015-66

SM-2015-61

Family 1 Hits

O

O

NH

O

OBnO

OO

O

O

O

H

H

NH

O

OBnO

OO

O

41

0

0.5

1

1.5

2

DMSO 110 115 IWR-1

Fo

ld C

ha

ng

e

LEF

0

1

2

3

4

5

6

7

DMSO 110 115 IWR-1

Fo

ld c

ha

ng

e

TCF4

Wnt Transcription-related Genes

RT-PCR Analysis: Small Molecule Treated Tumorsphere

(G2) from HCT 116 (contd)

0

1

2

3

4

5

DMSO 61 66 IWR-1

Fo

ld c

ha

ng

e

LEF

0

1

2

3

4

5

6

7

DMSO 61 66 IWR-1

TCF4

SM-61 SM-66

O

O

NH

O

MeOBnO

O

R1

O

O

O

NH

O

MeOH

H

BnO

O

Bn

O

SM-110

SM-115

June 25, 2019 42

RT-PCR Analysis: Small Molecule Treated Tumorsphere

(G2) from HCT 116

Metastasis

0

2

4

6

DMSO 110 115 IWR-1

Fo

ld c

ha

ng

e

VIMENTIN

0

2

4

6

DMSO 61 66 IWR-1

VIMENTIN

0

5

10

DMSO 110 115 IWR-1

Fo

ld c

ha

ng

e

CD44

0

5

10

DMSO 61 66 IWR-1

Fo

ld c

ha

ng

e

CD44

0

0.5

1

1.5

DMSO 110 115 IWR-1

Fo

ld c

ha

ng

e

CD133

0

0.5

1

1.5

DMSO 61 66 IWR-1

Fo

ld c

ha

ng

e

CD133

Stemness

0.000

0.500

1.000

1.500

2.000

2.500

Fo

ld c

ha

ng

e

SNAIL

0.000

0.500

1.000

1.500

2.000

Fo

ld C

ha

ng

e

SNAIL

0

1

2

3

4

DMSO 110 115 IWR-1

Fo

ld C

ha

ng

e

HEF1

0

1

2

3

4

DMSO 61 66 IWR-1

Fo

ld c

ha

ng

e

HEF1

Migration and Invasion

June 25, 2019 43

Effect on Tumorspheres (G2*) from Patient Buccal Mucosa (Indian Patient 001) G2 = 2nd generation

DM

SO

IWR

-1S

M-6

6S

M-1

10

SM

-115

0 Hr 72 Hr 144 Hr 168 Hr

O

O

H

H

NH

O

OBnO

OO

O

SM-66

SM-110

SM-115

44100 µm

Our Working Hypothesis

CBP

Our small molecules as transcription

inhibitors

RNA seq with 110/115

Quant proteomics with66, 110 and 115

Evaluation on patient-derived organoids

Target pull-down studies

Medicinal chemistry // Protein degradation direction

Ongoing Studies

June 25, 2019 45

Filing two US provisional patents related to macrocyclic inhibitors of Wnt pathway / b-catenin-CBP-TCF interactions

Academic (India) - Graduate Students (2009-2018): Madhu AeluriSrinivas Chamakuri Ravikumar Jimmidi Shiva Krishna Reddy Bhanudas Dasari Srinivas Jogula Saidulu Konda Mahender KhatravathNaveen KumarJagan Gaddam

Our Biotech Team:Cell signaling/stem cell tech: Raveendra Babu Vamshi KrishnaAnusha KolusuSamarpita TarafderManjushriSubhadra DravidaNGS: Madhu MohanChem toolbox/med chem: Jagan GaddamNaveen Kumar Mahender KhatravathAnand Neha KardamPrabhat Arya