Helquat derivatives, unique G-quadruplex (c-myc ... · Helquat derivatives, unique G-quadruplex (c-myc) stabilizing anticancer molecules Dr. Filip Teplý May 11th 2015 Institute of

Helquat derivatives,

unique G-quadruplex (c-myc)

stabilizing anticancer molecules

Dr. Filip Teplý May 11th 2015

Institute of Organic Chemistry and Biochemistry AS CR

https://www.biovaria.org/

Institute of Organic Chemistry and Biochemistry AS CR Dr. Filip Teplý 2

Helquats downregulate c-Myc

Our target is c-Myc


Our target is c-Myc

Transcription factor Oncogene



Our target is c-Myc


angiogenesis

glucose and glutamine metabolism initial phase of cell growth

senescence activation of ribosomal protein genes

nontranscriptionally stimulating translation

activation of the miR-17-92 cluster

mitochondrial biogenesis

energy metabolism

DNA replication

cell-cycle control

amplification

chromosomal translocation

downstream of WNT, Hedgehog, Notch, TGF-β, RTK signaling pathways

sustained increase in Myc protein expression

cell size increase

circadian gene response

terminal differentiation of HSC, pro B – cells and skin stem cells

chromatin modification

tumor maintenance

cell adhesion

apoptosis

protein synthesis

pluripotency

biomass accumulation

cell proliferation

deregulated Notch and WNT pathways

microRNAs and lncRNAs

tumor initiation

epithelial-mesenchymal transition

miR-9

metastasis

increased genomic instability

sustained ROS induction

downstream of PI3K

nucleolar hypertrophy

glycolysis under hypoxia


telomere function

chromosomal rearrangements

insulin signaling

sustained tumor growth

ARF and p53 checkpoint

DNA damage

reprogrammed metabolism

replicative stress

cell migration NAD+ synthesis




angiogenesis



nontranscriptionally stimulating translation



energy metabolism

DNA replication

cell-cycle control

amplification




cell size increase




tumor maintenance

cell adhesion

apoptosis

protein synthesis

pluripotency


cell proliferation



tumor initiation


miR-9

metastasis



downstream of PI3K




telomere function


insulin signaling



DNA damage

reprogram metabolism

replicative stress


MYC does everything


Our target is c-Myc


Our target is c-Myc


angiogenesis



nontranscriptionaly stimulating translation



energy metabolism

DNA replication

cell-cycle control

amplification




cell size increase




tumor maintenance

cell adhesion

apoptosis

protein synthesis

pluripotency


cell proliferation



tumor initiation


miR-9

metastasis



downstream of PI3K




telomere function


insulin signaling



DNA damage


replicative stress



MYC is involved

in all cancers

unless proven otherwise

Tansey 2014 NJS

MYC does everything


Our target is c-Myc


angiogenesis



nontranscriptionaly stimulating translation



energy metabolism

DNA replication

cell-cycle control

amplification




cell size increase




tumor maintenance

cell adhesion

apoptosis

protein synthesis

pluripotency


cell proliferation



tumor initiation


miR-9

metastasis



downstream of PI3K




telomere function


insulin signaling



DNA damage


replicative stress



MYC is involved

in all cancers

unless proven otherwise

Tansey 2014 NJS

Elevated expression of c-Myc is associated with

tumor aggression and poor clinical outcome

including increased rates of metastasis,

recurrence, and mortality

MYC does everything


Lin et al. 2012 Cell

How a single protein can have such

profound effect in so many and varied types

of cancer?



Lin et al. 2012 Cell

How a single protein can have such

profound effect in so many and varied types

of cancer?

c-Myc expression promoters and enhancers of

all active genes

amplifying the active genes’ transcription

cells bloated with excessive RNAs

and proteins

Oncogenic c-Myc is an amplifier, not a specifier, of gene expression

in cancer cells



What may be the Achilles’ heel of cancer cells that

overproduce c-Myc?

Their reliance on c-Myc; they are “Myc-addicted”

Tuning down the levels of transcription might be catastrophic

for the Myc-addicted cancer cells

c-myc gene

Lin et al. 2012 Cell, Brooks & Hurley 2009 NRC



We produced a library of G-quadruplex stabilizers

Our screening strategy:

1. FRET assay & competition with DNA duplex

2. Luciferase reporter assay

luciferase G4 c-myc gene

3. Viability tests – XTT assay HUVEC and „CA-46 test“

4. RT qPCR Myc mRNA expression

5. Protein expression WB & MS

7. Best ligands mouse experiments

6. Cell Cycle analysis


Siddiqui-Jain et al. 2002 PNAS


Siddiqui-Jain et al. 2002 PNAS

We produced a library of G-quadruplex stabilizers

Our screening strategy:

1. FRET assay & competition with DNA duplex

2. Luciferase reporter assay

luciferase G4 c-myc gene

3. Viability tests – XTT assay HUVEC and „CA-46 test“

4. RT qPCR Myc mRNA expression

5. Protein expression WB & MS

7. Best ligands mouse experiments

6. Cell Cycle analysis


Δ Tm > 7°C Downregulation to 70% of the control

IC50

Downregulation

Downregulation

Accumulation in G1

MTD


Results


Perfect cell penetration

Compound 5

5. c-Myc protein downregulation (24h & 48h) 3. IC50 for Raji and CA-46 cells (48h)

4. c-Myc mRNA downregulation (24h)

CA-46 Raji

Control Compound 3

G1 37% G1 41%

G1 53% G1 71%

6. Accumulation in G1 phase (24h)


R&D Status

Focused screen of 2nd round candidates • activity on 12 cell lines

Further study of cellular effects • activity profiling

In vivo studies • pharmacokinetics, pharmacodynamics, bioavailability and genotoxicity • disease mouse model – tumor xenograft

• lead optimization (> 1500 original compounds have been prepared and tested) • in vitro testing • preliminary toxicity (MTD data) • solubility is being optimized for some candidates • 4 lead structure candidates selected


Plans


Summary

• c-Myc downregulation is a hot target of cancer research

• original small molecules with strong IP position

• potential to become first-in-class G-quadruplex targeting molecules

• favourable general toxicity (mice MTD)


IP Status

• two patent applications (priority dates 2013 and 2014)

• further patent is expected

• the project is offered for co-development and/or licensing

Have a look at our poster for more results and discussion


Acknowledgements

Contacts Dr. Filip Teplý [email protected]

Dr. Jaromír Zahrádka [email protected]

Supported by

IOCB, AS CR, v.v.i. (RVO: 61388963)

Project InterBioMed LO1302 from Ministry of Education of the Czech Republic


Martin Fusek Jaromír Zahrádka Miroslav Hájek Pavlína Novotná

Paul Reyes Lukáš Severa Michael Jirásek Pradeep Devadig Harish Talele Manoj Sonawane Vishwas Joshi Tomáš Kapal

Erika Kužmová Jaroslav Kozák Veronika Komárková Martin Zavřel Markéta Šmídková Karolína Müllerová Jana Günterová Lenka Pohlová

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Helquat derivatives, unique G-quadruplex (c-myc ... · Helquat derivatives, unique G-quadruplex (c-myc) stabilizing anticancer molecules Dr. Filip Teplý May 11th 2015 Institute of