TRAFTAC
A generalizable method fortranscription factor degradation
230413
Literature seminar #1
M1 Takeuchi
1Contents

Contents◆Introduction:➢Transcription factor (TF) and disease➢Difficulty of direct-targeting TF strategy➢c-Myc activation in cancer◆Existing strategies for c-Myc inhibition◆New strategy for TF degradation: oligo-TRAFTAC➢c-Myc➢Brachyury◆Summary2

Contents

Contents◆Introduction:➢Transcription factor (TF) and disease➢Difficulty of direct-targeting TF strategy➢c-Myc activation in cancer◆Existing strategies for c-Myc inhibition◆New strategy for TF degradation: oligo-TRAFTAC➢c-Myc➢Brachyury◆Summary3

Introduction

Transcription factor (TF) and disease• TF coordinates the correct gene expression levels
for maintaining normal cellular functions.
• Dysregulation of TF activity implicates in numerous cancers.
4Introduction

c-Myc activation in cancer• c-Myc is a super-TF that regulate the transcription ofat least 15% of the entire genome.
• Gene amplification is one of the most frequentmechanisms for c-MYC activation in human cancers.
Chen, H. et. al. Signal Transduction and Targeted Therapy. 2018, 3(1), 1-7
Duffy, M. et. al. Cancer Treatment Reviews. 2020, 94, 102154-102160

5Introduction

Difficulty of direct-targeting TF strategy• Enzymes have ligandable pockets and small-molecule inhibitors.
• Development of small-molecule inhibitors for TFs is challenging.
(← lack of enzymeatic activity and ligandable pockets)
6

Contents

Contents◆Introduction:➢Transcription factor (TF) and disease➢Difficulty of direct-targeting TF strategy➢c-Myc activation in cancer◆Existing strategies for c-Myc inhibition◆New strategy for TF degradation: oligo-TRAFTAC➢c-Myc➢Brachyury◆Summary7

Existing strategies

Existing indirect c-Myc-targeting strategies

Upstreamfactors
Downstream
factors
Targeting
upstream/downstream
factors

Wang, C. et. al. Signal Transduction and Targeted Therapy. 2021, 6(1), 1-148

Existing strategies

Comparison of each indirect c-Myc-targeting strategy
TargetClassicCompoundadvantages

disadvantages

c-MYC gene

Smallmolecule

・Small molecule

・Off-target
・difficult finding ofsuitable compounds

c-MYC mRNAsiRNA・high selectivity
・longer duration of effect・faster discovery of
drug candidates

・low membrane
permeability

c-Myc-Max heterodimer

peptide /protein

・suitable for PPI inhibition ・low stability to peptidase・difficult finding ofsuitable compounds

Upstream / downstream
factors

Smallmolecule

・Small molecule

・Off-target
・difficult finding ofsuitable compounds

TF has few small molecule inhibitors.

Direct targeting of Myc protein,→ Indirect targeting of Myc

PROTAC is difficult.
9

Existing strategies

PROTAC (PROteolysis TArgeting Chimera)
・Structure of 1st PROTAC

Ligand for SCF(E3 ligase)・Structure of 1st small-molecule PROTAC
・Structure of VHL ligand PROTAC
AHPC: ligand for
VHL(E3 ligase)
Kd=150nM
Nutlin-3: ligand for
MDM2(E3 ligase)10

ContentsContents◆Introduction:➢Transcription factor (TF) and disease➢Difficulty of direct-targeting TF strategy➢c-Myc activation in cancer◆Existing strategies for c-Myc inhibition◆New strategy for TF degradation: oligo-TRAFTAC➢c-Myc➢Brachyury◆Summary

Samarasinghe, K. T. G. et. al. RSC Chemical Biology. 2022, 3(9), 1144-1153

11

oligo-TRAFTAC

Oligo-TRAFTAC (TRAnscription Factor TArgeting Chimera)・Mechanism

・E-box sequence (CACGTG) (c-Myc)

・Brachyury-binding DNA sequence

AHPC: VHL (E3 ligase) ligand

12

oligo-TRAFTAC (c-Myc)

Oligo-TRAFTAC induced c-Myc degradation.
WB
HeLa cell lysates

HeLa cells

13oligo-TRAFTAC (c-Myc)

Neddylation inhibition disrupts cullin RING E3 ligase function.

NAE: NEDD8 Activating Enzyme

VHL-CUL2-RBX1 complex

Neddylation inhibition↓Disruption of cullin RING

E3 ligase function
AHPC: VHL (E3 ligase) ligand
Vriend, J., & Nachitigal, M. W. Cancers. 2021, 13(11)Fouad, S. et. al. Frontiers in Physiology. 2019, 10(October)14

oligo-TRAFTAC (c-Myc)

OT7 induced c-Myc degradation via the proteasomal pathway.
HEK293T cells

1 µM

HEK293T cells

10 µM← Neddylation inhibitor

Neddylation inhibition↓Disruption of cullin RING
E3 ligase function(CUL2)

VHL ligand

Modification site

OT75‘

OT103‘15

oligo-TRAFTAC (c-Myc)

OT7-mediated c-Myc degradation inhibited cell proliferation.
HeLa cells, 48 h16

oligo-TRAFTAC (brachyury)

Oligo-TRAFTAC induced brachyury degradation.
WB

Brachyury-GFP expressing HEK293T cell lysates

Brachyury-GFP expressing HEK293T cells

17

oligo-TRAFTAC (brachyury)

OT3 induced brachyury degradation via the proteasomal pathway.
10 µM

Brachyury-GFP expressing HEK293T cells
1 µM← Neddylation
inhibitor

Neddylation inhibition↓

Disruption of cullin RING
E3 ligase function(CUL2)

VHL ligand18

oligo-TRAFTAC (brachyury)

Xeno Nucleic Acid (XNA)

Increased stability
against nucleases

19oligo-TRAFTAC (brachyury)

Catalytic brachyury degradability by OT17

UM-Chor1 cells:
Human clival chordoma cell
(ヒト斜台脊索種細胞)

20

oligo-TRAFTAC (brachyury)

Microinjection of OT17 induced tail deformation.
Zebrafish embryos

48 hNTLA: Brachyury homolog in zebrafish

21

ContentsContents◆Introduction:➢Transcription factor (TF) and disease➢Difficulty of direct-targeting TF strategy➢c-Myc activation in cancer◆Existing strategies for c-Myc inhibition◆New strategy for TF degradation: oligo-TRAFTAC➢c-Myc➢Brachyury◆Summary22

SummarySummary• PS-modified oligo-TRAFTAC
improved its in vivo stabilityand demonstrated oligo-

TRAFTAC activity in zebrafish.

• Oligo-TRAFTAC can be rapidly
designed for many nonligandable DNA-binding TFs.

(Chemical biology tool andpotential therapeutic strategy)

Samarashnghe, K. T. G. et. al. RSC Chemical Biology. 2022, 3(9), 1144-1153

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