Conditional control of protein
degradation with small molecule

2017.10.21
M2 Yamaji Kyohei
1Contents

1. introduction
2. Tools for conditional protein degradation
1. Peptidic PROTACs
2. Destabilizing Domain system
3. Auxin-Inducible Degron system
4. Hydrophobic Tagging
5. Small-molecule PROTACs
3. Summary2Overview of the technology and future application
Chemical tools to degrade and recover the protein of interest artificially
recoverydegradation

Small molecule

POI

Blue lightPOI

wash outPOIdarkness

How can this technology be applied to Catalysis medicine?
On/Off Switching System of the Catalyst with artificial stimuli
goalCatalyst

OFFON
Liganddegradation

Small molecule
Catalyst

LigandBlue light
substrate

substrate3

Two strengths of the technology
Chemical tools to degrade and recover the protein of interest artificially
recoverydegradation

Small molecule

POI

Blue light

POI

wash outdarkness

POI

Why was this technology invented?
✔Powerful tool for biological research✔new concept of potent drug
4

Tools to control protein degradation conditionally
No Genetic manipulation

1st generation PROTACs
2001 Protac-1

Genetic manipulation

Dr. Crews
Dr. Wandless

peptideDD system based

2004 Protac-4
2nd generation PROTACs
2008 MDM2based Small-molecule
HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based
Small-molecule2006 DD and Shld-1
Small-molecule

AID system

2009 AID system
Small-molecule2011 LID system
Small-molecule2013 B-LID system
Blue light

Dr. Kanemaki5Tools to control protein degradation conditionally
No Genetic manipulation

1st generation PROTACs
2001 Protac-1

Genetic manipulation

Dr. Crews
Dr. Wandlesspeptide
DD system based

2004 Protac-4
2nd generation PROTACs
2008 MDM2based Small-molecule
HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based
Small-molecule2006 DD and Shld-1
Small-molecule

AID system

2009 AID system
Small-molecule2011 LID system
Small-molecule2013 B-LID system
Blue light

Dr. Kanemaki
6Ubiquitin/Proteasome System (UPS)
Ubiquitin/Proteasome System
Ubiquitin is recycled
Ubiquitin activation by E1
Substrate recognition

Degradation byproteasome

Ubiquitin ligation to
substrate by E3
E1: ubiquitin-activating enzyme --- only 1 class in many organisms
E2: ubiquitin-conjugating enzyme--- several families
E3: ubiquitin ligase--- hundreds of kinds of ligases→substrate specificity

Ubiquitin is conjugated to E27

Works of Craig Crews; PROTACsPROTACs bridges E3 ligase and substrate to promote ubiquitination
Substrate recognition

PROTACPROTACThe design of first PROTAC
Target: MetAP-2

The structure of Protac-1
Ovalicin (OVA):
inhibits MetAP-2 covalently
IPP: IκBα phosphopeptide(β-TrCP subtrate)

E3: SCF E2
complex

Sakamoto, K M, et al. Proc. Natl. Acad. Sci. U.S. A. 2001, 98:8554

8

Short Summary of Peptidic PROTACs
No Genetic manipulation

1st generation PROTACs
2001 Protac-1

Dr. Crews

Genetic manipulation

Dr. Wandlesspeptide
DD system based

2004 Protac-4
2nd✔proof-of-concept
generation PROTACs
2008 MDM2based Small-molecule
×High molecular weight×poor cell permeability
HyT system
×metabolic instability
2011 cIAP-1 based 2011 Adamantane
small-molecule-inducedprotein
VHL based2012 (Boc)3Arg
degradation would be better2006 DD and Shld-1
Small-molecule

2009 AID system
Small-molecule2011 LID system
Small-molecule2013 B-LID system
Blue light2015 CRBN based
Small-molecule

AID system

Dr. Kanemaki
9artificial degron stabilized by small molecules
Concept of Small-molecule responsive destabilizing domains (DD)
Banaszynski, L. A., et al. Cell, 2006, 126:995

POI fused to DD is destabilized in
the absence of ligand

POI fused to DD is stabilized
in the presence of ligand

POI: protein of interest
DD: destabilizing domain
Concept of Ligand Inducible Degradation (LID) system

Shld-1

FKBP(DD)

10

Short Summary of Dr. Wandless’ work
No Genetic manipulation✔high specificity
Dr. Crews✔temporal and dose-dependent
1st generation PROTACs✔recovery of protein
2001✔lowProtac-1
toxicity peptide
2004 Protac-4
×genetic manipulation
×continued presence of Shld-1 (DD)
2nd generation PROTACs
Small-molecule driven technology
Small-molecule
2008without
MDM2based
genetic manipulation
waspursued.
HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based
Small-molecule

Genetic manipulation

Dr. Wandless

DD system based
2006 DD and Shld-1
Small-molecule

AID system

2009 AID system
Small-molecule2011 LID system
Small-molecule2013 B-LID system
Blue lightDr. Kanemaki
11

Short Summary of Dr. Wandless’ work
No Genetic manipulation✔high specificity
Dr. Crews✔temporal and dose-dependent
1st generation PROTACs✔recovery of protein
2001✔low
Protac-1
toxicity peptide
2004 Protac-4
×genetic manipulation
×continued presence of Shld-1 (DD)
Small-molecule driven technology
Small-molecule
2008without
MDM2based
genetic manipulation
waspursued.
2nd generation PROTACs HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based

Small-molecule

Genetic manipulation

Dr. Wandless

DD system based
2006 DD and Shld-1
Small-molecule

AID system

2009 AID system
Small-molecule2011 LID system
Small-molecule2013 B-LID system
Blue light

Dr. Kanemaki
12Development of HyT system
Concept of Hydrophobic Tagging (HyT) technology

Chaperone recognizes exposed hydrophobic regions of unfolded protein
Neklesa, T, K, et al. Nature 2012, 487, 308-9
and causes its degradation
recognize

RecruitHydrophobic tagging by small molecule may lead to protein degradation
Adamantyl group(Boc)3Arg
13

Development of HyT system
Hydrophobic tags destabilizing Halo Tag fusion proteinsHaloTag reactive linker(binds covalently to HaloTag)
By changing R, the protein fused to HaloTag is easily studied
HyT5
HA-HaloTag-luciferase
HyT12HyT16HyT13

High stability
High cell permeability

HyT21

HyT22
Neklesa, T, K, et al. Nat. Chem. Biol 2011, 7, 538-43

14Development of HyT system
HaloTag reactive linker(binds covalently to HaloTag)
HyT13
HyT13 destabilizes HA-EGFP-HaloTag
24hTime course of destabilization of HAEGFP-HaloTag by HyT13
1uM

Maximal degradation: 100nM
8h for full degradation, 1.5h for 50%
HyT13 can destabilize various proteins regardless of C- or
N-terminus HaloTag is fused
Neklesa, T, K, et al. Nat. Chem. Biol 2011, 7, 538-43
1uM, 24h15

Development of HyT system
HyT13 suppresses HaloTag-HRas1(G12V)-driven tumor
HyT13 destabilizes HA-HaloTagHRas1(G12V) in cell

HyT13 suppresses the formation of foci, a marker ofHRas1(G12V) tumor in cell

1uM, 24h

Negative controlfoci

HyT13 suppresses the tumor growth in vivo
Day 0

HA-HaloTag-HRas1(G12V)
expressing NIH3T3 cellsvehicle

Nude mouse

HyT13

Neklesa, T, K, et al. Nat. Chem. Biol 2011, 7, 538-43

16

Development of HyT system
Her3 ligand-amantadine conjugate can target Her312hTX2-121-1

Downstream of Her3
Xie, T, et al. Nat. Chem. Biol 2014, 10, 1006-12

HyT compound for Androgen receptor
RU59063
SARD279

SARD033
Gustafson, J, L, et al. Angew. Chem. 2015, 127, 9595-8

Linker length is important17

Short summary of HyT system
No Genetic manipulation

1st generation PROTACs
2001 Protac-1

Dr. Crews

Genetic manipulation

Dr. Wandlesspeptide
DD system based

2004 Protac-4
2nd generation PROTACs
2008 MDM2based Small-molecule
HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based
Small-molecule2006 DD and Shld-1
Small-molecule
AID system✔temporal and dose-dependent✔recovery of protein2009 AID system✔in vivo application Small-molecule×longhigh dose2011
LIDandsystem
Small-molecule
2013
B-LID
system to small
PROTACis improved
Blue lightmolecule inducedtechnology
Dr. Kanemaki
without genetic manipulation18

overview of 2nd generation PROTACs
No Genetic manipulation

1st generation PROTACs
2001 Protac-1

Dr. Crews

peptideFour types have been developed basedon the kind of E3 ligase.
Potent ligand for E3 ligase is necessary.

2004 Protac-4
2nd generation PROTACs
2008 MDM2 based

Small-molecule

HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg

MDM2 or cIAP-1 based PROTACs wereeffective at >1uM order.
Selectivity among proteins in the samefamily as POI differed between VHL andCRBN based PROTACs.
Linker length and position is important

VHL based PROTACs are mainly
explained here.

2015 CRBN basedSmall-molecule

First example of small-molecule PROTACs

E3

Ligand-ligand
conjugate

POIdegradation

E3POI

Small molecule ligands were selected for both of E3 and POI

SARM: Androgen
SARM-nutlin PROTAC
Receptor ligand Ki for AR=4nM
Weak degradation activity
10uM, 7h

Nutlin-3: ligand for
IC50: 13.6uM or
0.09uM(enantiomer) MDM2(E3 ligase)

Higher affinity ligands were sought after
Schneekloth, A. R., et al. Bioorganic & Medicinal
Chemistry Letters 2008, 18, 5904-820

improved small-molecule PROTACs (VHL based)
PROTACs targeting ERRα and RIPK2 were designed
Bondeson, D, P, et al. Nat. Chem. Biol 2015, 11, 611-617

ERRα: estrogen related
receptor

PROTAC_ERRαComp. 29: binds to ERRαE3 ligase

RIPK2: serine/threonine kinase

VHL ligand
Kd=320nM

Vandetanib: tyrosine kinase inhibitor
PROTAC_RIPK221

improved small-molecule PROTACs (VHL based)
PROTAC_RIPK2 can dose-dependently downregulate RIPK216h
Suggests the need of ternarycomplex formation

Dmax=10nM
DC50=1.4nM

Ternary complex formation and proteasome Time-course of degradation and recovery
is necessary for downregulation of RIPK230nM
Epimer: negative control

30nM
1uM
30nM
30nM

16h
Bondeson, D, P, et al. Nat. Chem. Biol 2015, 11, 611-617

22improved small-molecule PROTACs (VHL based)
PROTAC_RIPK2 induces ubiquitination of RIPK2 Catalytic nature of PROTAC_RIPK2
dose-dependently and time-dependently

Bondeson, D, P, et al. Nat. Chem. Biol 2015, 11, 611-617

23

improved small-molecule PROTACs (VHL based)
PROTAC_ERRα can dose-dependently downregulate ERRαNegative control

Proteasome
inhibitor

PROTAC_ERRα can target ERRα in vivo

Bondeson, D, P, et al. Nat. Chem. Biol 2015, 11, 611-617
24

HaloPROTACs (VHL based)
HaloTag can expand the variety of POI

Buckley, D, L, et al. ACS Chem. Biol. 2015, 10, 1831-7

HaloTag structure with ligand

Close-up25

HaloPROTACs (VHL based)
Linker position and length are important for HaloPROTACs’ activity
Buckley, D, L, et al. ACS Chem. Biol. 2015, 10, 1831-7

best26

HaloPROTACs (VHL based)
Proteasome inhibitor suppresses thedegradation caused by HaloPROTAC3

Buckley, D, L, et al. ACS Chem. Biol. 2015, 10, 1831-7

Time-course of GFP-HaloTag7 degradation

Proteasomeinhibitor

Recovery of GFP-HaloTag
after washout

Microscopic data

24h27

HaloPROTACs (VHL based)
HaloPROTAC3 can degrade GFP-HaloTag7
faster and more

HaloPROTAC3 can degrade other fusion
proteins with HaloTag7

HyT36

Buckley, D, L, et al. ACS Chem. Biol. 2015, 10, 1831-728

Short summary of small-molecule PROTACs
No Genetic manipulation

1st generation PROTACs
2001 Protac-1

Dr. Crews

Genetic manipulation

Dr. Wandlesspeptide

2004 Protac-4
2nd generation PROTACs
2008 MDM2based Small-molecule
HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based
Small-molecule

DD system✔temporal
andbaseddose-dependent✔recovery
protein2006
DD andofShld-1✔catalyticSmall-molecule
AID system✔application to various
proteins(HaloPROTACs)
2009 AID system✔in vivo application
Small-molecule×liganddependent
2011
LID system
Small-molecule2013 B-LID system
Blue light

Dr. Kanemaki29

Summary
・conditional protein degradation is powerful in context of bothbiological research and drug discovery
Peptidic PROTACs✔proof of concept×difficulty in practical use
DD and Shld1 based systems/AID✔high specificity due to genetic manipulation
×limited application due to genetic manipulation
HyT✔in vivo application, low toxicity
×relatively high dose
Small-molecule PROTACs✔in vivo application, quite low dose・ room for optimization30

Tools to control protein degradation conditionallyNo Genetic manipulation

1st generation PROTACs
2001 Protac-1

Genetic manipulation

Dr. Crews
Dr. Wandlesspeptide
DD system based

2004 Protac-4
2nd generation PROTACs
2008 MDM2based Small-molecule
HyT system
2011 cIAP-1 based 2011 Adamantane
VHL based2012 (Boc)3Arg
2015 CRBN based
Small-molecule2006 DD and Shld-1
Small-molecule

AID system

2009 AID system
Small-molecule2011 LID system
Small-molecule2013 B-LID system
Blue light

Dr. Kanemaki
31