Proteolysis targeting chimeras (PROTACs) offer a fast and reversible chemical knock-down approach to control protein function. The impact of PROTAC platform has changed the landscape of drug discovery and development[1-3]. Medicilon’s PROTAC drug discovery technology platform covers the currently popular target protein ligands. We have established a linker system with an extensive collection of bifunctional linkers. Together with our expanding E3 ubiquitin ligase binder library, we can efficiently synthesize a substantial amount of highly active PROTAC bispecific small molecules, which would have the potential to significantly facilitate ithe drug discovery and development process. In addition, Medicilon has established as well as improved the PROTAC biological screening and testing platform throughout the pre-clinical stages. Medicilon’s strong technical expertise and flexible service models allow individualized and customized projects ranging from sole chemical synthesis to in vitro and/or in vivo service, and to more comprehensive integrated package support. Our laboratories are US FDA and China NMPA accredited, and we will soon receive the European OECD GLP accreditation as well. We have successfully filled over 150 IND submissions worldwide. Medicilon is confident in providing efficient, cost-effective, and professional services to support our clients in reaching their drug development milestones. https://www.medicilon.com/platform/protac-drug-discovery-research-platform/

1. References
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www.medicilon.com
Results
Efficacy Studies and PK/PD of PROTAC Drugs In Vitro &Vivo
Abstract
Zhuo Mao, Xingquan Ma, and Xuedong Dai
Chemistry & Biology Department, Medicilon, China
Corresponding Author: Xuedong Dai Email: xuedongdai@medicilon.com.cn
Proteolysis targeting chimeras (PROTACs) offer a fast and reversible chemical knock-down approach to
control protein function. The impact of PROTAC platform has changed the landscape of drug discovery
and development[1-3]
. Medicilon’s PROTAC drug discovery technology platform covers the currently popular
target protein ligands. We have established a linker system with an extensive collection of bifunctional
linkers. Together with our expanding E3 ubiquitin ligase binder library, we can efficiently synthesize a
substantial amount of highly active PROTAC bispecific small molecules, which would have the potential
to significantly facilitate ithe drug discovery and development process. In addition, Medicilon has established
as well as improved the PROTAC biological screening and testing platform throughout the pre-clinical
stages. Medicilon’s strong technical expertise and flexible service models allow individualized and
customized projects ranging from sole chemical synthesis to in vitro and/or in vivo service, and to more
comprehensive integrated package support. Our laboratories are US FDA and China NMPA accredited,
and we will soon receive the European OECD GLP accreditation as well. We have successfully filled over
150 IND submissions worldwide. Medicilon is confident in providing efficient, cost-effective, and
professional services to support our clients in reaching their drug development milestones.
Case: PROTAC In Vitro Evaluation
Case: Plasma Stability Studies of PROTAC
Medicilon Case:
[1] Si-Min Qi, et al. PROTAC: An Effective Targeted Protein Degradation Strategy for Cancer Therapy.
Front Pharmacol. 2021 May 7;12:692574.
[2] Galen Andrew Collins, et al. The Logic of the 26S Proteasome. Cell. 2017 May 18;169(5):792-806.
[3] Jared A M Bard,et al. Structure and Function of the 26S Proteasome. Annu Rev Biochem. 2018 Jun
20;87:697-724.
[4] Xin Han, et al. Strategies toward Discovery of Potent and Orally Bioavailable Proteolysis Targeting
Chimera Degraders of Androgen Receptor for the Treatment of Prostate Cancer. J Med Chem. 2021
Sep 9;64(17):12831-12854. doi: 10.1021/acs.jmedchem.1c00882.
Summary
PROTACs offer a fast and reversible chemical knock-down approach to control protein function in the
cell. The impactof the PROTAC platform has changed the landscape of drug discovery and develop-
ment. Medicilon’s PROTAC drug discovery technology platform covers many of the popular target pro-
tein ligands. We have established an extensive collection of bifunctional linkers. Together with our ex-
panding E3 ubiquitin ligase binder library, we can efficiently synthesize highly active PROTAC bispecific
small molecules, which have the potential to significantly facilitate the drug development process. In
addition, Medicilon has established as well as improved the PROTAC biological screening and testing
platform throughout the pre-clinical stages. Medicilon’s strong technical expertise and flexible service
models allow individualized and customized projects ranging from sole chemical synthesis to in vitro
and/or in vivo service, and to more comprehensive integrated package support. As of the end of 2022,
Medicilon has successfully assisted in the clinical approval of 3 PROTAC drugs by NMPA and/or FDA
and has more than 20 PROTAC projects under development.
Medicilon has accumulated rich PROTAC experience working on a wide range of popular target pro-
teins with high affinity small molecules and small molecule fragment compound libraries, a wide range
of E3 ubiquitin ligase binders, and an extensive collection of bifunctional linkers. We leverage our broad
chemistry capabilities and capacity with fully integrated biological and pre-clinical validation of the candi-
date PROTAC molecules.
Case: PROTAC In Vivo Evaluation
Western blot of anti-IRAK4 on
OCI-LY10 cells treated with
IRAK4 degrader-1.
Percentage degradation of
HiBiT-IRAK4in HiBiT-IRAK4
overexpression OCI-LY10 cells.
O C I-L Y 1 0 c e lls -W e s te rn b lo t
C o n c e n tr a tio n o f c o m p o u n d ( µ M )
In
h
i
b
it
io
n
R
a
t
e
(
%
)
1 0 -5
1 0 -4
1 0 -3
1 0 -2
1 0 -1
1 0 0
1 0 1
-2 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IR A K 4 d e g ra d e r-1
D C 5 0 = 3 6 n M
O C I-L Y 1 0 c e lls -H iB iT -IR A K 4
C o n c e n tr a tio n o f c o m p o u n d ( μ M )
D
e
g
r
a
d
a
t
io
n
o
f
H
ib
iT
-
IR
A
K
4
(
%
)
1 0 - 5
1 0 - 4
1 0 - 3
1 0 - 2
1 0 - 1
1 0 0
1 0 1
-2 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IR A K 4 d e g ra d e r-1
D C 5 0 = 4 2 n M
Background
Proteolysis targeting chimeras (PROTACs), also known as bivalent
chemical protein degraders, are heterobifunctional molecules that
degrade specific endogenous proteins through the E3 ubiquitin
ligase
pathway. A PROTAC molecule structurally connects the protein of
interest (POI)-binding ligand and the E3 ubiquitin ligase (E3) ligand
through an appropriate linker.
PROTAC technology is an effective tool to degrade endogenous
target proteins through the ubiquitin-proteasome system (UPS)[1-3]
.
POI
POI
POI
Crosslinker
PROTAC
E3 Ligase
E3 Ligase
E3 Ligase
Proteasome
PROTAC
PROTAC
Ub
Ub
Ub
Ub
Ub
Ub
Ub
POI
Ligand
E3
Ligand
E2
E2
E2
Pharmacology Models
In vivo efficacy study of HCC827 model (EGFR degrader-1 & positive control Gefitinib)
0.00
5.00
10.00
15.00
20.00
25.00
30.00
3 6 9 12 15 18 21 24 27 30 33 36 39 42
Body
Weight(g)
Days Post Treatment
HCC827 Xenograft Tumor Model in Female Nude Mice
Body Weight (g) (Mean±SEM)
0
200
400
600
800
1000
1200
1400
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42
Tumor
Volume
(mm
3
)
Days Post Treatment
HCC827 Xenograft Tumor Model in Female Nude Mice
Tumor Volume (mm
3
) (Mean±SEM)
Group 1 Vehicle PO QD x 3weeks
Group 2 Gefintinib 3mg/kg PO QD x 3weeks
Group 3 EGFR degrader-1 2mg/kg PO QD x 3weeks
Group 4 EGFR degrader-1 1mg/kg PO QD x 3weeks
Group 5 EGFR degrader-1 0.5mg/kg PO QD x 3weeks
Group 6 EGFR degrader-1 0.2mg/kg PO QD x 3weeks
Group 7 EGFR degrader-1 3mg/kg PO BIW x 3weeks
species
mouse
rat
dog
monkey
human
plasma stability (T1/2, min)
>120
>120
>120
>120
>120
Evaluation of ARD-2128 for Its Plasma Stability:
PROTAC AR degrader ARD-2128 was evaluated
for its plasma stability in mouse, rat, dog,
monkey, and humans. ARD-2128 has excellent
plasma stability in all the five species.
Plasma Stability of ARD-2128 in Five Species (Human, Mouse, Rat, Dog, and Monkey)
Case: PK Studies of PROTAC
compound route
dose (mg/kg) T1/2 (h)
AUC0–t
(h·ng/ml)
Cl
(ml/min/kg)
Vss (L/kg)
route
dose (mg/kg) T1/2 (h) Tmax (h) Cmax (ng/ml)
AUC0–t
(h·ng/ml)
F (%)
26 IV 2 17.8 11,035 1.9 2.7 PO 5 12.0 4.0 1389 20,600 75
27 IV 2 11.5 15,759 1.7 1.5 PO 5 11.2 4.0 980 14,588 37
28 (ARD-2128) IV 2 27.6 13,299 1.2 2.7 PO 5 18.8 4.7 1304 22,361 67
33 IV 1 21.0 4334 2.2 3.8 PO 3 12.4 6.0 207 3127 24
34 IV 1 25.5 2565 3.2 6.8 PO 3 67.8 4.7 134 2550 33
Medicilon PROTAC Technology Platform
Scaffold: > 150 E3 ligands available including Cereblon, VHL, MDM2, IAP, etc.
Team: > 300 dedicated well-trained chemists
Know-how: > 6 years PROTACs experience Comprehensiveness: > 20 ongoing projects
Building Block: > 300 advanced linkers Available
Method
Biochemical assays Cellular Assays
Study binary/ternary complex formation
Target ubiquitination & degradation assay
Biophysics approach with SPR
Target-E3 ligase interaction assay
Target degradation assay(DC50)
Pomalidomide(CRBN binder) competition
experiment show that IRAK4 degrader-1
degrades IRAK4 through CRBN.
MG132 inhibition experiment show that
IRAK4 degrader-1 degrades
IRAK4 through proteasome pathway.
μ
μ
In cell Western of BRM show that ACBI1(BRM degrader)
degrades BRM in dose response manner.
C o n c e n tr a tio n o f c o m p o u n d ( μ M )
G
r
o
w
t
h
i
n
h
i
b
it
io
n
o
f
O
C
I
-
L
Y
1
0
c
e
l
ls
(
%
)
1 0 - 4
1 0 - 2
1 0 0
1 0 2
-2 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
IC50
IRAK4 degrader-1
0.3796
Growth inhibition effect of IRAK4
degrader-1 on OCI-LY10 cells.
50
150 Xenograft models
35 Orthotopic models
30 Syngeneic models
30 Humanized models
5 GEM models
50 CNS disease models
20 Cardiovascular & metabolic disease models
10 Inflammatory & immune diseases models
10 Digestive system disease models
5 Ocular diseases models
5 Other disease models
The pharmacokinetics (PK) of five highly potent AR degraders (compounds 26, 27, 28, 33, and 34) are
evaluated in mice with both intravenous and oral administration.
Summary of PK Data for Compounds 26, 27, 28, 33, and 34 in Male ICR Mice
We have successfully established a series of in vitro protein-based assays for PROTACs screening.
We also have successfully generated a series of cell lines based on different POI.
We also have developed a series of cell line-based validation assays.
We hope that our detection system can speed up the development of new drugs and contribute to an-
ti-tumor therapy.