1. Targeting Drug Resistance Mutations: Discovery of EUD-GK-91 Chemical Series
Fabio Zuccotto1, Luca Mologni2, Anna Bernardi3, Francesco Airaghi3, McDonald Neil4, Rakhee Chauhan4, Kevin Read5, Ola Opemolu5 and Mauro Angiolini1
1Eudendron Srl – Italy; 2Università Bicocca – Italy; 3Università degli Studi, Milano – Italy; 4Francis Crick Institute, London – UK; 5Drug Discovery Unit, Dundee – UK
Eudendron has developed innovative Molecular Designs to generate highly designed ATP competitor compounds that prevent the insurgence of drug resistance mutations by addressing both gain-of-function mutation and mutation potentially
interfering with ligand recognition. Eudendron molecular design strategy does not rely on specific target features and is not chemotype dependent hence has the potential to be a kinome-wide approach applicable to different chemotypes. Taking
advantage of its specific know-how, Eudendron is developing a new generation of more effective anti-cancer agents for targeted therapy. An example of Eudendron successful molecular design is represented by the EUD-GK chemical series. The
lead compound, EUD-GK-91, is highly selective with high in vitro antiproliferative activity against several cancer cell lines driven by the oncogenic kinase targets and their resistant mutated forms. This set of kinase targets plays a crucial role in
several human cancers and, in particular, in non-small cell lung cancer. Eudendron approach has achieved proof-of-concept in cell.
ALK
Antiproliferative IC50 nM
COMPANY DRUG
BaF3
NPM-
ALK WT
RESISTANCE MUTATIONS (BaF3 NPM-ALK CELLS) ALK-
HL60L1196M C1156Y L1152R G1269A S1206Y F1174L
EUDENDRON EUD-GK-91 3 4 2 2 5 17 21 >16000
PFIZER CRIZOTINIB 42 459 307 121 490 460 250 430
NOVARTIS CERITINIB 14 42 71 44 9 23 69 nd
ROCHE ALECTINIB 20 32 38 47 104 25 70 nd
IGNYTA ENTRECTINIB 12 20 5 4 240 157 200 nd
EUD-GK-91 MoA in BaF3 NPM-ALK L1196M
NPM/ALK+
NPM/ALK-
23 nM
36 nM
52 nM
17’000 nM
2’900 nM
1’430 nM
EUD-GK-91 antiproliferative IC50 in lymphoma cell lines
BACKGROUND RET
EUD-GK-5 and EUD-GK-69 MoA
in TT & MZ-CRC cells compared
to Vandetanib
Xray structure RET:EUD-GK-91
EORTC 2016 – Poster 194
• Following a chemocentric approach Eudendron
develops highly designed lead-like compounds
with a focus on drug resistance and improved
selectivity
• The chemical series EUD-GK was developed
from the scaffold reported in Figure 1 presenting
an unprecedented pattern of substitution
• Preliminary data (Table 1) highlighted a
favourable activity profile against oncogenic
kinase targets involved in a specific sub-set of
non-small cell lung cancer patients: RET, ROS1,
DDR2, TRK and ALK
X
N
N
HR2
R3
R5
R1
R4 n
Figure 1: General formula of
EUD-GK chemical series
n=0,1
Ki values of EUD-GK-91 for
selected kinases (nM)
Enzymatic profile of EUD-GK-91 against a panel of selected
kinases
ALK 14
IC50 > 1 µM: VEGFR2, ITK, FGFR1, MERTK, BTK, PKCα, EPHB4, ABL,
EPHA2, AURORAB, CDK6, CDK4, PKA, FLT3, FGFR3, IR, IGF1R, EGFR,
EPHA8, ERBB2, MELK, FAK, CDK2, AXL, FGFR4, KIT, VEGFR1, VEGFR3,
RON, JNK1, MAP4K4, PDK1, PYK2, ZAK, ZAP70, LRRK2, MEK2, PKCβ,
EPHB3, GRK7, IRAK4
ALK L1196M 22
ALK F1174L 23
TRKB 44
RET 60
RET V804M 42
RET M918T 60 IC50 > 5 µM: TTK, S6K, PLK1, PLK3, PIM2, PAK4, PAK6, PAK7, P38α,
MEK1, MARK1, JNK1, JNK3, GSK3α, ERK2, EEF2K, TYRO3, AKT1, AKT2,
NIK, TEC, EPHA7, SGK2, TBK1, MET, ASK1, CK1α, CK2α, BRAF, ERK1,
HIPK2, CHK1, CHK2, CAMK4, PDGFRα, WEE1, AKT3, MARK2, MARK3,
MARK4, mTOR, P38β, ROCK1, PIM3, MAP3K1, IKKβ, MYLK
DDR2 75
DDR2 T654M 42
ROS1 80
Potency on target. When tested in antiproliferative experiments EUD-GK-91
showed activity in the low nM range against ALK+ lymphoma cell lines whereas
the compound was inactive against ALK- cell lines
EUD-GK-91 MoA in Karpas299 cell line
• Potent activity was also observed against mutated forms of the kinase
targets
• At the light of this positive data the series was further developed and
investigated. Typically R4 was used to modulate the activity against one
of the targets whereas R1 and R3 were exploited for molecular
properties optimisation.
Drug Resistance Mutations.
Compounds were evaluated in engineered BaF3 cell
lines harbouring the NPM-ALK L1196M drug
resistance mutation. A well defined SAR was
observed and a number of additional analogues
displayed single digit nM potency similar to EUG-
GK-91 (Table 2). Similar results were obtained in
Karpas299 cell line. EUD-GK-91 showed superior
activity against resistance mutations when
compared to known ALK inhibitors (Table 3)
Table 1: Biochemical Activity Profile. EUD-GK-91 is a potent inhibitor
displaying a favourable selectivity profile
ROS1-DDR2-TRK
Favourable developability profile. EUD-GK series is characterised by
good solubility, good in-vivo clearance, acceptable half-life time and a
moderate volume of distribution
Mechanism of Action. EUD-GK-91 showed a potent inhibition of ALK
phosphorylation in both Karpas299 and BaF3-NMP-ALK L1196M cell lines
STRUCTURAL BIOLOGY
Biochemical Activity. EUD-GK compounds are
very potent RET inhibitors with a remarkable
selectivity against the gatekeeper mutation
V804M (Table 4)
EUDENDRON
IC50 RET
V804M (nM)
IC50 RET
WT (nM)
WT/V804M
selectivity
EUD-GK-5 2 57 29
EUD-GK-3 3 155 52
EUD-GK-1 6 66 11
EUD-GK-37 4 179 45
EUD-GK-69 6 39 7
PONATINIB 10 18 2
EUDENDRON
TPC1
(CCDC6-RET)
TT (RET
C634W)
MZ-CRC
(RET M918T)
LUNG -
THYROID
THYROID
MEN2A
THYROID
MEN2B
EUD-GK-5 23 31 50
EUD-GK-69 37 18 34
EUD-GK-1 26 83 65
EUD-GK-3 56 161 130
EUD-GK-4 109 38 64
Cellular Activity Profile. Compounds retained a
strong potency in RET driven cancer cell lines
(lung and thyroid) with a verified mechanism of
action. Interestingly some potent dual RET/ALK
inhibitors were identified (e.g. EUD-GK-5) (Table 5)
EUD-GK-69 antiproliferative
IC50 in RET+ cell lines
EUD-GK compounds bind in the ATP pocket. The
protein displays a DFG-in conformation and a
folded P-loop
EUD-GK chemical
series
Antiproliferative IC50
BaF3 NPM-ALK L1196M
(nM)
EUD-GK-3 2.4
EUD-GK-119 2.4
EUD-GK-91 4
EUD-GK-6 4.1
EUD-GK-103 5.5
EUD-GK-97 6
To decipher EUD-GK chemical series activity against ROS1, DDR2 and TRK,
research is ongoing with particular focus on novel drug resistance mutations
emerging from clinical development
Radiometric protein kinase assay
3-H thymidine incorporation assay; 72h incubation, duplicate
Table 3.
Table 2.
Table 5.
Table 4.
SUMMARY
We identified a novel chemical series of potent and selective inhibitors targeting
relevant oncogenic drivers like ALK, RET, TRK, DDR2, ROS1 with uncommon
activity against mutations responsible for drug resistance. Novel inhibitors were
characterized in biochemical and cellular assays. The chemical series showed
acceptable stability and solubility properties. These data strongly support further
development in pre-clinical animal models.
0
2000
4000
6000
8000
0 120 240 360 480
Wholebloodconcentration(ng/mL)
Time-point (min)
The pharmacokinetics of EUD -GK-91 following single
intravenous administration at 3mg free base/kg to the
female Balb/c mouse
EUD-GK-91 IV administration 3mg/kg
T1/2 (hr) 2
AUC0-24 (ng-min/mL) 443406
Clb (mL/min/kg) 7
Vdss (L/kg) 1