Identification and characterization of highly … and characterization of highly potent and...
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Internal candidates Commercial MKIs Introduction Identification and characterization of highly potent and selective RET inhibitors for the treatment of RET-driven cancers Barbara J. Brandhuber 1 , Nisha Nanda 2 , Julia Haas 1 , Karyn Bouhana 1 , Lance Williams 1 , Shannon Winski 1 , Michael Burkard 1 , Brian Tuch 2 , Kevin Ebata 2 , Jennifer Low 2 , Robin Hamor 1 , Francis Sullivan 1 , Lauren Hanson 1 , Tony Morales 1 , Guy Vigers 1 , Jessica Gaffney 1 , Ross D. Wallace 1 , James Blake 1 , Yutong Jiang 1 , S. Michael Rothenberg 2 , Steven Andrews 1 . 1 Array BioPharma, Boulder, CO; 2 Loxo Oncology, Stamford, CT 15-A-489-AACR RET in Cancer Activating mutations and oncogenic fusions of the RET receptor tyrosine kinase have been identified in multiple tumor types, including thyroid, lung, breast and colon carcinoma. Furthermore, tyrosine kinase inhibitors (TKIs) with anti-RET activity have produced clinical responses in patients whose tumors harbor RET alterations. However, currently available RET inhibitors were initially developed to target kinases other than RET and are only moderately potent against RET, inhibit multiple kinases other than RET or poorly inhibit secondary resistance mutations (e.g. gatekeeper mutations) common to other TKIs. We have identified a series of potent and selective RET inhibitors with high oral bioavailability and favorable PK properties in animals. Several demonstrated potent inhibition of RET in enzyme and cellular assays, with minimal activity against highly related kinases. In an NIH3T3-KIF5B-RET allograft model, Compound R002 effectively inhibited phospho-RET and caused dramatic tumor growth inhibition without significant toxicity. The identification of potent and selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of currently available inhibitors with anti-RET activity. Summary Methods • R001 and R002 were identified as potent and selective RET inhibitors with high oral bioavailability and favorable pharmacokinetic (PK) properties in animals. • In vitro and in vivo evaluations, including enzyme and cell-based assays, PK/PD (pharmacodynamic) correlations, drug metabolism characterization, and non- clinical safety evaluation were performed using standard methods. • Tumor growth inhibition and PK/PD studies were carried out using subcutaneous allografts of NIH-3T3 cells expressing KIF5B-RET in nude mice in accordance with IACUC guidelines and the Guide for Laboratory Animal Care and Use. • R001 and R002 were compared to two clinically approved, commercially available multikinase inhibitors (MKIs) with anti-RET activity, in vitro and in vivo. Dose-dependent inhibition of phospho-RET and tumor growth in NIH3T3-KIF5B-RET allografts Results We have discovered novel, potent and selective RET inhibitors. The resulting compounds exhibit nanomolar potency against wild type RET and select RET mutants, including the KIF5B-RET fusion and V804M gatekeeper mutation, in both enzyme and cellular assays, with minimal activity against highly related kinases. The activity of representative compounds and that of related analogs in relevant in vitro and in vivo models is presented. Wild-type RET Extracellular mutation C634R Intracellular mutation M918T Oncogenic fusion KIF5B-RET CCD6-RET Medullary thyroid cancer Papillary thyroid cancer Lung cancer Colorectal cancer Breast cancer Myelofibrosis/CMML RET mutations in human cancers lead to ligand-independent RET dimerization (e.g. C634R), kinase activation through mutation (e.g. M918T) or fusion-induced dimerization (e.g. KIF5B-RET). Figure modified from Mulligan, Nat Rev Cancer (2014) 14;3: 173-185. X-ray crystal structures of RET (left) and KDR (VEGFR2—right). Potent KDR inhibition is a common feature of multikinase inhibitors (MKIs) that target RET and a primary cause of their dose-limiting toxicities. The C-helix of each enzyme is highlighted in yellow. X-ray crystal structures of RET and KDR RET Crystal Structure C-helix (yellow) KDR Crystal Structure C-helix (yellow) 100-fold selectivity for KDR (VEGFR2) can be achieved without loss of gatekeeper mutant potency High potency and selectivity for RET can be achieved Left IC50s for phospho-RET and phospho-KDR inhibition in HEK293 cells expressing KIF5B-RET or KDR, respectively, were determined by ELISA assay of protein lysates. Compounds (colored circles) shifted up and to the left are more potent against KIF5B-RET than KDR. Right Similar analysis for wild-type RET and V804M RET in enzyme assays at 1mM ATP. R001 and R002 (lower left quadrant) maintain potency against V804M RET within 7- and 2-fold of wild-type RET, respectively, while two commercially available MKIs with anti-RET activity demonstrate loss of inhibitory activity. Phospho-RET (% vehicle) Compound (µg/ml) in plasma P-KIF5B-RET [compound] in blood [compound] in tumor R002 IC 90 10 mpk 30 mpk 100 mpk 4h 8h 12h 24h 4h 8h 12h 24h 4h 8h 12h 24h Time after single dose administration 10x Equivalent activity (1x) 100x Better gatekeeper coverage R001 R002 MKI-1 10x 100x More selective MKI-1 R001 R002 Equivalent activity (1x) Insensitive Sensitive 10 100 1000 Cell Count EC50 (nM) RET fusion/mutation positive (n=2) No RET alteration (n=76) Loxo candidates Inhibitors are selectively cytotoxic to RET-mutant cells Each cell line was treated with 10 concentrations of each inhibitor in triplicate for 72 hours, following by DAPI staining and cell counting. Upper pRET levels in tumor lysates after a single dose of R002 was determined by immunoblot (bars) and overlaid with blood and tumor drug levels (left). Dose-dependent PK exposures in vivo (right). Middle Daily treatment caused dose-dependent inhibition of tumor growth comparable to a clinically approved MKI (left), without decreasing body weight (right). Lower Maximum tumor growth inhibition and regression. 2500 Tumor volume (mm 3 ) 0 500 1000 1500 2000 vehicle R002 3 mpk 2 R002 10 mpk 2 R002 30 mg/kg 2 MKI-1 60 mg/kg 1 Dosing 11 1 3 5 7 9 13 15 Day of Study 20 21 22 23 24 25 Body weight (g) Dosing 11 1 3 5 7 9 13 15 Day of Study ATP Site ATP Site Treatment Max. growth inhibition Max. Regression MKI-1 91% 53% R002 97% 67% vehicle R002 3 mpk 2 R002 10 mpk 2 R002 30 mg/kg 2 MKI-1 60 mg/kg 1 MKI-2 MKI-2 10 100 1000 10000 10 100 1000 10000 RET V804M Enzyme IC 50 (nm) RET Enzyme IC 50 (nm) 10 10 100 400 4000 100 1000 400 40 40 4 1 RET cell IC 50 (nm) KDR cell IC 50 (nm) Upper In vitro parameters for compounds R001, R002 and two clinically approved MKIs. Selectivity is shown in parentheses as the ratio of IC50s for the indicated kinase compared to wild-type RET. Lower ≥ 100-fold selectivity was observed for >95% of 228 purified kinase domains in radiometric kinase assays, including additional kinases (shown) commonly inhibited by MKIs. % inhibition compared to control is shown. A value ≥ 50 is equivalent to ≥ 100-fold selectivity. Kinase R001 % control R002 % control Ret 0.5 0 EGFR 99 94.5 KIT 99 77 Met 86.5 88.5 PDGFRalpha 90.5 115.5 PDGFRbeta 95 97 Tie2 89 50 0.001 0.01 0.1 1 10 0 4 8 12 16 20 24 10 mg/kg PO 30 mg/kg PO Time after single dose administration Compound (µg/ml) in plasma R002 IC 90 R002 IC 50 Parameter R001 R002 MKI-1 MKI-2 RET enzyme (fold selectivity) WT Km ATP IC 50 0.4 0.3 6.5 9.5 M918T Km ATP IC 50 0.5 (1.25x) 0.2 (0.67x) 210 (32x) 4.6 (0.5x) V804l Km ATP IC 50 0.3 (0.75x) 0.5 (1.67x) 367 (56x) 5032 (530x) V804M Km ATP IC 50 0.6 (1.5x) 2.0 (6.67x) -- 3775 (397x) WT 1mM ATP IC 50 12 5.6 -- -- V804M 1mM ATP IC 50 79 (6.6x) 12 (2.1x) -- -- KIF5B-RET cell cell IC 50 (nM) 12.5 3.3 74.6 1231 Aurora AurA enz Km ATP IC 50 (nM) 76 (191x) 59 (197x) 1270 (195x) 10000 (1053x) (fold selectivity) AurB enz Km ATP IC 50 (nM) 15 (38x) 13 (43x) 176 (27x) -- Aur H3 mitotic cell IC 50 (nM) 920 (74x) 781 (237x) 5944 (80x) >10000 KDR KDR cell IC 50 (nM) 752 (60x) 195 (59x) 1.7 (0.02x) 327 (0.3x)
Transcript of Identification and characterization of highly … and characterization of highly potent and...
Internal candidates Commercial MKIs
Introduction
Identification and characterization of highly potent and selective RET inhibitors for the treatment of RET-driven cancers
Barbara J. Brandhuber1, Nisha Nanda2, Julia Haas1, Karyn Bouhana1, Lance Williams1, Shannon Winski1, Michael Burkard1, Brian Tuch2, Kevin Ebata2, Jennifer Low2, Robin Hamor1, Francis Sullivan1, Lauren Hanson1, Tony Morales1, Guy Vigers1, Jessica Gaffney1, Ross D. Wallace1, James Blake1, Yutong Jiang1, S. Michael Rothenberg2, Steven Andrews1. 1Array BioPharma, Boulder, CO; 2Loxo Oncology, Stamford, CT
15-A-489-AACR
RET in Cancer Activating mutations and oncogenic fusions of the RET receptor tyrosine kinase have been identified in multiple tumor types, including thyroid, lung, breast and colon carcinoma. Furthermore, tyrosine kinase inhibitors (TKIs) with anti-RET activity have produced clinical responses in patients whose tumors harbor RET alterations. However, currently available RET inhibitors were initially developed to target kinases other than RET and are only moderately potent against RET, inhibit multiple kinases other than RET or poorly inhibit secondary resistance mutations (e.g. gatekeeper mutations) common to other TKIs.
We have identified a series of potent and selective RET inhibitors with high oral bioavailability and favorable PK properties in animals. Several demonstrated potent inhibition of RET in enzyme and cellular assays, with minimal activity against highly related kinases. In an NIH3T3-KIF5B-RET allograft model, Compound R002 effectively inhibited phospho-RET and caused dramatic tumor growth inhibition without significant toxicity. The identification of potent and selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of currently available inhibitors with anti-RET activity.
Summary
Methods
• R001 and R002 were identified as potent and selective RET inhibitors with high oral bioavailability and favorable pharmacokinetic (PK) properties in animals.
• In vitro and in vivo evaluations, including enzyme and cell-based assays, PK/PD (pharmacodynamic) correlations, drug metabolism characterization, and non-clinical safety evaluation were performed using standard methods.
• Tumor growth inhibition and PK/PD studies were carried out using subcutaneous allografts of NIH-3T3 cells expressing KIF5B-RET in nude mice in accordance with IACUC guidelines and the Guide for Laboratory Animal Care and Use.
• R001 and R002 were compared to two clinically approved, commercially available multikinase inhibitors (MKIs) with anti-RET activity, in vitro and in vivo.
Dose-dependent inhibition of phospho-RET and tumor growth in NIH3T3-KIF5B-RET allografts
Results
We have discovered novel, potent and selective RET inhibitors. The resulting compounds exhibit nanomolar potency against wild type RET and select RET mutants, including the KIF5B-RET fusion and V804M gatekeeper mutation, in both enzyme and cellular assays, with minimal activity against highly related kinases. The activity of representative compounds and that of related analogs in relevant in vitro and in vivo models is presented.
Wild-type RET Extracellular mutation
C634R
Intracellular mutation M918T
Oncogenic fusion KIF5B-RET CCD6-RET
Medullary thyroid cancer
Papillary thyroid cancer Lung cancer
Colorectal cancer Breast cancer
Myelofibrosis/CMML
RET mutations in human cancers lead to ligand-independent RET dimerization (e.g. C634R), kinase activation through mutation (e.g. M918T) or fusion-induced dimerization (e.g. KIF5B-RET). Figure modified from Mulligan, Nat Rev Cancer (2014) 14;3: 173-185.
X-ray crystal structures of RET (left) and KDR (VEGFR2—right). Potent KDR inhibition is a common feature of multikinase inhibitors (MKIs) that target RET and a primary cause of their dose-limiting toxicities. The C-helix of each enzyme is highlighted in yellow.
X-ray crystal structures of RET and KDR
RET Crystal Structure C-helix (yellow)
KDR Crystal Structure C-helix (yellow)
100-fold selectivity for KDR (VEGFR2) can be achieved without loss of gatekeeper mutant potency
High potency and selectivity for RET can be achieved
Left IC50s for phospho-RET and phospho-KDR inhibition in HEK293 cells expressing KIF5B-RET or KDR, respectively, were determined by ELISA assay of protein lysates. Compounds (colored circles) shifted up and to the left are more potent against KIF5B-RET than KDR. Right Similar analysis for wild-type RET and V804M RET in enzyme assays at 1mM ATP. R001 and R002 (lower left quadrant) maintain potency against V804M RET within 7- and 2-fold of wild-type RET, respectively, while two commercially available MKIs with anti-RET activity demonstrate loss of inhibitory activity.
Phos
pho-
RET
(% v
ehic
le)
Compound (µg/m
l) in plasma
P-KIF5B-RET [compound] in blood [compound] in tumor
R002 IC90
10 mpk 30 mpk 100 mpk
4h 8h 12h 24h
4h 8h 12h 24h
4h 8h 12h 24h
Time after single dose administration
10x
Equivalent activity (1x)
100x Better
gatekeeper coverage
R001
R002
MKI-1
10x
100x More
selective
MKI-1
R001
R002
Equivalent activity (1x)
Insensitive
Sensitive
10
100
1000
Cell
Coun
t EC
50 (
nM)
RET fusion/mutation positive (n=2) No RET alteration (n=76)
Loxo candidates
Inhibitors are selectively cytotoxic to RET-mutant cells
Each cell line was treated with 10 concentrations of each inhibitor in triplicate for 72 hours, following by DAPI staining and cell counting.
Upper pRET levels in tumor lysates after a single dose of R002 was determined by immunoblot (bars) and overlaid with blood and tumor drug levels (left). Dose-dependent PK exposures in vivo (right). Middle Daily treatment caused dose-dependent inhibition of tumor growth comparable to a clinically approved MKI (left), without decreasing body weight (right). Lower Maximum tumor growth inhibition and regression.
2500
Tum
or v
olum
e (m
m3 )
0
500
1000
1500
2000
vehicle R002 3 mpk2 R002 10 mpk2
R002 30 mg/kg2 MKI-1 60 mg/kg1
Dosing
11 1 3 5 7 9 13 15 Day of Study
20
21
22
23
24
25
Body
wei
ght
(g)
Dosing
11 1 3 5 7 9 13 15 Day of Study
ATP Site ATP Site
Treatment Max. growth inhibition Max. Regression
MKI-1 91% 53%
R002 97% 67%
vehicle R002 3 mpk2 R002 10 mpk2
R002 30 mg/kg2 MKI-1 60 mg/kg1
MKI-2
MKI-2
10 100 1000 10000
10
100
1000
10000
RET
V804
M E
nzym
e IC
50 (
nm)
RET Enzyme IC50 (nm)
10
10 100 400
4000
100
1000
400
40
40 4 1 RET cell IC50 (nm)
KDR
cell
IC50
(nm
)
Upper In vitro parameters for compounds R001, R002 and two clinically approved MKIs. Selectivity is shown in parentheses as the ratio of IC50s for the indicated kinase compared to wild-type RET. Lower ≥ 100-fold selectivity was observed for >95% of 228 purified kinase domains in radiometric kinase assays, including additional kinases (shown) commonly inhibited by MKIs. % inhibition compared to control is shown. A value ≥ 50 is equivalent to ≥ 100-fold selectivity.
Kinase R001 % control
R002 % control
Ret 0.5 0EGFR 99 94.5KIT 99 77Met 86.5 88.5
PDGFRalpha 90.5 115.5PDGFRbeta 95 97
Tie2 89 50
0.001
0.01
0.1
1
10
0 4 8 12 16 20 24
10 mg/kg PO
30 mg/kg PO
Time after single dose administration
Com
poun
d (µ
g/m
l) in
pla
sma
R002 IC90
R002 IC50
Parameter R001 R002 MKI-1 MKI-2
RET enzyme (fold
selectivity)
WT Km ATP IC50 0.4 0.3 6.5 9.5 M918T Km ATP IC50 0.5 (1.25x) 0.2 (0.67x) 210 (32x) 4.6 (0.5x) V804l Km ATP IC50 0.3 (0.75x) 0.5 (1.67x) 367 (56x) 5032 (530x)
V804M Km ATP IC50 0.6 (1.5x) 2.0 (6.67x) -- 3775 (397x) WT 1mM ATP IC50 12 5.6 -- --
V804M 1mM ATP IC50 79 (6.6x) 12 (2.1x) -- -- KIF5B-RET cell cell IC50 (nM) 12.5 3.3 74.6 1231
Aurora AurA enz Km ATP IC50 (nM) 76 (191x) 59 (197x) 1270 (195x) 10000 (1053x) (fold
selectivity) AurB enz Km ATP IC50 (nM) 15 (38x) 13 (43x) 176 (27x) --
Aur H3 mitotic cell IC50 (nM) 920 (74x) 781 (237x) 5944 (80x) >10000 KDR KDR cell IC50 (nM) 752 (60x) 195 (59x) 1.7 (0.02x) 327 (0.3x)
