SUPPLEMENTAL INFORMATION FOR:

Repotrectinib (TPX-0005) is a next generation ROS1/TRK/ALK inhibitor that potently

inhibits ROS1/TRK/ALK solvent front mutations

AUTHORS

Alexander Drilon,1* Sai-Hong Ignatius Ou,2* Byoung Chul Cho,3 Dong-Wan Kim,4 Jeeyun

Lee,5 Jessica J. Lin,6 Viola W. Zhu,2 Myung-Ju Ahn,5 D. Ross Camidge,7 Judy Nguyen1,

Dayong Zhai,8 Wei Deng,8 Zhongdong Huang,8 Evan Rogers,8 Juliet Liu,8 Jeff Whitten,8

John K. Lim,8 Shanna Stopatschinskaja,8 David M. Hyman1, Robert C. Doebele,7 J. Jean

Cui,8& Alice T. Shaw6&

AUTHORS’ AFFLIATION

1Memorial Sloan Kettering Cancer Center, Weill Cornell Medical College, New York,

NY, USA; 2Chao Family Comprehensive Cancer Center, University of California Irvine

School of Medicine, Orange, CA, USA; 3Yonsei Cancer Center, Severance Hospital,

Yonsei University College of Medicine, Seoul, Republic of Korea; 4Seoul National

University Hospital, Seoul, Republic of Korea; 5Samsung Medical Center,

Sungkyunkwan University School of Medicine, Seoul, Republic of Korea; ;

6Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;

7University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA 8TP

Therapeutics Inc, San Diego, CA, USA

1

*contributed equally, &corresponding authors

CONTENTS

SUPPLEMENTARY METHODS

SUPPLEMENTARY FIGURES S1-10

SUPPLEMENTARY TABLES S1-7

SUPPLEMENTARY REFERENCES

SUPPLEMENTARY METHODS

Design of repotrectinib

Structures of repotrectinib and selected ALK, ROS1, and TRK TKIs approved for clinical

use or in late clinical development are shown in Figure S1 (1).

Structural models of mutant kinases were derived from X-ray co-crystal structures in

PDB databank: PDB ID: 2XP2 and 4CLJ for ALK G1202R; PDB ID: 4UXL for ROS1

G2032R and D2033N; PDB ID: 4AOJ for TRKA G595R; PDB ID: 4AT3 for TRKB

G639R and PDB ID: 4YMJ for TRKC G623R. The binding of repotrectinib with ALK,

ROS1, TRKA, TRKB, and TRKC were modeled with Mastro (Schrodinger Release

2017-4 or early release versions) using Prime MM-GBSA method. The substitution of

2

the mutant residues were predicted with the Prime module, using the default settings

without modifications to the backbone conformations.

The ATP adenosine binding pocket spans an area from the gatekeeper residue at the

β5 sheet (e.g. L1196 for ALK) to a conserved glycine residue (e.g. G1202 for ALK)

before the solvent front αD helix (Figure S2). Most kinase inhibitors are oversized, and

often have extra motifs either cross over the gatekeeper to the hydrophobic back pocket,

or pass through the conserved glycine residue to the solvent front area, to gain extra

interactions with kinases. Type I 1/2 and II kinase inhibitors often have additional motifs

close to or across kinase gatekeeper residue that render them vulnerable to the clinical

gatekeeper mutations, such as EGFR T790M and ABL T315I. The hinge C-terminal

conserved glycine residue forms a hydrophobic sandwich with a residue at the β1 sheet

(e.g. G1202 with L1122 for ALK), and kinase inhibitors often use an aromatic or a flat

motif crawling through this narrow sandwich region to deliver a connected basic or polar

group to the solvent area for interactions with acid, basic or polar residues located at the

kinase solvent front of the αD helix, e. g. crizotinib bound in ALK kinase (Figure S2).

ALK, ROS1, and TRKA/B/C inhibitors that are either approved or in the clinic are ATP-

competitive type I kinase inhibitors (Figure S1). These inhibitors have an ATP adenine-

equivalent kinase hinge binder (colored as red color in the structures) and also have an

extra motif that extend to the solvent area (colored as blue color in the structures).

Alterations at the conserved glycine (e.g. ALK G1202R) and mutations that result in

substitutions at the solvent front of the αD helix (e.g. ALK D1203N, S1206Y/C, and

E1210K) will therefore sterically hinder the binding of many inhibitors to the ALK, ROS1,

3

or TRKA/B/C kinase, as exampled with crizotinib with a large basic motif sterically

clashed with ALK G1202R mutation (Figure S3).

To systematically overcome these resistant mutations, repotrectinib was designed to

target resistance-driven kinase active conformation with a compact strucure that is

completely located inside the ATP binding boundary to avoid steric clash with common

clinical mutations (Figure S4). Structurally, the drug is a rigid three-dimensional

macrocycle that precisely anchors the molecule in the adenine binding site with a

bioactive binding conformation predefined to avoid the entropy penalty after binding.

Furthermore, repotrectinib is smaller in size (molecular weight of 355.37, Figure S1)

compared to currently available ALK, ROS1, and TRKA/B/C inhibitors. This novel

design was aimed to target both wildtype and clinical resistant mutations, especially

solvent front mutations. Structural modeling suggests that repotrectinib can

accommodate the bulky, positively-charged arginine side chain in the solvent front

without any steric clashes. These include ROS1 G2032R, TRKA G595R, TRKB G639R,

TRKC G623R, and ALK G1202R (Figure 1).

ROS1 and ALK belong to the same receptor tyrosine kinase subfamily with 77% amino

acid homology in the ATP binding sites of the tyrosine kinase domain (2). Crizotinib,

ceritinib, brigatinib and ensartinib are dual inhibitors of ALK and ROS1 kinases based

on the data presented here. However, ROS1 and ALK proteins demonstrate different

properties in the X-ray crystal structures of unphosphorylated catalytic kinase domain as

illustrated with the X-ray cocrystal structures of lorlatinib with ALK (PDB ID 4CLI) and

4

ROS1 (PDB ID 4UXL) (Figure S5) (3, 4). The unphosphorylated ALK revealed a

partially inactive protein kinase conformation with the A-loop in an inhibitory pose and a

novel N-terminal -turn motif that block the C helix movement to more inactive

conformation and restrict the inhibitor extend to the back pocket (5). Therefore, most of

ALK inhibitors are type I kinase inhibitors. Although ROS1 demonstrated a similar

conformation in the cocrystal structures as ALK when complexed with crizotinib or

lorlatinib, the ALK specific characteristics of autoinhibitory proximal A-loop helix (AL)

and N-terminal -turn motif are not present in the ROS1 crystal structures, that imply the

ROS1 hydrophobic back pocket is more accessible for type II kinase inhibitors.

Therefore, the type II kinase inhibitor cabozantinib is a potent ROS1 inhibitor, but not an

ALK inhibitor. Furthermore, the solvent front D helix is more rigid in ROS1 kinase with

one more helix turn than ALK, that will make the solvent front mutations of ROS1 kinase

are more efficient to block the inhibitor binding and develop resistance. Many ALK

inhibitors have a bulky positively charged motif in the solvent area to benefit the

interaction with the negatively charged residue ALK E1210. The large basic argenine

ALK G1202R mutation is relatively tolerable becuase the large positively charged

argenine can rotate to interact with the acidic ALK E1210 residue, especially when the

inhibitor motif in the solvent area is small, e.g. lorlatinib is still active against ALK

G1202R due to a relative small N-methylpyrazole group, that is just located above ALK

G1202R residue (Figure S5). However, a basic lysine residue ROS1 K2040 at the

ROS1 D helix is located at the same position as the acidic ALK E1210 and will repell

with the positively charged arginine side chain from ROS1 G2032R mutation (Figure

S6). Therefore, the rigid ROS1 solvent front D helix and the positively charged ROS1

5

K2040 residue will make the solvent front mutation ROS1 G2032R much leass tolerable

by ROS1 inhibitors that have an extra motif above or across G2032 position, even the

small N-methylpyrazole group in lorlatinib is not as tolerable as in ALK. Lorlatinib is

much less active against ROS1 G2032R mutation. Repotrectinib is designed to be

located completely inside ATP adenine binding site with no motif above or across ROS1

G2032 residue (Figure S4) and will have less impact from ROS1 G2032R mutation.

A successful small molecule drug candidate is highly associated with good “drug-like”

properties, that refer to the molecule’s physicochemical, absorption-distribution-

metabolism-excretion (ADME), and toxicological properties. Lacking optimal drug-like

properties often caused the drug candidates to fail in preclinical or clinical development.

Repotrectinib has demonstrated preferred physiochemical, ADME, and toxicological

properties for oral drug development (Table S1). Brain metastases have been a major

challenge in advanced non-small cell lung cancer and contribute significantly to

decrement in survival and quality of life. The development of small molecules that cross

the blood-brain barrier (BBB) is highly desired. The physiochemical parameters

including lipophilicity, polar surface area, molecular weight, hydrogen bond donor, and

the charge are important factors in determination of the ability of a small molecule

across human BBB based on analyses of 119 marketed CNS drugs (6). Therefore,

Central Nervous System Multiparameter Optimization (CNS MPO) Desirability score

system was proposed with a range of 0-6. 74% of the marketed CNS drugs have CNS

MPO scores >4 (6). Repotrectinib was designed with a favorable CNS MPO score of

4.65 (Table S2A), although it had a moderate pgp efflux ratio of 7.3 (Table S1) and low

6

mice brain penetration (4% at steady state, Table S2B). The human brain penetration of

repotrectinib need to be further determined in clinical studies, even though CNS

antitumor activity was observed in the subject in case 2 study.

Repotrectinib synthesis

A series of molecules that are structurally-distinct, potent, and selective kinase inhibitors

against both wildtype and mutant ROS1, TRK, and ALK were designed, synthesized

and assayed by in vitro kinase inhibitory assays. From the effort, repotrectinib (TPX-

0005) was selected as a clinical candidate for further evaluation. Please reference US

Patent No. 9,714,258 B2, Example 93 for repotrectinib and references therein (7).

Enzyme assays

Kinase activity inhibition

The enzymatic kinase activity inhibition of repotrectinib against wildtype ROS1/TRK/ALK

and ROS1/TRK/ALK with solvent-front mutations was evaluated at Reaction Biology

Corporation using the radiolabeled HotSpot kinase assay platform (8) (Table S3). The

individual substrate for each kinase was prepared in freshly made Reaction Buffer with

the subsequent addition of required cofactors if needed, followed by addition of the

individual kinase and gentle mixing. Repotrectinib in DMSO was added into the kinase

reaction mixture utilizing acoustic technology (Echo 550), and then 33P-ATP (specific

7

activity 0.01 µCi/µl final) was delivered into the reaction mixture to initiate the reaction.

The kinase reaction was incubated for 120 minutes at room temperature. Reactions

were then spotted onto P81 ion exchange paper (Whatman # 3698-915), which was

washed extensively in 0.75% Phosphoric acid. The radioactive phosphorylated

substrate remaining on the filter paper was measured. Repotrectinib was tested in a 10-

dose IC50 mode with 3-fold serial dilution starting at 1 μM, and the control compound,

staurosporine, was tested in both a 10-dose IC50 mode with 4-fold serial dilution starting

at 20 μM, and a 10-dose IC50 mode with 3-fold serial dilution starting at 0.1 μM. All of the

reactions were carried out in the presence of 10 μM ATP concentration. Kinase activity

data were expressed as the percent remaining kinase activity in test samples compared

to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve fits were obtained using

Prism4 Software (GraphPad).

Additional inhibitory activity against clinical relevant non-solvent front mutations of ALK

was also tested (Table S4).

Broad kinase panel profiling

Repotrectinib kinase selectivity was first evaluated using the KINOMEscan® site-

directed competition binding assay against 456 human kinases and mutants at

DiscoveRx in duplicate at a repotrectinib concentration of 100 nM which was more than

1000 fold higher than repotrectinib ROS1 IC50 value (0.071 nM) (9). The screening hits

were further evaluated for kinase inhibition IC50 values at Reaction Biology Corporation

using the radiolabeled HotSpot kinase assay platform (Table S5).

8

Cell lines

Human lung cancer cell line NCI-H2228 was obtained from ATCC (2014, Manassas,

VA). Cell lines NIH3T3 and Ba/F3 were purchased from DSMZ (2015, German

Collection of Microorganisms and Cell Culture, Braunschweig, Germany). Karpas-299

cell line was purchased and licenced from Sigma (2015, Cambridge). KM12 cell line

was obtained from NCI. (2015, Frederick Cancer DCTD Tumor Cell Repository). NCI-

H2228, Karpas-299, and KM12 cells were authenticated by confirmation of the

presence of each fusion (EML4-ALK, NPM-ALK or TPM3-TRKA). NIH3T3 and Ba/F3

cells were not authenticated. Cell lines were confirmed to be mycoplasma-free

(Biomiga) and were been used between 3-10 passages. NIH3T3 was maintained in

DMEM medium supplemented with 10% fetal bovine serum and 100 U/mL of

penicillin/streptomycin. Karpas-299 and H2228 were maintained in RPMI-1640

supplemented with 10% fetal bovine serum with 100 U/mL of penicillin/streptomycin.

Ba/F3 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum,

10% (Vol/Vol) conditioned media from the WIHI-3B myelomonocytic IL-3 secreting cells

and 100 U/mL of penicillin/streptomycin. Ba/F3 stable cell lines were maintained in

RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 0.5

µg/mL puromycin solution.

Cloning and Ba/F3 or NIH3T3 stable cell line creation

9

The genes of EML4-ALK (variant 1), CD74-ROS1, LMNA-TRKA , ETV6-TRKB, ETV6-

TRKC, and the solvent front mutant genes of EML4-ALK G1202R, CD74-ROS1

G2032R, CD74-ROS1 G2033N, LMNA-TRKA G595R, ETV6-TRKB G639R, ETV6-

TRKC G623R, and ETV6-TRKC G623E were synthesized at GenScript and cloned into

pCDH-CMV-MCS-EF1-Puro plasmid (System Biosciences, Inc). The corresponding

Ba/F3 cells were generated by transducing Ba/F3 cells with lentivirus containing the

desired fusion gene or mutant. Stable cell lines were selected by puromycin treatment,

followed by IL-3 withdrawal. Briefly, 5X106 Ba/F3 cells were transduced with lentivirus

supernatant in the presence of 8µg/mL protamine sulfate. The transduced cells were

subsequently selected with 1 µg/mL puromycin in the presence of IL3-containing

medium RPMI1640, plus 10% FBS. After 10-12 days of selection, the surviving cells

were further selected for IL3 independent growth. NIH3T3 stable cell lines were

generated by transducing the cells with lentivirus containing the corresponding fusion

gene or the fusion mutant gene. The transduced cells were subsequently selected with

1 µg/mL puromycin.

Immunblotting for cellular kinase phosphorylation assays

NSCLC cell line H2228 (harboring endogenous EML4-ALK fusion gene), Karpas-299

cells (harboring endogenous NPM-ALK fusion gene) or SET-2 (harboring endogenous

JAK2V617F activating mutation) were cultured in RPMI medium, and KM12 (harboring

endogenous TPM3-TRKA fusion gene) cell line was cultured in DMEM medium, both

supplemented with 10% fetal bovine serum and 100 U/mL of penicillin/streptomycin.

Ba/F3 and NIH3T3 cells stably expressing ALK or ROS1 (WT or mutant) were culture

10

as mentioned above. Half a million cells per well were seeded in 24 well plate for 24 hrs,

and then treated with compounds for 4 hours. Cells were collected after treatment and

lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5%

Deoxycholate, 0.1% SDS) supplemented with 10 mM EDTA, 1X Halt protease and

phosphatase inhibitors (Thermo Scientific). Protein lysates (approximately 20 µg) was

resolved on 4–12% Bolt Bis-Tris precasted gels with MES running buffer (Life

Technologies), transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer

System (Bio-Rad) and detected with antibodies targeting phosphorylated ALK Y1604

(Cell Signaling Technology), total ALK (Cell Signaling Technology), phosphorylated

ROS1 and total ROS1 (Cell Signaling Technology), phosphorylated TRK A/B (Cell

Signaling Technology), total TRKA antibody ( Santa Cruz Biotechnogy), phosphorylated

STAT5, total STAT5 (Cell Signaling Technology), phosphorylated SRC, total SRC (Cell

Signaling Technology), and Tubulin (Sigma). Antibodies were typically incubated

overnight at 4 oC with gentle shake, followed by washes and incubation with the

appropriate HRP-conjugated secondary antibodies. Membranes were incubated with

chemiluminescent substrate for 5 min at room temperature (SuperSignal West Femto,

Thermo Scientific). The chemiluminescent images were acquired with a C-DiGit Imaging

System (LI-COR Biosciences). The relative density of the chemiluminescent bands

were quantified via Image Studio Digits from LICOR. The half inhibitory concentration

(IC50) value is calculated using non-linear regression analysis through GraphPad Prism

software (GraphPad, Inc., San Diego, CA).

The inhibitory effect of repotrectinib on phosphorylation of ROS1 in NIH3T3 CD74-

ROS1 cells, TRKA in KM12 cells with TPM3-TRKA fusion, ALK in Karpas-299 cells

11

having NPM-ALK fusion, JAK2 substrate STAT5 in SET2 cells having JAK2 V617F

mutation, and SRC in H2228 cells having EML3-ALK v3 fusion was determined,

respectively (Figure S7)

Cell proliferation assays:

Two thousand cells per well were seeded in 384 well white plate for 24 hrs, and then

treated with compounds for 72 hours (37 oC, 5% CO2). Cell proliferation was measured

using CellTiter-Glo luciferase-based ATP detection assay (Promega) following the

manufactures’s protocol. IC50 determinations were performed using GraphPad Prism

software (GraphPad, Inc., San Diego, CA).

The antiproliferation activity of repotrectinib and other kinase inhibitors in the stable

Ba/F3 cell lines engineered with corresponding fusion gene or mutated fusioin gene was

determined (Figures S8A-S8C and Tables S6A-S6C).

Animal care

All animal studies were conducted in accordance with the guidelines as published in the

Guide for the Care and Use of Laboratory Animals. Female athymic nude mice and

SCID/Beige mice (5-8 weeks of age) were obtained from Charles River Laboratory and

housed in the vivarium of Explore BioLabs, Inc. Mice were maintained and used in

accordance with the animal protocol EB15-013 (approved by Explora BioLabs’

Institutional Animal Care and Use Committee). Mice were housed in Innovive IVC

12

disposable cages on HEPA filtered ventilated racks with ad libitum access to rodent

chow and water.

Subcutaneous Xenograft Models in Immune Compromised Mice

Five million cells in 100 L serum-free medium supplemented with 50% Matrigel

(Corning, Inc.) were implanted subcutaneously in the right flank region of the mouse.

Ba/F3 EML4-ALK wild type and G1202R mutant cells, Ba/F3 CD74-ROS1 wild type and

G2032R mutant cells were implanted into the SCID/Beige mice. NIH3T3 LMNA-TRKA

wild type and G595R mutant cells, and NIH3T3 ETV6-TRKC G623E mutant cells were

implanted into the athymic nude mice, respectively. Tumor size and body weight were

measured on designated days. Tumor size was measured with an electronic caliper

and tumor volume was calculated as the product of length * width2 * 0.5. Mice were

randomized by tumor size into treatment groups when tumor volume reached about

100-200 mm3 and repotrectinib were administered orally (BID) at determined dosage.

Tumor growth inhibition (TGI) was calculated according to the following formula:

If TV t>TV 0 , TGI=100%×(1− TV t−TV 0CV t−CV 0

)

If TV t<TV 0 , TGI=100%×(2− TV tTV 0

)

where TV0 was the tumor volume in the treatment group at the beginning of the study,

TVt was the tumor volume in the treatment group at the end of the study, CV0 was the

tumor volume in the control group at the beginning of the study, and CVt was the tumor

volume in the control group at the end of the study.

13

Repotrectinib was dosed as a suspention solution containing 0.5% CMC and

1% Tween-80. In the mouse tumor models with ROS1 or ALK fusion gene, a regular

crystalline repotrectinib was used in the suspension formulation. Repotrectinib is a

highly crystalline small molecule with low solubility. To increase the absorption of

repotrectinib in in vivo studies, repotrectinib was micronized to particle size <10 micro-

meter. The micronized repotrectinib was used in the mouse tumor models with NTRK

fusion gene. The antitumor activities of repotrectinib in the mouse tumor models were

illustrated in Figures 3A-3F. The body weights in each animal study were recorded in

Figures S9A-S9C and the mean trough plasma concentration in Table S7.

Source of TKIs

Crizotinib, ceritinib, alectinib and larotrectinib were purchased from Selleckchem;

brigatinib and ensartinib were purchased from MedChem Express, and entrectinib were

purchased from MedKoo Bioscience.

14

Supplemental Figures S1-10

Figure S1.

Figure S1. Structures of repotrectinib (TPX-0005) and ALK, ROS1 and TRK inhibitors

approved or in clinical trials. The red-colored motifs represent the kinase inhibitors’

hinge binder, a mimic of adenine at ATP, and the blue-colored motifs extend to the

kinase solvent front area, that are not present at ATP and represent a shared liability in

the setting of on-target resistance mediated by the acquisition of kinase domain

substitutions.

15

Figure S2.

L1122

L1196

G1202

D1203S1206E1210

D Helix

Figure S2. ALK protein in complex with crizotinib (PDB ID: 2XP2) labeled with key

contact residues and solvent front residues.

Figure S3.

E1210

G1202R

L1196M

Figure S3. Modeling of crizotinib sterically clashing with ALK G1202R and ALK L1196M

mutant residues (PDB ID 2XP2).

16

Figure S4.

G2032

D2033

L2026

Figure S4. Modeling of repotrectinib bound in ROS1 kinase domain (PDB ID 4UXL).

Figure S5

D Helix D Helix

AL Helix

N-Terminal-turn

N-Terminal

ALK ROS1

Figure S5. Comparison of the unphosphorylated ALK and ROS1 kinase cocrystal

structures: ALK kinase in complex with lorlatinib (PDB ID 4CLI) and ROS1 kinase in

complex with lorlatinib (PDB ID 4UXL).

17

Figure S6

ALK ROS1

E1210

G1202R

K2040

G2032R

Figure S6. Modeling of the impacts of ALK G1202R and ROS1 G2032R mutations on

lorlatinib binding (PDB ID 4CLI for ALK and 4UXL for ROS1).

Figure S7

Karpas-299 cells with NPM-ALK

SET2 cells with JAK2 V617F mutation

Repotrectinib Repotrectinib Repotrectinib

Repotrectinib Repotrectinib

Figure S7. The effect of repotrectinib on phosphorylation of ROS1 in NIH3T3 CD74-

ROS1 cells, TRKA in KM12 cells with TPM3-TRKA fusion, ALK in Karpas-299 cells

having NPM-ALK fusion, JAK2 substrate STAT5 in SET2 cells having JAK2 V617F

mutation, and SRC in H2228 cells having EML3-ALK v3 fusion, respectively.

18

Figure S8A

Ba/F3 CD74-ROS1 WT

-2 0 2 4 60

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

)

CrizotinibRepotrectinibLorlatinibEntrectinibCeritinibBrigatinib

EnsartinibCabozantinib

-2 0 2 4 60

20

40

60

80

100

120

140

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol) Crizotinib

RepotrectinibLorlatinibEntrectinibCeritinibBrigatinib

EnsartinibCabozantinib

Ba/F3 CD74-ROS1 G2032R

-2 0 2 4 60

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

)

CrizotinibRepotrectinibLorlatinibEntrectinibCeritinibBrigatinib

EnsartinibCabozantinib

Ba/F3 CD74-ROS1 D2033N

Figure S8A. Anti-proliferation activity of repotrectinib, crizotinib, lorlatinib, entrectinib,

ceritinib, brigatinib, cabozantinib and ensartinib against Ba/F3 cells engineered with

CD74-ROS1 WT, CD74-ROS1 G2032R, or CD74-ROS1 D2033N fusion proteins,

respectively.

19

Figure S8B

-4 -2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cell

Viab

ility

(% c

ontro

l)

RepotrectinibEntrectinibLarotrectinib

Ba/F3 LMNA-TRKA WT Ba/F3 LMNA-TRKA G595R

-2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cell

Viab

ility

(% c

ontro

l) RepotrectinibEntrectinibLarotrectinib

Ba/F3 ETV6-TRKB WT Ba/F3 ETV6-TRKB G639R

-2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

)

RepotrectinibEntrectinibLarotrectinib

-2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

) RepotrectinibEntrectinibLarotrectinib

Ba/F3 ETV6-TRKC WT Ba/F3 ETV6-TRKC G623R

-2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

)

RepotrectinibEntrectinibLarotrectinib

-2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

)

RepotrectinibEntrectinibLarotrectinib

KM12

-3 -2 -1 0 1 2 30

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol)

RopotrectinibEntrectinibLarotrectinib

Ba/F3 ETV6--TRKC G623E

-2 0 2 40

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol

)

RepotrectinibEntrectinibLarotrectinib

Figure S8B. Anti-proliferation activity of repotrectinib, entrectinib and larotrectinib

against Ba/F3 cells engineered with LMNA-TRKA WT, LMNA-TRKA G595R, ETV6-

TRKB WT, ETV6-TRKB G639R, ETV6-TRKC WT, ETV6-TRKC G623R, or ETV6-TRKC

20

G623E fusion proteins, respectively and KM12 cells with endogenous expression of

TPM3-TRKA.

Figure S8C

Ba/F3 EML4-ALK WT

-2 0 2 4 60

20

40

60

80

100

120

Compounds Log (nM)

Cel

l Via

bilit

y (%

con

trol)

CrizotinibRepotrectinibCeritinibAlectinibBrigatinibLorlatinib

Ba/F3 EML4-ALK G1202R

-2 0 2 4 60

20

40

60

80

100

120

Compounds Log (nM)C

ell V

iabi

lity

(% c

ontro

l)

CrizotinibRepotrectinibCeritinibAlectinibBrigatinibLorlatinib

Karpas-299 with NPM-ALK

-2 0 2 4 60

20

40

60

80

100

120

Compounds Log (nM)

Cell

Viab

ility

(% c

ontro

l)

CrizotinibRopotrectinibCeritinibAlectinibBrigatinibLorlatinib

Figure S8C. Anti-proliferation activity of repotrectinib, crizotinib, ceritinib, alectinib,

brigatinib and lorlatinib against Ba/F3 cells engineered with EML4-ALK WT or EML4-

ALK G1202R fusion proteins, respectively and Karpas-299 cells with endogenous

expression of NPM-ALK.

21

Figure S9A

8 10 12 14 16 18 2014

16

18

20

22

The Change of Body Weight in RepotrectinibTreated MiceBearing BaF3 CD74-ROS1 WT Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID15 mg/kg, BID75 mg/kg, BID

8 10 12 14 16 18 20 2214

16

18

20

22

Change of Body Weight in RepotrectinibTreated MiceBearing CD74-ROS1 G2032R Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID15 mg/kg, BID75 mg/kg, BID

Figure S9A. The change of body weight in repotrectinib treated mice bearing Ba/F3

CD74-ROS1 WT tumors or Ba/F3 CD74-ROS1 G2032R tumors.

Figure S9B

4 6 8 10 1215

20

25

30

The Change of Body Weights in Repotrectinib Treated MiceBearing NIH3T3 LMNA-TRKA WT Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID3 mg/kg, BID, Repotrectinib15 mg/kg, BID, Repotrectinib15 mg/kge, BID, Entrectinib

4 6 8 10 1215

20

25

30

Change of Body Weight in Repotrectinib Treated Mice Bearing NIH3T3 LMNA-TRKA G595R Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID3 mg/kg, BID, Repotrectinib15 mg/kg, BID, Repotrectinib60 mg/kg, BID, Repotrectinib60 mg/kg, BID, Entrectinib

Figure S9B. The change of body weight in repotrectinib treated mice bearing NIH3T3

LMNA-TRKA WT tumors or NIH3T3 LMNA-TRKA G595R tumors.

22

Figure S9C

8 10 12 14 16 18 20 22 24 2614

16

18

20

22

Change of Body Weight in Repotrectinib Treated MiceBearing BaF3 EML4-ALK WT Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID15 mg/kg, BID75 mg/kg, BID

12 14 16 18 20 22 24 26 28 30 32 3414

16

18

20

22

Change of Body Weight in Repotrectinib Treated MiceBearing BaF3 EML4-ALK G1202R Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID15 mg/kg, BID75 mg/kg, BID

Figure S9C. The change of body weight in repotrectinib treated mice bearing Ba/F3

EML4-ALK v1 WT tumors or Ba/F3 EML4-ALK v1 G1202R tumors.

Figure S10A

ETV6-TRKC G623EBa/F3 cell IC90

Figure S10A. Single dose and multiple dose plasma PK profiles of patient 1002-001

having MASC with ETV6-TRKC G623E solvent front mutation taking repotrectinib at 40

mg once daily. Once daily dose PK exceeded the IC90 needed to inhibit cell proliferation

of Ba/F3 cells with ETV6-TRKC G623E over 24 hours period.

23

Figure S10B

4 6 8 10 120

300

600

900

1200

1500

Anti-tumor Activity of Repotrectinib inNIH3T3 ETV6-TRKC G623E Tumors

Day Post Inoculation

Tum

or V

olum

e (m

m3 )

Vehicle, BID3 mg/kg, BID15 mg/kg, BID60 mg/kg, BID

TGI

87%127%153%

-3

-2

-1

0

1

2

3

4

-3

-2

-1

0

1

2

3

4

Anti-tumor Activity of Repotrectinib inthe NIH3T3 ETV6-TRKC G623E Tumors

Log 2

Fol

d Ch

ange

in T

umor

Vol

ume

% Change in Tum

or Volume

(Day 5 to D

ay 11)

+100

+300

+700

-50

-75

-87.5

0

Vehicle Repotrectinib3 mg/kg, BID

Repotrectinib15 mg/kg, BID

Repotrectinib60 mg/kg, BID

+1500

4 6 8 10 1215

20

25

30

The Change of Body Weight of Repotrectinib Treated Mice BearingNIH3T3 ETV6-TRKC G623E Tumors

Day Post Inoculation

Bod

y W

eigh

t (g)

Vehicle, BID3 mg/kg, BID15 mg/kg, BID60 mg/kg, BID

Figure S10B. Anti-tumor efficacy, change in mice body weight, and mouse plasma

exposure of repotrectinib in the NIH3T3 ETV6-TRKC G623E xenograft model in athymic

nude mice. Each waterfall plot represents the degree of xenograft response in one

mouse.

24

Figure S10C

CD74-ROS1 G2032RBa/F3 cell IC90

Figure S10C. Single dose plasma PK profile of patient 1001-011 having NSCLC with

CD74-ROS1 G2032R solvent front mutation taking repotrectinib at 160 mg once daily.

Once daily dose PK exceeded the projected IC90 needed to inhibit cell proliferation of

Ba/F3 cells with CD74-ROS1 G2032R over 24 hours period.

25

SUPPLEMENTARY TABLES S1-7

Table S1. Physiochemical and absorption, distribution, metabolism, and excretion

(ADME), and cardio-safety properties of repotrectinib

MW LogP

Thermal Solubility at pH 7.0 (g/mL)

Human plasma protein binding

Human microsome CLint (L/mg protein)

Caco-2 Papp

A→B / Papp B→A (x10-

6cm/s)

Pgp efflux ratio

hERG in CHO cells IC50 (M)

hNav1.5 and hCav1.2 in HEK293 cells IC50 (M)

355.37 3.65 5.51 95.41% 7.85 5.7 / 41.4 7.3 18 both >30

Table S2A. CNS MPO Scores and Individual Transformed Scores (T0)

Comp T0_ClogPa T0_ClogDb T0_TPSAc T0_MWd T0_HBDe T0_pKaf CNS MPOg

crizotinib 0.36 1.0 1.0 0.357 0.333 0.365 3.42

lorlatinib 1.0 1.0 0.43 0.671 0.500 1.0 4.60

Repotrectini

b1.0 0.15 1.0 1.00 0.50 1.0 4.65

aTransformed score for calculated partition coefficient (ClogP). bTransformed score for calculated distribution coefficient at pH = 7.4 (ClogD). cTransformed score for molecular weight (MW). dTransformed score for topological polar surface area (TPSA). eTransformed score for number of hydrogen bond donors (HBD). fTransformed score for most basic center (pKa). gCentral nervous system multiparameter optimization (CNS MPO) (range 0-6).

Table S2B. Mice brain penetration of repotrectinib

Dosing Scheme Mouse ID

Repotrectinib Concentration Brain/Plasma Ratio

Plasma (ng/mL) Brain (ng/g) Ratio Mean SD

Single Dose at 75 mg/kg

585 6930 400 0.0577

0.0516 0.0060586 9010 464 0.0515

587 9010 412 0.0457

Repeated Dose, QD at 75 mg/kg for 7

days

592 4050 162 0.0400

0.0381 0.0093593 5510 155 0.0281

594 3540 164 0.0463Additional Information: Brain tissue-to-plasma ratio for repotrectinib was approximately 5.2% and 3.8%

26

after single and repeated dosing, respectively.

Table S3. Reaction Biology In vitro HotSpot Enzymatic Kinase inhibitory assay of

repotrectinib against wildtype (WT) and solvent front mutations of ROS1, TRKA, TRKB,

TRKC, and ALK.

Kinase IC50 (nM) at 10 M ATPROS1 TRKA TRKB TRKC ALK

WT G2032R D2033N WT G595R WT G639R

WT G623R

WT G1202R

0.0706 0.456 0.236 0.53

3 2.67 0.297 2.66 0.211 4.46 1.04 1.21

Table S4. Reaction Biology In vitro HotSpot Enzymatic Kinase inhibitory assay of

repotrectinib against clinically relevant ALK resistance mutations.

Kinase Inhibition at 10 M ATP IC50 (nM)ALK-NPM1 1.23 ALK (C1156Y) 0.932 ALK (1151T Ins) 2.16

ALK (L1196M) 1.08 ALK (S1206R) 0.525 ALK (T1151M) 0.491ALK (F1174L) 1.46 ALK (L1152R) 1.23 ALK (G1269A) 5.50ALK (F1174S) 1.02 ALK (R1275Q) 2.79 ALK (G1269S) 14.1

27

Table S5. Repotrectinib selectivity for ROS1 over selected kinases.

28

Target IC50 (nM) at 10 μM ATPSelectivity Index (SI: Kinase

IC50/ROS1 IC50)

ROS1 0.071 1.0

TRKC 0.211 3.0

TRKB 0.297 4.2

TRKA 0.533 7.5

ALK 1.04 14.6

JAK2 1.04 14.6

FYN 1.05 14.8

LYN 1.66 23.4

YES1 2.15 30.3

FGR 3.05 43.0

TXK 3.17 44.6

ARK5 4.46 62.8

SRC 5.3 74.6

DDR1 5.7 80.3

FAK 6.96 98.0

LCK 18.6 262.0

JAK1 19 267.6

TYK2 21.6 304.2

LTK 21.8 307.0

DDR2 23 324.0

BTK 23.7 333.8

EPHA1 25.0 352.1

RET 47.1 663.4

JAK3 50 704.2

EPHA8 50.2 707.0

IGFR 111 1563

AXL 149 2099

MARK3 512 7211

Table S6A. Inhibitory activity (IC50, nM) of repotrectinib and other ROS1 inhibitors in

Ba/F3 cell proliferation assays

ROS1 inhibitors Ba/F3 cells CD74-ROS1 Anti-proliferation IC50 (nM)

WT G2032R D2033NRepotrectinib < 0.2 3.3 1.3Crizotinib 14.6 266.2 200.9Lorlatinib 0.2 160.7 3.3Entrectinib 10.5 1813 169.2Ceritinib 42.8 1391 535.4Brigatinib 21 1172 128.4Cabozantinib 0.5 11.3 0.2Ensartinib 39.5 371.8 401.9

Table S6B. Inhibitory activity (IC50, nM) of repotrectinib and other TRK inhibitors in the

cell proliferation assays of engineered Ba/F3 cells and KM12 cells

TRK inhibitorsBa/F3 cells LMNA-TRKAAnti-proliferation IC50 (nM) KM12

(TPM3-TRKA)WT G595RRepotrectinib < 0.2 0.2 0.2

Entrectinib 0.5 705 9.2Loratrectinib 4 1024 12.3

TRK inhibitorsBa/F3 cells ETV6-TRKB

Anti-proliferation IC50 (nM)WT G639R

Repotrectinib < 0.2 0.6Entrectinib 0.5 1834

Larotrectinib 10.9 3000

TRK inhibitorsBa/F3 cells ETV6-TRKC

Anti-proliferation IC50 (nM)WT G623R G623E

Repotrectinib < 0.2 0.39 1.4Entrectinib 0.6 1623 1351Larotectinib 10.2 3293 742.3

Table S6C. Inhibitory activity (IC50, nM) of repotrectinib and other ALK inhibitors in the

cell proliferation assays of engineered stable Ba/F3 cells and Karpas-299 cells

ALK inhibitors

Ba/F3 cells EML4-ALK v1 Anti-proliferation IC50 (nM)

Karpas-299IC50 (nM)

H2228 IC50 (nM)

Parental Ba/F3 cells with IL3

IC50 (nM)WT G1202RRepotrectinib 27 63.6 23.7 100 1268Crizotinib 55.7 400 40 1200 2464Ceritinib 7.1 965 7.3 1000 3057

29

Alectinib 11.6 417.2 25.6 ND > 1000Brigatinib 10.9 190.5 10.9 ND NDLorlatinib 0.5 41.5 1.6 ND 10000

ND: not determined

Table S6D. Inhibitory activity (IC50, nM) of repotrectinib in the cell proliferation assays of

cell lines with unrelated oncogene drivers

Cell lines Cell proliferation IC50s (nM) Cellular system

SET2 168.9 JAK2 V617F

H460 2215 KRAS mutant

NCI-H1975 >3000 EGFR (L858R/T790M)

HCC827 1500 EGFR (exon 19 delE746_ A750)

HT1080 3000 NRAS(Q61K)

MKN45 5000 c-MET gene amplification

TT 3000 RET (C634W)

Table S7. The mean trough plasma concentration (at 12 hour) in mice tumor studies

Tumor Model Repotrectinib for Formulation

Dose(mg/kg BID) Ctrough (ng/mL) Ctrough,free

(nM)All ROS1 and ALK crystalline 15 19 2.2All ROS1 and ALK crystalline 75 112 13.3

NTRK micronized crystalline 3 29 3.5NTRK micronized crystalline 15 191 22.7NTRK micronized crystalline 60 1460 173.5

30

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