A novel orally active inverse agonist of estrogen-related receptor … · 2019. 4. 20. · Jae-Eon Lee5, Sang6 Bong Lee3, Heeseok Yoon3, Ji Hoon Yu3, Sang Kyoon Kim5, GhilSuk 7 Yoon

1

A novel orally active inverse agonist of estrogen-1

related receptor gamma (ERRγ), DN200434, a 2

booster of NIS in anaplastic thyroid cancer 3 4

Thoudam Debraj Singh1,2,‡, Jaeyoung Song3,‡, Jina Kim3,‡, Jungwook Chin3,‡, Hyun Dong Ji4, 5

Jae-Eon Lee5, Sang Bong Lee3, Heeseok Yoon3, Ji Hoon Yu3, Sang Kyoon Kim5, GhilSuk 6

Yoon6, Hayoung Hwang3, Ho Won Lee4, Ji Min Oh4, Sang-Woo Lee2,4, Jaetae Lee4, Hueng-7

Sik Choi10, Soon-Young Na10, Won-Il Choi2, 11, Young Joo Park7, Young Shin Song7, Young-8

A Kim8, In-Kyu Lee2,9,*, Sung Jin Cho2,3,*, Yong Hyun Jeon2,5,* 9

1Department of Medical Oncology Laboratory, All India Institute of Medical Sciences 10

(AIIMS), Ansari Nagar, New Delhi, India; 2Leading-edge Research Center for Drug 11

Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National 12

University Hospital, Daegu, South Korea; 3New Drug Development Center, Daegu-13

Gyeongbuk Medical Innovation Foundation, Daegu, South Korea; 4Department of Nuclear 14

Medicine, School of Medicine, Kyungpook National University Hospital, Daegu, South 15

Korea; 5Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, 16

Daegu, South Korea; 6Department of Pathology, School of Medicine, Kyungpook National 17

University Hospital, Daegu, South Korea; 7Department of Internal Medicine, Seoul National 18

University College of Medicine, Seoul, South Korea; 8Department of Pathology, Borame 19

Medical Center 20, Boramae-ro 5-gil, Dongjak-gu, Seoul, South Korea; 9Department of 20

Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook 21

National University Hospital, Daegu, South Korea; 10National Creative Research Initiatives 22

Center for Nuclear Receptor Signals, School of Biological Sciences and Technology, 23

Chonnam National University, Gwangju, South Korea; 11Bio-Medical Research Institute, 24

Kyungpook National University Hospital, Daegu, South Korea 25 ‡Authors contributed equally to this work. 26 1To whom correspondence should be addressed. Email: [email protected], 27

[email protected], [email protected] 28

29

Running Title: DN200434 as a NIS booster in ATC 30

31

Keywords: Sodium iodide symporter (NIS); Anaplastic thyroid cancer (ATC); Estrogen-32

related receptor gamma (ERRγ); Radioiodine therapy 33

Additional Information: 34 1To whom correspondence should be addressed. Email: [email protected], 35

[email protected], [email protected] 36

37

Conflict of Interest: All authors have declared that no conflict of interest exists. 38

Research. on June 9, 2021. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 22, 2019; DOI: 10.1158/1078-0432.CCR-18-3007

http://clincancerres.aacrjournals.org/

2

1

Statement of Translational Relevance: Approximately 30% of patients with thyroid cancer 2

are refractory to radioiodine therapy consequent to insufficient sodium iodide symporter (NIS) 3

expression and function in anaplastic thyroid cancers (ATCs). In the present study, we report 4

the discovery as well as the structural, biological, and functional characterization of DN200434, 5

which, to our knowledge, is the most cell-active and first orally bioavailable inverse agonist of 6

ERRγ. DN200434 demonstrated a unique co-crystal structure with ERRγ and enhanced 7

radioiodine avidity in vitro as well as in CAL62 tumors in an ATC mouse model in vivo. 8

Notably, this enhancement allowed successful radioiodine therapy of CAL62 tumors that were 9

refractory to conventional radioiodine therapy. Thus, our findings highlight the DN200434 10

potential for clinical application in the diagnosis and treatment of ATCs as well as other poorly 11

differentiated thyroid cancers. 12

13

Abstract 14

15

Background: New strategies to restore sodium iodide symporter (NIS) expression and 16

function in radioiodine therapy-refractive anaplastic thyroid cancers (ATCs) are urgently 17

required. Recently, we reported the regulatory role of estrogen-related receptor gamma (ERRγ) 18

in ATC cell NIS function. Herein, we identified DN200434 as a highly potent (functional IC50 19

= 0.006 μM), selective, and orally available ERRγ inverse agonist for NIS enhancement in 20

ATC. 21

Experimental Design: We sought to identify better ERRγ-targeting ligands and explored the 22

crystal structure of ERRγ in complex with DN200434. After treating ATC cells with 23

DN200434, the change in iodide-handling gene expression as well as radioiodine avidity was 24

examined. ATC tumor-bearing mice were orally administered with DN200434, followed by 25

124I-positron emission tomography/computed tomography (PET/CT). For radioiodine therapy, 26

ATC tumor-bearing mice treated with DN200434 were administered 131I (beta ray-emitting 27

therapeutic radioiodine) and then bioluminescent imaging was performed to monitor the 28

therapeutic effects. Histological analysis was performed to evaluate ERRγ expression status in 29

normal tissue and ATC tissue, respectively. 30

Results: DN200434-ERRγ complex crystallographic studies revealed that DN200434 binds to 31

key ERRγ binding pocket residues through four-way interactions. DN200434 effectively 32




3

upregulated iodide-handling genes and restored radioiodine avidity in ATC tumor lesions, as 1

confirmed by 124I-PET/CT. DN200434 enhanced ATC tumor radioiodine therapy 2

susceptibility, markedly inhibiting tumor growth. Histological findings of patients with ATC 3

showed higher ERRγ expression in tumors than in normal tissue, supporting ERRγ as a 4

therapeutic target for ATC. 5

Conclusions: DN200434 shows potential clinical applicability for diagnosis and treatment of 6

ATC or other poorly differentiated thyroid cancers. 7

8

Keywords: Sodium iodide symporter (NIS); Anaplastic thyroid cancer (ATC); Estrogen-9

related receptor gamma (ERRγ); Radioiodine therapy 10

11

Introduction 12

13

Sodium iodide symporter (NIS) constitutes a transmembrane glycoprotein that induces iodide 14

uptake in thyroid follicular cells [1,2] consequent to increased thyroid stimulating hormone 15

stimulation. Ablative radioiodine therapy and nuclear medicine imaging of NIS are widely used 16

in clinical settings for treatment and diagnosis of well differentiated thyroid cancers and their 17

metastasis with minimal adverse effects. However, their practical application to anaplastic 18

thyroid cancer (ATC), a poorly differentiated thyroid cancer exhibiting progressive 19

dedifferentiation, cancer treatment resistance, and aggressive lung, bone, and regional lymph 20

node metastasis, is hampered by insufficient NIS expression and function. Thus, restoring NIS 21

expression and function in ATC and less differentiated thyroid cancers has been attempted 22

through gene delivery or demethylating agent, nuclear receptor agonist, kinase inhibitor, or 23

histone deacetylase inhibitor treatment to render cells responsive to radioiodine [2,3], albeit 24

without satisfactory therapeutic outcomes. 25

Estrogen-related receptors (ERRs) including ERRα, ERRβ, and ERRγ comprise 26

constitutively active nuclear receptors belonging to the NR3B nuclear receptor superfamily. 27

ERR isoforms are expressed in the brain, heart, pancreas, and liver. ERRγ, encoded by Esrrg, 28

plays a key role in tissue metabolism. The peripheral circadian clock regulates ERRγ in muscle, 29

white or brown adipose tissue, and liver [4]; in turn, ERRγ enhances uncoupling protein 1 30

expression and fatty acid oxidation in brown adipose tissues [5]. ERRγ is an essential factor 31

for functional glucose-responsive β-cell maturation [6] but is also closely associated with 32




4

cancer progression including breast and prostate cancers [7,8]. ERRγ is reported as a promising 1

biomarker for evaluating breast cancer prognosis [7]. Conversely, ERRγ leads to both 2

androgen-sensitive and insensitive prostate cancer cell suppression. Thus, ERRγ may be an 3

attractive therapeutic target and biomarker for metabolic disorders and cancer. 4

We have recently reported that the selective ERRγ inverse agonist GSK5182 enhances NIS-5

mediated radioiodine uptake in ATC cells with either KRAS or BRAF mutations, promoting 6

enhanced radioiodine therapy responsiveness in vitro [9]. Toward discovering novel ERRγ 7

inverse agonists, we also reported several lead optimization studies including 4-8

hydroxytamoxifen analogue synthesis and biological evaluation [10,11]. This finding provided 9

a rationale for exploring new ERRγ inverse agonists that effectively enhance NIS function in 10

vivo and show potential for clinical translation to patients with ATC. Herein, we report the 11

discovery of DN200434, a biocompatible, highly selective, and orally bioavailable ERRγ 12

inverse agonist that acts as a NIS booster as well as its applicability for ATC treatment in vitro 13

and in vivo. 14

15

Materials and Methods 16

17

Metabolic stability assay of liver microsomes 18

Metabolic stability assays were performed via incubation of human and selected animal liver 19

microsomes (most often dog, rat, and mouse) at 37 °C with a test compound at a final 20

concentration of 1 μM in the presence of 0.5 mg/mL microsomal protein and NADPH 21

regeneration system in a total volume of 100 μL of 100 mM phosphate buffer (pH 7.4). The 22

incubation was started by the addition of the NADPH regeneration system and terminated with 23

40 μL ice-cold acetonitrile after 0 and 30 min. Precipitated proteins were removed by 24

centrifugation at 10,000 × g for 5 min at 4 °C. Aliquots of the supernatant were injected into a 25

liquid chromatography-tandem mass spectrometry (LC-MS/MS; Thermo Fisher Scientific, 26

Waltham, MA) system. Incubations terminated prior to the addition of NADPH regeneration 27

system (time point, 0 min) were used as standards and defined as 100%. Percent of the parent 28

compound remaining was calculated by comparing peak areas. 29

30

CYP inhibition assay 31




5

All incubations were performed in duplicate and the mean values were used for analysis. 1

Phenacetin O-deethylase, tolbutamide 4-hydroxylase, S-mephenytoin 4-hydroxylase, 2

dextromethorphan O-demethylase, and midazolam 1′-hydroxylase activity assays were 3

determined as probe activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A, 4

respectively, using cocktail incubation and MS/MS. Briefly, the incubation reaction was 5

performed with 0.25 mg/mL human liver microsomes in a final incubation volume of 100 μL. 6

The incubation medium contained 100 mM phosphate buffer (pH 7.4) with probe substrates. 7

The incubation mixture containing various inhibitors (10 μM) was pre-incubated for 5 min, 8

after which an NADPH regenerating system was added. After incubation at 37 °C for 15 min, 9

the reaction was stopped by placing the incubation tubes on ice and adding 40 μL ice-cold 10

acetonitrile. The incubation mixtures were then centrifuged at 10,000 × g for 5 min at 4 °C. 11

Aliquots of the supernatant were injected into an LC-MS/MS system. CYP-mediated activity 12

in the presence of inhibitors was expressed as percentages of the corresponding control values. 13

14

Parallel artificial membrane permeability assay (PAMPA) 15

PAMPA was conducted with lipid tri-layer PVDF membranes according to the manufacturer’s 16

instructions (Gentest; Corning Inc., New York, NY). Briefly, 300 L of test compound diluted 17

to 10 M in PBS (pH7.4) was added to the bottom wells of a 96-transwell plate while 200 L 18

PBS was added to the upper wells. After incubation for 5 h at 25 C, 20 L aliquots from each 19

well (bottom and upper wells) were transferred into new tubes and mixed with 80 L 20

acetonitrile (Sigma-Aldrich, St. Louis, MO) containing 4 M chlorpropamide (Sigma-Aldrich) 21

as internal standard. Then, test compound concentrations were measured by LC-MS/MS 22

(Thermo Fisher Scientific, Waltham, MA) and permeability rates were calculated using the 23

equations previously reported. 24

25

Human ether-à-go-go-related gene (hERG) assays 26

hERG channel binding assays were performed using the predictor hERG fluorescence 27

polarization assay (Cat. no. PV5365; Invitrogen, Carlsbad, CA) according to manufacturer’s 28

instructions. For measuring IC50, compounds were serially diluted (16 points, 3-fold), followed 29

by reactions for 4 h at 25 °C in a reaction mixture containing hERG membrane, fluorescence 30

tracer red dye, and fluorescence polarization buffer. Fluorescence intensity (excitation at 530 31

nm, emission at 590 nm) was measured using the multi-mode microplate reader Synergy Neo 32




6

(BioTek, Winooski, VT). E-4031 was used as the reference positive standard (IC50 = 10–90 1

nM). 2

3

Ames microplate format (MPF) mutagenicity assay 4

The Ames MPF 98/100 Mutagenesis Assay Kit (Xenometrix, Allschwil, Switzerland) was used 5

to test mutagenic activity and contains the following components, 2-nitrofluorene(2-NF, 4-6

nitroquinolone-N-oxide(4-NQO),2-aminoanthracene(2-AA) as positive control, aroclor 1254-7

induced lyophilized rat liver S9 fraction as the exogenous metabolic activation, ampicillin (50 8

mg/mL), salmonella growth medium, exposure medium, and indicator medium. The 9

TA98/TA100 Salmonella strain was used to inoculate the bacterial culture medium and 10

cultured overnight. The Salmonella test strain was then exposed to six concentrations of the 11

test compound (including positive and negative controls). Compounds were prepared in 96-12

well plates in sterile DMSO (Sigma-Aldrich). Cultured bacteria, test compound, and S9 13

mixture are incubated for 90 min at 37 °C in a shaking incubator. Next, the mixture solution 14

was added with indicator medium and dispensed into 384-well plates. The plates were then 15

incubated at 37 °C for 48 h, after which the number of revertant wells in each section was 16

counted. 17

18

Pharmacokinetic studies 19

Sprague-Dawley rats were purchased from Korean Animal Technology (Koatech, Pyeongtaek, 20

South Korea) and maintained in a specific pathogen-free facility (Laboratory Animal Center, 21

Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea) with unlimited 22

access to water and food. Rats weighing 250–300 g and 7 weeks old were fasted for 16 h and 23

subsequently used for experiments. Before compound administration, blood was collected from 24

the jugular vein and used as blank control. For oral administration, four rats received the 25

compound suspended in 10% DMSO, 15% water, and 75% PEG400 at a dose of 10 mg/kg via 26

oral gavage. For intravenous administration, the compound was injected at a dose of 1 mg/kg 27

via the caudal vein. Dosing volume of the compound was 600 µL for oral and 200 µL for 28

intravenous administration. Blood from the jugular vein was collected into heparinized tubes 29

at 0.08, 0.25, 0.5, 1, 2, 4, 6, and 8 h after compound administration. Plasma was prepared from 30

blood samples by centrifugation at 12,000 rpm for 15 min, after which 20 µL plasma was mixed 31

with 80 µL acetonitrile (Sigma-Aldrich) containing internal standard and centrifuged at 14,000 32




7

rpm for 5 min. Collected plasma supernatants were loaded into triple quadrupole LC-MS/MS 1

(Triple Quad 5500; Applied Biosystems, Foster City, CA) to measure the compound 2

concentrations. The standard curve range was 5 to 1000 ng/mL and the lower limit of 3

measurement quantification was 5 ng/mL. Pharmacokinetic parameters were analyzed with 4

non-compartmental analysis using Phoenix WinNolin ver 6.4 (Pharsight, St. Louis, MO). 5

6

Protein production and crystallization 7

Protein expression and purification 8

The gene fragment of human ERRγ LBD (222–458) was designed and codon optimized, after 9

which it was ordered from Integrated DNA Technologies (Coralvile, IA). The DNA fragment 10

was cloned into PGEX-6P1 vector as a GST-tagged protein and expressed in Escherichia coli 11

BL21(DE3) cells. The overnight culture from a single plate colony was used to inoculate LB 12

media containing 100 μg/mL ampicillin, after which the cells were grown for 1–2 h to reach 13

an A600 = 0.7 at 37 °C. The temperature was lowered to 25 °C and 1 mM isopropyl-1-thio-β-D-14

galactopyranoside was added to start protein expression. Cells were harvested after 4 h and 15

stored at −80 °C. Cell pellets were lysed by resuspension and sonication in PBS buffer 16

containing 1 tablet per 50 mL buffer of protease inhibitor cocktail (Roche, Basel, Switzerland) 17

and 1 mM PMSF (Sigma-Aldrich). The lysate was clarified by centrifugation and the protein 18

solution was filtered and loaded onto a 4 mL Glutathione Sepharose 4B column (GE Healthcare, 19

Chicago, IL) with gravity flow. Bound protein was washed with PBS buffer and eluted with 50 20

mM Tris (pH 8.0) and 10 mM reduced glutathione. The ERRγ-containing fractions were pooled 21

according to SDS-PAGE analysis. Pooled protein solutions were dialyzed against 50 mM Tris 22

(pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT at 4 °C overnight. PreScission protease 23

(GE Healthcare) was added to the protein solution for cleavage of the GST tag with rocking 24

incubation at 4 °C for 4 h, after which the protein solution was loaded onto the 4 mL 25

Glutathione Sepharose 4B column to separate tag-free ERRγ from GST-tagged ERRγ. Flow-26

through containing tag-free ERRγ was pooled and then diluted 5-fold with 50 mM Tris (pH 27

8.0) and 1 mM DTT. Diluted protein was loaded onto a 5 mL Hitrap Q HP (GE Healthcare) 28

and eluted with a 75 mM to 600 mM NaCl gradient over 10 column volumes. The ERRγ-29

containing fractions were pooled according to SDS-PAGE analysis. The pooled material was 30

further purified using a Superdex 200 with an elution buffer containing 25 mM Tris (pH 7.5), 31

150 mM NaCl, and 1 mM DTT. Peak fractions were collected by SDS-PAGE analysis and 32




8

concentrated to 10 mg/mL. The final material was aliquoted, flash frozen in liquid N2, and 1

stored at −80 °C until further use. 2

3

Crystallization 4

Purified ERRγ in Superdex 200 buffer was complexed with a 3-fold molar excess of the 5

compound DN200434 for 1 h on ice, followed by incubation at room temperature for 30 min. 6

Any precipitated material was removed by centrifugation prior to initial screening trials. Initial 7

crystallization trials using commercial screens for protein complex were carried out using 8

mosquito liquid handler (TTP LabTech, Melbourn, UK) followed by additive screening. 9

Optimal crystals were grown in a buffer containing 0.2 M NH4F (pH 6.2), 20% polyethylene 10

glycol 3350, and 0.01 M ethylenediaminetetraacetic acid disodium salt dihydrate at 18 °C. For 11

drop size up, the hanging drop vapor diffusion technique was performed, in which drops 12

containing 1 μL protein and 1 μL mother liquor were equilibrated above a 500 μL mother liquor 13

reservoir. 14

15

X-ray data collection and processing 16

Co-crystals of ERRγ with DN200434 were directly mounted onto cryoloops and quickly frozen 17

in liquid N2. Glycerol concentration of the well buffer was slowly increased in a stepwise 18

fashion for cryopreservation of the crystals from a concentration of 8% to 32%, followed by 19

mounting the crystals onto cryoloops and quickly freezing in liquid N2. All data were collected 20

at the 5C beamline of the Pohang Accelerator Laboratory (PAL), Republic of Korea. Data 21

processing and scaling were carried out using the HKL2000. 22

23

Structure determination and refinement 24

The ERRγ crystal structure in complex with DN200434 was solved by molecular replacement 25

using CCP4 program suite. Model building and refinement were carried out using COOT7 and 26

Refmac58, respectively. Water molecules were added automatically with the ARP/wARP 27

function in Refmac5, then manually examined for reasonable hydrogen bonding possibilities. 28

29

Chemistry 30

(E)-4-(5-hydroxy-1-(4-(4-isopropylpiperazin-1-yl)phenyl)-2-phenylpent-1-en-1-yl)phenol 1H 31

NMR (CD3OD, 400 MHz) δ 7.18–7.09 (m, 5 H), 7.04 (d, J = 8.6 Hz, 2 H), 6.83–6.77 (m, 4 H), 32




9

6.69 (d, J = 8.8 Hz, 2 H), 3.78 (d, J = 13.7 Hz, 2 H), 3.55 (m, 3 H), 3.42 (t, J = 6.8 Hz, 2 H), 1

3.23 (m, 2 H), 2.94 (t, J = 11.8 Hz, 2 H), 2.53 (m, 2 H), 1.55 (m, 2 H), 1.40 (d, J = 6.7 Hz, 6 2

H). 13C NMR (CD3OD, 100 MHz) δ 156.07, 145.18, 142.60, 139.74, 138.73, 138.59, 134.39, 3

131.69, 130.26, 129.44, 127.61, 125.81, 116.27, 114.60, 61.62, 58.46, 32.10, 31.64, 15.64. 4

HRMS (EI+) m/z calcd for C30H36N2O2 457.2777; found 457.28412. 5

6

Clinical tissue sample analysis 7

For histological analyses of human thyroid tissues, we constructed tissue microarrays (TMAs) 8

consisting of 38 normal thyroid samples, 96 papillary thyroid carcinoma (PTC), and 26 poorly-9

differentiated or anaplastic thyroid carcinoma (PD/ATC) samples. Representative areas of the 10

cancer lesions were carefully selected on hematoxylin and eosin-stained sections and two tissue 11

cores (2-mm in diameter) were obtained from each paraffin block. Immunohistochemical (IHC) 12

staining of the TMAs was performed using anti-human ERR gamma (mouse monoclonal 13

antibody; PP-H6812-00; dilution 1:100; R&D systems, Minneapolis, MN), NIS (goat 14

polyclonal antibody; sc-48052; dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), 15

thyrotropin receptor (TSHR goat polyclonal antibody; sc-7816; dilution 1:100; Santa Cruz 16

Biotechnology), thyroglobulin (rabbit polyclonal antibody; A0251; dilution 1:5000; Dako, 17

Glostrup, Denmark), and thyroid peroxidase (TPO rabbit polyclonal antibody; NBP1-80670; 18

dilution 1:200; Novus Biologicals, Littleton, CO). This study was approved by the 19

Institutional Review Board of the Seoul National University Hospital and was conducted in 20

accordance with the Declaration of Helsinki (approved ID: H-1107-060-369). 21

22

Immunohistological analysis 23

Histological data were obtained from formalin-fixed, paraffin-embedded tumor samples. IHC 24

staining using a Benchmark XT Slide Stainer (Ventana Medical Systems, Inc., Tucson, AZ) 25

was performed according to manufacturer’s instructions. Anti-hNIS (Thermo Fisher Scientific, 26

Waltham, MA) was applied to whole-sectioned tumor slides. The stained sections were then 27

reviewed without any knowledge of the experimental data. 28

29

Animals 30

Specific pathogen-free, 6-week-old, female BALB/c nude mice were obtained from SLC, Inc. 31

(Shizuoka, Japan). All animals were maintained and used in accordance with the Guidelines 32




10

for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Center, 1

Daegu-Gyeongbuk Medical Innovation Foundation. The animal studies were conducted after 2

approval by the institutional reviewer board (IRB) on the Ethics of Animal Experiments of the 3

Daegu-Gyeongbuk Medical Innovation Foundation (approval number: DGMIF-17120802-00). 4

5

Cells 6

Anaplastic thyroid cancer cells, CAL62 and BHT101, were purchased from Deutsche 7

Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). CAL62 8

and BHT101 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM high 9

glucose) supplemented with 10% fetal bovine serum (Gibco FBS; Thermo Fisher Scientific) 10

and 1% antibiotic-antimycotic (HyClone; Thermo Fisher Scientific) at 37 ºC in a 5% CO2 11

atmosphere. Mycoplasma testing was regularly performed every month using a BioMycoX 12

Mycoplasm PCR Detection Kit (CellSafe, Seoul, South Korea) 13

14

Plasmids 15

pFR-luc (Stratagene, San Diego, CA) was used as a Gal4-driven luciferase reporter. Expression 16

vectors for the wild-type ERRγ ligand binding domain (LBD) and the mutants D273A, E275A, 17

Y326A, and N346A were constructed in pCMX-Gal4DBD (DNA binding domain). ERRγ 18

mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). 19

20

Cell culture, transient transfection, and luciferase assay 21

HEK 293T cells were cultured in DMEM supplemented with 10% FBS. Cells were then 22

transiently transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to 23

manufacturer’s instructions. Luciferase activity was measured after treatment with vehicle, 24

GSK5182 (1 µM), and DN200434 for 18 h and normalized to -galactosidase activity. 25

26

Radioiodine uptake assay 27

The change in radioiodine avidity was determined as previously described [9]. 28

29

18F-FDG uptake assay 30

Cells seeded in 24-well plates were treated with DN200434 for 24 h at a concentration of 0, 6, 31

and 12 μM. After aspirating the drug-containing medium, cells were washed with 1 mL Hank’s 32




11

balanced salt solution (HBSS) and incubated in 500 µL HBSS containing 0.5% bovine serum 1

albumin (bHBSS) and 74 kBq of 18F-FDG per milliliter for 30 min at 37 °C. The cells were 2

then washed twice with ice-cold bHBSS and lysed in 500 μL 2% sodium dodecyl sulfate (SDS). 3

Radioactivity was measured using a gamma counter (Packard Cobra II Gamma Counter; 4

PerkinElmer, Waltham, MA). Cell radioactivity was normalized using total protein 5

concentrations determined with a Pierce BCA kit (Thermo Fisher Scientific). 6

7

Quantitative RT-PCR 8

Total RNA was extracted using TRIzol reagent (Invitrogen) and then 2 µg of total RNA was 9

reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher 10

Scientific). Quantitative RT-PCR was carried out with the SYBR Green PCR master mix 11

(Applied Biosystems, Foster City, CA) using a ViiA 7 Real-Time PCR System (Applied 12

Biosystems) with the following primer sets, NIS forward, 5ʹ-CTG CCC CAG ACC AGT ACA 13

TGC C-3ʹ and reverse, 5ʹ-TGA CGG TGA AGG AGC CCT GAA G-3ʹ; TSHR forward, 5ʹ-14

ACC CTG ATG CCC TCA AAG AGC-3ʹ and reverse, 5ʹ-GCT TCA GTG TCA AGG TTT 15

CAT TGC-3ʹ; TPO forward, 5ʹ-CCT CTG CAA AGA TGT GAA CGA-3ʹ and reverse, 5ʹ-TCC 16

CGG AGT CTA CGC AGG TT-3ʹ; TG forward, 5ʹ-TCT AAC CGA TGC TCA CCT CTT 17

CTG-3ʹ and reverse, 5ʹ-AGA TGA TGG CAC CTC CTT GAA CC-3ʹ; and acidic ribosomal 18

protein 36B4 forward, 5ʹ-CCA CGC TGC TGA ACA TGC T-3ʹ and reverse, 5ʹ-TCG AAC 19

ACC TGC TGG ATG AC-3ʹ. Target genes were normalized to the endogenous reference gene 20

36B4 and relative mRNA expression levels were calculated using the test and control samples. 21

22

Western blotting 23

The change in membrane NIS and other protein levels was evaluated as previously described 24

[9]. 25

26

Cell viability assay using in vitro bioluminescent imaging (BLI) 27

To evaluate the effects of the reporter gene on cell proliferation, cell proliferation assays were 28

performed using the Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Tokyo, Japan). 29

CAL62/effluc cells were plated at 1 × 104 cells per well in 96-well plates. After 24 h, 30

DN200434 at various concentrations was used to treat the cells. At 24 h after treatment, D-31




12

luciferin solution was added to each well and then in vitro BLI was conducted using IVIS 1

Lumina III. 2

3

Cell cycle analysis 4

Washed and fixed cells were stained in the dark with 0.5 mL propidium iodide/RNase staining 5

buffer for 15 min at room temperature. DNA content, cell cycle profiles, and forward scatter 6

were analyzed using a Becton Dickinson LSRFortessaTM (Franklin Lakes, NJ) with emission 7

detection at 488 nm (excitation) and 575 nm (peak emission). Data were analyzed using 8

FlowJoTM (BD Biosciences, San Jose, CA). 9

10

Apoptosis analysis 11

Cells were washed with PBS containing 1% horse serum and stained with the FITC-Annexin 12

V Apoptosis Detection Kit I (BD Biosciences) at room temperature for 30 min in the dark. 13

DNA content, cell cycle profiles, and forward scatter profiles were determined using the Becton 14

Dickinson LSRFortessaTM flow cytometer and analyzed with FlowJoTM software. 15

16

Clonogenic assay 17

Cells were plated on 6-well plates and incubated for 48 h. After treatment with 12 μM 18

DN200434 for 24 h, the drug-containing medium was discarded, and cells were washed twice 19

with PBS. The medium was then replaced with DMEM in the presence or absence of 50 μCi 20

131I (KIRAMS, Seoul, Korea) for 6 h. Cells were washed with cold bHBSS and incubated in 21

complete culture medium for a time duration corresponding to six doublings. Finally, cells 22

were fixed in 4% paraformaldehyde (PFA) solution and stained with 0.05% crystal violet. 23

Control and treated colonies with more than 50 cells were counted. 24

25

Biodistribution analysis and 124I-positron emission tomography/computed tomography 26

(PET/CT) imaging 27

Tumor model establishment, drug treatment, and administration of radioactive iodide 28

Mice were subcutaneously challenged with CAL62/effluc cells (5 × 106 cells per mouse). When 29

tumor formation was detected via inspection and palpation, tumor-bearing mice were orally 30

administered vehicle (polyethylene glycol) or DN200434 (100 and 200 mg/kg) dissolved in 31

solution (10% ethanol, 10% cremphol, and 80% PEG) once a day for 6 days. CAL62/effluc 32




13

tumor-bearing mice received radioactive 125I (10 uCi per mouse for the biodistribution 1

experiment) or 124I (50 uCi per mouse for PET imaging) via the tail vein. After 2 h of circulation 2

time, biodistribution analysis or PET/CT imaging was performed. 3

4

Biodistribution with radioactive 125I 5

For biodistribution analysis, organs including tumors, liver, lung, heart, kidney, intestine, and 6

others were removed, weighed, and tested for radioactivity using a gamma counter. The results 7

were expressed as the percentage of injected dose per gram of tissue (%ID/g). 8

9

PET/CT imaging with radioactive 124I 10

For PET/CT imaging, 20-min scans were performed using the Triumph II PET-CT System 11

(LabPET8; Gamma Medica-Ideas, LA, CA). All mice were anesthetized using 1–2% isoflurane 12

gas during imaging. CT scans were performed using an X-ray detector immediately following 13

the acquisition of PET images. PET images were reconstructed by 3D-OSEM iterative image 14

reconstruction and CT images were reconstructed using filtered back-projections.. PET images 15

were co-registered with anatomical CT images using 3D image visualization and analysis 16

software VIVID (Gamma Medica-Ideas). To measure uptake for the volumes of interest (VOIs), 17

the VOIs from each image were manually segmented from co-registered CT images using 18

VIVID. 19

20

In vivo therapy 21

Tumor-bearing mice were divided into four groups as follows, group 1: vehicle; group 2: 200 22

mg/kg DN200434 daily for 6 days; group 3: single dose of 1 mCi 131I; and group 4: 200 mg/kg 23

DN200434 daily for 6 days followed by a single dose of 1 mCi 131I. After the final DN200434 24

treatment, tumor-bearing mice received radioactive 131I. In vivo bioluminescence imaging (BLI) 25

was performed regularly at the indicated time intervals to monitor therapy response. Mouse 26

body weight was also monitored at the same time. 27

For the BHT101 xenograft model, in vivo therapy was conducted as described for the CAL62 28

xenograft model when the tumor was detectable via inspection and palpation. Tumor size was 29

measured with a caliper at the indicated time points and tumor volume (mm3) was calculated 30

using the following formula, tumor volume (mm3) = d2 × D/2, where d and D are the shortest 31

and longest diameter in mm, respectively. 32




14

1

In vivo bioluminescence imaging 2

Tumor-bearing mice received D-luciferin via intraperitoneal injection and then BLI was 3

performed 10 min after substrate injection using the IVIS Lumina III In Vivo Imaging System 4

(PerkinElmer). All mice were anesthetized using 1–2% isoflurane gas during imaging. 5

Grayscale photographic images and bioluminescent color images were superimposed using 6

LIVINGIMAGE (version 2.12; PerkinElmer) and IGOR Image Analysis FX software 7

(WaveMetrics, Lake Oswego, OR). BLI signals were expressed in units of photons per cm2 per 8

second per steradian (P/cm2/s/sr). 9

10

Statistical analysis 11

All data are expressed as the mean ± standard deviation (SD) of at least three representative 12

experiments, and statistical significance was determined using an unpaired Student’s t-test. P-13

values of < 0.05 were considered statistically significant. To determine the significance of in 14

vivo radioiodine avidity (PET/CT imaging experiment) between the vehicle and DN200434 15

groups, paired Student’s t-test was adopted. To compare trends of ERRγ, NIS, TSHR, 16

thyroglobulin, and TPO expression in normal thyroid samples with PTC and PD/ATC samples, 17

the linear-by-linear association test was used. 18

19

Results 20

21

ERRγ as a therapeutic target in ATC 22

We evaluated ERRγ and NIS protein expression in human ATC carcinomas and normal thyroid 23

tissues via immunoblotting and found that only the former demonstrated strong ERRγ protein 24

expression (Supplementary Fig. S5); conversely, only the normal thyroid tissues exhibited 25

strong NIS expression. Moreover, western blotting showed that ERRγ expression was detected 26

in two of five normal thyroid tissues. To confirm our results, we attempted to examine as many 27

tumor tissues as possible via immunoblotting; however, it is difficult to obtain a sufficient 28

number of fresh frozen tissues and thus we performed immunohistochemistry to confirm the 29

differences in ERRγ protein expression levels according to pathologic type using TMA 30

consisting of 38 normal, 96 PTC, and 26 PDC/ATC tissues. As shown in Fig. 1a and b, high 31

ERRγ protein expression in poorly differentiated ATC and samples from patients with thyroid 32




15

cancer was confirmed by TMA analysis with an ERRγ-specific antibody (Supplementary 1

Table S1). On the other hand, ERRγ expression was downregulated in normal thyroid tissue 2

and papillary thyroid carcinoma (PTC). As expected, iodide-handling gene expression, 3

including NIS, TPG, and TSHR, was lower in poorly-differentiated or anaplastic thyroid 4

carcinomas (PD/ATC) than in normal tissues or PTC carcinomas. These results indicate an 5

ERRγ association with ATC pathophysiological characteristics, supporting our hypothesis of 6

ERRγ as a promising ATC therapeutic target in vivo. 7

8

ERRγ targeting and DN200434 binding 9

We synthesized an approximately 300-compound focused library based on structural motifs of 10

the hit compound GSK5182, an active metabolite of tamoxifen, an FDA-approved drug 11

optimized for ER+ breast cancer. Lead optimization of this series toward DN200434 will be 12

published separately. Basic structure-activity relationships were tracked using a combination 13

of biochemical assays with time-resolved fluorescence resonance energy transfer (TR-FRET) 14

for binding potency and subtype selectivity, and cell-based functional assays using co-15

transfection systems for cellular activity. 16

We first performed a quick screening based on ERRγ binding potency and then checked 17

binding selectivity over related subtypes. For the selected compounds, inverse agonistic 18

characteristics were validated in cell-based assays including functional IC50. Between-assay 19

values showed relatively good correlations (Fig. 2a). Briefly, analogues distal to the N,N-20

dimethylaminoethyloxy group were mainly examined, yielding significant binding selectivity 21

and cellular potency as well as in vitro/in vivo absorption, distribution, metabolism, excretion, 22

and toxicity (ADMET) property improvements. After several iterative design cycles, 23

DN200434 (Fig. 2b and Supplementary Fig. S1–4) was identified as the best tool compound 24

for subsequent biological evaluations including X-ray crystallography and in vivo therapy. 25

DN200434 exhibited promising ERRγ binding affinity (IC50 = 0.040 μM) and comparable 26

subtype selectivity with no prominent ERRα, ERRβ, or ERα affinity (IC50 > 10, 1.330, and 27

1.240 μM, respectively) in in vitro binding assays (Fig. 2c–f). DN200434 was the most potent 28

ERRγ inverse agonist with a functional IC50 of 0.006 μM activity, 12-fold more than that of 29

GSK5182 (Fig. 2g). 30

DN200434 showed comparable in vitro safety profiles to those of the hit compound, 31

including representative 1A2, 2C9, 2C19, 2D6, and 3A4 in CYP subtype inhibition, human 32




16

ether-à-go-go-related gene (hERG) binding and current inhibition, and mini-Ames test 1

(Supplementary Table S2 and S3). Additional in vitro ADMET studies, such as parallel 2

artificial membrane permeability assays (Supplementary Table S2), Caco-2 cell permeability, 3

plasma protein binding, plasma stability, and P-glycoprotein inhibition, indicated DN200434 4

as the new lead compound for treating ERRγ-related disorders (data not shown). Although in 5

vitro microsomal stability data were indicative of a metabolically soft nature (Supplementary 6

Table S2), it showed appropriate in vivo pharmacokinetics profiles for subsequent in vivo 7

efficacy validation, including an extended half-life (3.8 h) as well as enhanced p.o. area under 8

the curve (AUC; 0.81 μMh) and bioavailability (21.4%; Supplementary Table S4). As the 9

oral bioavailability of GSK5182 was only 8.4%, we theorized that the improved bioavailability 10

of DN200434 resulted from a small change in the C-ring attachment, producing one of the key 11

differences in the drug-like properties of the new lead (Supplementary Table S4) [11]. 12

To address ligand binding to the ERRγ ligand binding domain (LBD; 222–458), we solved 13

the ERRγ crystal structure in complex with DN200434 at 2.5 Å resolution by molecular 14

replacement using a search model (PDB ID: 1S9Q). The collected data were refined and 15

converged to final Rwork = 19.93% and Rfree = 24.79% using Coot [12] and Phenix [13]. All 16

figures were generated by Chimera (version 1.11.2, build 41376) [14]. Structure analysis 17

showed a ERRγ-DN200434 binding mode similar to that of ERRγ-GSK5182 [15] (Fig. 2h). 18

Compared with GSK5182 (key protein residue distance to ligand, TYR326: 2.56 Å , ASN346: 19

2.55 Å, GLU275: 2.6 Å), the DN200434 binding affinity in the ERRγ ligand binding pocket 20

was enhanced owing to closer key residue (Y326, N346, E275) ligand proximity. A trimer 21

state was observed in the solved structure labelled as A, B, and C chains. In A and B chains, 22

DN200434 alcohol functionality interacts with TYR326 at 2.4 Å and ASN346 at 2.5 Å via 23

hydrogen bonding. Phenol interacts with GLU275 at 2.4 Å and the basic chain interacts with 24

ASP273. HIS434 and PHE435 movement and DN200434 OH tip tilting in a different 25

orientation from that of GSK5182 occurs only for the C chain (Fig. 2i); this induces binding 26

pocket movement near the ligand and tighter ligand packing into the binding pocket, 27

respectively. Additionally, PHE435 in ERRγ-DN200434 moves toward the ligand benzene 28

ring at 3.95 Å , compared with that at 5.25 Å in ERRγ-GSK5182, and among the three chains, 29

only one shows π-H bonding between the DN200434 benzene ring and the PHE435 hydrogen. 30

Hence, π-H bonding likely enhances (33% probability) DN200434 binding affinity, yielding 31

higher DN200434 potency than for GSK5182. Consequently, the four-way DN200434 32




17

interactions with key ERRγ binding pocket residues via hydrogen, ion, and π-H bonding 1

enhance DN200434 potency. 2

According to the crystal structure in complex with DN200434 and ERRγ, E275, Y326, and 3

N346 were the key residues interacting with DN200434. To investigate the role of these 4

residues, we substituted each amino acid with alanine and performed luciferase reporter assays 5

with wild type ERRγ and its mutants E275A, Y326A, N346A, and additional mutant D273A. 6

As depicted in Fig. 2j, DN200434 exerted an inhibitory effect on E275A, Y326A, and N346A, 7

whereas the inhibitory effect of GSK5182 was dependent on E275A and Y326A. Specifically, 8

ERRγ D273A was not inhibited by GSK5182 and DN200434 indicating that the substitution 9

of D273 with alanine considerably impeded the interaction with GSK5182 and DN200434 10

compared with that of other substitutions. This finding suggests that the ion interactions of 11

D273 with the basic chain of both GSK5182 and DN200434 is stronger than the hydrogen 12

bonding interactions of E275, Y326, and N346. These results also indicate that π-H bonding 13

between the DN200434 benzene ring and PHE435 hydrogen may play a key role in the 14

interaction, enhancing the inhibitory effect of DN200434. 15

16

In vitro ATC radioiodine avidity 17

We examined the effects of DN200434 on the proliferation of CAL62 cells expressing 18

luciferase as a bioluminescent reporter gene. In vitro BLI showed DN200434 dose-dependent 19

growth inhibition (Supplementary Fig. S6). With 11% and 29% relative growth inhibition at 20

24 h after treatment with 6 and 12 μM DN200434, respectively; these concentrations were 21

selected for subsequent experiments. As DN200434 led to dose-dependent growth inhibition 22

of CAL62 cells, we examined whether DN200434 can induce apoptosis. Western blot analysis 23

showed that DN200434 increased both expression of cleaved PARP and cleaved caspase-3 24

after treatment with 6 μM and 12 μM DN200434 (Supplementary Fig. S7a). We further 25

employed flow cytometry to demonstrate whether DN200434 results in apoptosis. Flow 26

cytometry analysis with Annexin V/PI staining showed that treatment with DN200434 resulted 27

in an increase in the number of cells undergoing apoptosis in a dose-dependent manner 28

(Supplementary Fig. S7b). Consistently, a dose-dependent increase in the sub-G1 cell 29

population was also uncovered for DN200434-treated CAL62 cells (Supplementary Fig. S7c). 30

Furthermore, DN200434 significantly increased CAL62 cell radioiodine uptake in a dose-31

dependent fashion, with a 1.5 and 2.1-fold relative increase at 6 and 12 μM DN200434, 32




18

respectively, compared with that of vehicle-treated cells (Fig. 3a). Conversely, 6 μM GSK5182 1

did not effectively increase radioiodine uptake (Fig. 3b), indicating that DN200434 is more 2

potent at conferring ATC cell radioiodine avidity. As dose-dependent analysis did not detect 3

increased radioiodine uptake above 12 μM DN200434, this concentration was used for 4

assessing time-dependent radioiodine uptake. Significantly increased iodide uptake occurred 5

at 3 h (~1.4-fold), reaching a peak at 24 h (~2.7-fold; Fig. 3c). Specific NIS inhibition by 6

potassium perchlorate (KClO4) completely inhibited DN200434-stimulated radioiodine uptake 7

to basal levels (Fig. 3d), suggesting that increased radioiodine uptake is associated with 8

DN200434-mediated NIS function modulation. However, vehicle and vehicle+KClO4-treated 9

cells exhibited similar radioiodine uptake, revealing the lack of NIS-mediated radioiodine 10

uptake in CAL62 cells. 11

Similar to GSK5182, DN200434 also significantly decreased endogenous CAL62 cell ERRγ 12

expression as evidenced by immunoblotting with an ERRγ-specific Ab (Fig. 3e and 13

Supplementary Fig. S8a). Additionally, as GSK5182-induced pERK1/2 activation is directly 14

associated with enhanced NIS function, DN200434 also dose-dependently increased pERK1/2 15

levels in CAL62 cells (Fig. 3f and Supplementary Fig. S8b). Furthermore, the selective MEK 16

inhibitors PD98059 or U0126 inhibited DN200434-stimulated CAL62 cell radioiodine uptake 17

to basal levels, whereas neither inhibitor alone promoted increased iodide uptake 18

(Supplementary Fig. S9a). Accordingly, western blot analysis also revealed that PD98059 or 19

U0126 inhibited DN200434-upregulated p-ERK1/2 in CAL62 cells (Supplementary Fig. S9b, 20

c). Collectively, these results indicate that enhanced NIS function through DN200434-21

mediated ERRγ protein downregulation as well as MAP kinase activation are directly involved 22

with radioiodine avidity stimulation. 23

24

Expression of genes involved in iodine metabolism in ATC 25

Quantitative PCR analysis demonstrated that DN200434 dose-dependently increased NIS, 26

TSHR, TPO, and TG mRNA expression levels in CAL62 cells (Fig. 3g). TG, TPO, and TSHR 27

protein expression similarly increased compared with that of vehicle-treated cells (Fig. 3h and 28

Supplementary Fig. S10), although a slight decrease in TG levels occurred under 12 μM 29

DN200434. DN200434-treated CAL62 cells showed total and membranous NIS protein 30

upregulation with fully (approximately 95 kDa) and partially (approximately 50 kDa) 31

glycosylated forms (Fig. 3i and Supplementary Fig. S11), revealing a 1.9 and 2.4-fold relative 32




19

increase in the fully glycosylated membrane-NIS form at 6 and 12 µM DN200434, respectively. 1

Conversely, U0126 or PD98059 reversed the increase in membrane NIS protein expression to 2

basal levels (Supplementary Fig. S12), suggesting that MAK kinase is important for 3

enhancing DN200434-mediated NIS expression and functional activity in ATC cells. Thyroid-4

regulating gene expression kinetics at various time points after DN200434 treatment 5

demonstrated a time-dependent increase in protein expression (Fig. 3j and Supplementary 6

Fig. S13), although expression peaked at different time points. 7

8

Glucose metabolism and gene expression 9

Among key glucose metabolism proteins, DN200434-treated CAL62 cells showed a dose-10

dependent decrease in GLUT-1 and GLUT-4 (Fig. 3k and Supplementary Fig. S14). In vitro 11

F-18-FDG uptake assays also showed a dose-dependent decrease in glucose uptake in 12

DN200434-treated CAL62 cells with a 1.5 and 1.7-fold relative decrease at 6 and 12 μM 13

DN200434, respectively (Fig. 3l). Together, these results indicate that DN200434 exhibits a 14

unique ability to redifferentiate ATC by restoring thyroid-regulating genes and downregulating 15

glucose metabolism. 16

17

In vivo ATC radioiodine avidity and therapy 18

Biodistribution and 124I-PET/CT imaging analyses were performed in an ATC tumor mouse 19

model following oral DN200434 administration (Fig. 4a and Supplementary Fig. S15a). 20

DN200434 dose-dependently increased CAL62 tumor radioiodine incorporation 21

(Supplementary Fig. S15b). No differences were observed in other organs between vehicle-22

treated and DN200434-treated groups (Supplementary Fig. S15c). 23

To non-invasively and quantitatively evaluate DN200434-mediated CAL62 tumor 24

radioiodine avidity changes in vivo, we conducted 124I-PET/CT imaging using 200 mg/kg 25

DN200434 as the concentration yielding the highest radioiodine incorporation level from 26

biodistribution studies (Fig. 4a). CAL62 tumors exhibited a distinct increase (relative 4.4-fold) 27

in DN200434-mediated radioiodine incorporation (Fig. 4b-d), whereas CAL62 tumors of 28

vehicle-treated mice showed no increase. Immunoblotting analysis with an NIS-specific 29

antibody revealed higher endogenous NIS protein expression in tumors from DN200434-30

treated than vehicle-treated mice (Fig. 4e). Similarly, IHC examination with an anti-hNIS 31

antibody demonstrated strong NIS membrane staining in DN200434-treated but not in vehicle-32




20

treated CAL62 tumors (Fig. 4f). These results indicate the feasibility of orally bioactive 1

DN200434 as a potent drug for inducing an effective increase in ATC radioiodine avidity by 2

enhancing NIS protein expression. Furthermore, in vitro clonogenic assays indicated that either 3

DN200434 or 131I alone induced a minimal cytotoxic effect (Supplementary Fig. S16a and 4

b). Conversely, marked cytotoxic effects were observed in the combination group. 5

We next performed in vivo therapy to determine whether DN200434-mediated radioiodine 6

avidity provides enhanced ATC susceptibility to therapeutic radioiodine therapy as described 7

in Fig. 4g. Marked tumor growth was observed in the vehicle-treated group. 131I-treated mice 8

also exhibited noticeable tumor growth resulting from lack of radioiodine avidity for CAL62 9

tumors (Fig. 4h). In DN200434-treated mice, slight tumor growth inhibition was apparent. In 10

vivo BLI demonstrated marked tumor growth inhibition in the combination group, unlike single 11

therapy. Furthermore, we investigated the therapeutic outcomes of a combination of 12

DN200434 and radioiodine therapy in another ATC cancer model—BHT101 harboring 13

BRAFV600E mutation (Supplementary Fig. S17a). Similar with CAL62 tumor-bearing mice, 14

mice receiving a single treatment of either 131I or DN200434 exhibited fewer therapeutic 15

effects (Supplementary Fig. S17b; gray and blue line). However, combination treatment led 16

to marked tumor growth reduction in the BHT101 xenograft model up to 32 days post-17

treatment (Supplementary Fig. S17b; gray and red line). Abnormal behavior or body weight 18

loss was not detected over the entire treatment course (Supplementary Fig. S18). 19

20

Discussion 21

22

ERRγ regulation represents a promising therapeutic target in various metabolic/cardiac 23

diseases and certain types of cancers [16-19]. Recently, we found that ERRγ modulation by its 24

inverse agonist GSK5182 enhances NIS function and radioiodine therapeutic effects in ATC 25

cells in vitro [9]. However, GSK5182 was not effective in an in vivo ATC model, possibly 26

owing to poor in vivo pharmacokinetic profiles (e.g., low bioavailability) and biocompatibility. 27

Thus, we searched for novel drugs to selectively and effectively modulate ERRγ, thereby 28

inducing effective radioiodine avidity and improving radioiodine therapeutic outcomes in in 29

vitro and in vivo ATC models. 30

Herein, we extended the clinical scope of ERR modulating activities by identifying a novel 31

ligand, DN200434, that enhances NIS function and is potently effective in ATC radioiodine 32




21

treatment in vivo. This newly developed ERR inverse agonist was highly selective over other 1

subtypes in binding assays and exhibited the most potent functional activity in a cell-based 2

assay system. DN200434 also showed improved in vitro/in vivo ADMET profiles in required 3

standard discovery studies including CYPs and hERG inhibition, mini-Ames test, in vivo PK, 4

and in vivo safety. Structural assessment confirmed specific DN200434 binding to ERRγ, 5

based on four-way interactions of DN200434 with corresponding residues at precise positions 6

within the binding pocket. For clinical application of DN200434 in ATC, identifying the 7

relationship between target protein expression levels (ERRγ) and thyroid cancer staging is 8

essential. Notably, we found a reverse relationship of ERRγ expression between patient-9

derived normal and ATC tumor tissues, strongly suggesting that ERRγ constitutes a novel 10

biomarker and therapeutic target for thyroid cancer management. 11

To evaluate the biological potential of DN200434, we determined its effects on radioiodine 12

uptake in CAL62 cells carrying KRAS gene mutations, which have been utilized to examine 13

the therapeutic efficacy of several agents, allowing cellular cytotoxicity induction and iodide-14

handling gene restoration [20-22]. DN200434 induced a dose and time-dependent increase in 15

radioiodine incorporation in CAL62 cells, which was inhibited to basal levels by KClO4 as an 16

NIS-specific inhibitor [1,23], suggesting that the increased radioiodine uptake was linked to 17

DN200434-modulated NIS function. Moreover, DN200434 was more potent at inducing 18

increased radioiodine avidity than did GSK5182. We therefore expected that enhanced 19

radioiodine avidity may also be due to iodide-handling gene restoration, including NIS, TPO, 20

TG, and TSHR. Accordingly, DN200434 drastically enhanced iodide-handling gene expression 21

at both mRNA and protein levels. 22

Poorly differentiated (or dedifferentiated) thyroid cancers exhibit high F-18-FDG uptake 23

with upregulated glucose transporter expression, whereas primary or metastatic differentiated 24

thyroid cancers demonstrate low F-18-FDG uptake and high radioiodine avidity [24-26], 25

indicating that glucose uptake is inversely proportional to thyroid cancer radioiodine avidity 26

or differentiation level [27]. Here, DN200434-treated cells exhibited downregulated Glut-1 27

and Glut-4 expression levels and decreased F-18-FDG uptake, suggesting that DN200434 28

exhibits a unique ability to re-induce differentiation of poorly or undifferentiated ATC cells. 29

For successful radioiodine therapy, effective β-ray-emitting radioiodine incorporation 30

should occur in thyroid cancer. The in vitro finding of DN200434-mediated increase in 31

radioiodine avidity prompted us to further evaluate its effects on in vivo radioiodine uptake 32




22

restoration in ATC tumors. To confirm the ability of DN200434 to enhance radioiodine avidity 1

in living mice, we used PET/CT imaging, which shows radioiodine incorporation levels in 2

thyroid tumors in a non-invasive and quantitative manner. DN200434-treated tumors exhibited 3

a striking conversion of lesions from negative to positive on 124I-PET/CT following DN200434 4

administration and accompanied by increased NIS protein expression, as assessed by 5

immunoblotting and immunohistochemistry. Thus, DN200434-mediated restoration of 6

radioiodine avidity may lead to potential therapeutic effects of radioiodine therapy in ATC 7

tumor-bearing mice. Accordingly, the restoration of radioiodine incorporation in DN200434-8

treated ATC tumors significantly inhibited tumor growth. Thus, ERRγ-modulated iodide-9

handling gene recovery by DN200434 may afford a reasonable strategy for achieving 10

radioiodine accumulation, allowing for acceptable radioiodine therapeutic outcomes. 11

According to TMA of 38 normal, 96 PTC, and 26 PDC/ATC tissues, ERRγ was also found 12

to be expressed in normal thyroid tissues but only in up to approximately 10% of cells; such 13

low expression levels may lead to a lack of responsiveness to ERRγ inverse agonists. On the 14

other hand, PD/ATC exhibit high expression levels of ERRγ (up to 40%), which may lead to 15

good responsiveness to ERRγ inverse agonists, thereby decreasing ERRγ functional activity. 16

Although we cannot fully explain the mechanism by which ERRγ appears to drive the 17

dedifferentiated phenotype of ATC, we presume that ERRγ inverse agonists may be effective 18

in ATC partially due to these reasons. 19

In conclusion, we reported the discovery of DN200434—which to our knowledge is the 20

most cell-active and first orally bioavailable inverse agonist of ERRγ—and uncovered its 21

unique co-crystal structure with ERRγ. Notably, we showed that in an ATC tumor model, 22

DN200434 enhanced ATC tumor radioiodine avidity, as determined by 124I PET/CT imaging, 23

via iodide-handling gene upregulation. Furthermore, radioiodine avidity enhancement by 24

DN200434 allowed successful radioiodine therapy of conventional radioiodine therapy-25

refractive ATC tumors. We are currently investigating whether DN200434 can also enhance 26

radioiodine avidity as well as radioiodine therapeutic effects in a papillary thyroid cancer 27

model (harboring RET/PTC rearrangements and BRAFV600E mutation), which accounts for 28

more than 90% of all thyroid cancer cases. 29

30

31

32




23

Acknowledgements 1

This study was supported by the following grants, HI16C1501, HT16C0001, 2

HT16C0002, 2017R1D1A1B03028340, 20110018305 (National Creative Research Initiatives 3

Grant), 2017R1C1B1005599, 2017M3A9G7073088, NRF-2017R1A2B3006406, and KDDF-4

201612-16. 5

6

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26. Matsuzu K, Segade F, Wong M, Clark OH, Perrier ND, Bowden DW. Glucose 37 transporters in the thyroid. Thyroid, 2005;15(6):545-50. 38

27. Feine U, Lietzenmayer R, Hanke J-P, Wöhrle H, Müller-Schauenburg W. 18FDG 39 whole-body PET in differentiated thyroid carcinoma. Flipflop in uptake patterns of 40 18FDG and 131I. Nuklearmedizin, 1995;34(4):127-34. 41

42

43

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25

Figure Legends 1

2

Figure 1. Endogenous iodide-handling genes and ERRγ expression in normal thyroid samples, 3

papillary thyroid carcinomas (PTCs), and poorly-differentiated or anaplastic thyroid 4

carcinomas (PD/ATCs). (A) Histological images of ERRγ and iodide-handling gene 5

expression in normal thyroid tissue, PTCs, and PD/ATCs. (B) Immunohistochemistry analysis 6

showing ERRγ, NIS, TPO, TG, and THSR expression levels in normal thyroid tissue, PTCs, 7

and PD/ATCs. Data are presented as the means ± SD. 8

9

Figure 2. Drug discovery of new ERRγ inverse agonist and structure characterization. (A) 10

Lead optimization and discovery of the best tool compound, DN200434: scattered 3D plot of 11

the approximately 300 compounds color-coded by hERG binding IC50 value and size-coded by 12

p.o. AUC value in the PK study. CLogP values are plotted against ERR cell-based functional 13

activity and ERR binding IC50. Compounds are colored based on their hERG binding screen 14

using a gradient from green (safe, IC50 >5) to red (problematic). Gray indicates that hERG 15

binding was not tested. (B) Chemical structure and in vitro GSK5182 and DN200434 16

pharmacological profiles. Selective DN200434 binding activity to ERR compared with other 17

related nuclear receptors. (C) ERR, (D) ERR, (E) ERR, and (F) ER binding activity 18

between the binding domain and PGC1 substrate measured by TR-FRET. Black line 19

represents the activity of gold standard compound (XCT790 for ERR, 4-OHT for ERR and 20

ER), blue line represents GSK5182, and red line represents DN200434 shown as % activity. 21

(G) Functional activity of DN200434 and GSK5182 in the presence of the tested compounds 22

is represented as normalized luciferase activity percentage. (H) Crystal structure of ERRγ LBD 23

(222–458) in complex with DN200434. Inset: binding pocket of ERRγ with bound ligand; 24

protein residues are in beige and the bound ligand in orange. The nitrogen of residues and 25

ligand is represented in dark blue and oxygen in red. The distance between the key protein 26

residues and ligand is designated and labeled in black. (I) Superimposed view of ERRγ LBD-27

DN200434 with ERRγ-GSK5182; ERRγ-DN200434 complex structure is in beige and ERRγ-28

GSK5182 in cyan. Compared with the ERRγ-GSK5182 complex structure, the chain 29

containing HIS434 and PHE435 in the ERRγ-DN200434 complex structure is oriented slightly 30

toward the benzene ring of the ligand, enhancing ligand binding affinity via the indicated π-H 31

bonding interaction. (J) Inhibitory effect of GSK5182 and DN200434 on wild-type ERRγ and 32




26

its mutants. HEK293T cells were transfected with the Gal4-luc reporter and the Gal4-ERRγ, 1

D273A, E275A, Y326A, or N346A expression vectors. Cells were treated with GSK5182 (1 2

μM) or DN200434 (1 μM) for 18 h, after which luciferase reporter assays were performed. 3

Error bars represent the mean ± SD. Compared with ERRγ-wt, *P < 0.05, **P < 0.001, ***P 4

< 0.0001. Con, control; N.S, not significant; wt, wild-type. 5

6

Figure 3. DN200434-mediated increase in the radioiodine avidity of CAL62 cells. (A) 7

Dose-dependent radioiodine uptake in DN200434-treated CAL62 cells. (B) Radioiodine 8

uptake in CAL62 cells by either GSK5182 or DN200434. (C) Time-dependent radioiodine 9

uptake in DN200434-treated CAL62 cells. (D) Modulation of DN200434-induced iodide 10

uptake in CAL62 cells by KClO4 as a NIS-specific inhibitor. (E) ERRγ protein expression in 11

DN200434-treated CAL62 cells and quantification of band intensity. (F) p-ERK1/2 levels in 12

DN200434-treated cells and quantification of band intensity. (G) mRNA expression levels of 13

NIS, TPO, TG, and TSHR in DN200434-treated cells. (H) Representative western blots 14

revealing TG, TSHR, and TPO protein expression levels in DN200434-treated CAL62 cells. 15

(I) Membranous and total NIS protein in DN200434-treated CAL62 cells. (J) Time-dependent 16

change in thyroid regulating genes in DN200434-treated CAL62 cells. (K) Representative 17

western blots illustrating the protein expression levels of GLUT-1 and GLUT-4. (L) F-18-FDG 18

uptake in DN200434-treated CAL62 cells. CPM: counts per minute. Data are presented as the 19

means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. 20

21

Figure 4. Recovery of radioiodine incorporation in CAL62 tumors in response to DN200434 22

treatment and in vivo evaluation of therapeutic response to 131I therapy with DN200434 23

treatment in CAL62/effluc tumor-bearing mice. (A) Schematic procedures for 124I-PET/CT 24

imaging. Prior to DN200434 treatment of tumor-bearing mice, 124I-PET/CT imaging was 25

performed to determine the basal uptake of radioiodine in CAL62 tumors. After imaging 26

acquisition, CAL62 tumor-bearing mice received either vehicle or 200 mg/kg DN200434 via 27

oral administration once a day for 6 days. After the last treatment, PET/CT imaging was 28

performed to determine the change in radioiodine avidity of tumors. (B) 3D-reconstructed 124I-29

PET/CT images of increased radioiodine avidity in CAL62 tumors following DN200434 30

treatment. Yellow arrow indicates the tumor. (C and D) Quantification of radioactivity in 31




27

tumors of vehicle or DN200434-treated mice before and after treatment. (E) Increase in NIS 1

protein expression in DN200434-treated CAL62 tumors. (F) IHC examination of NIS 2

expression status in treated tumors. Data are presented as the means ± SD. *P < 0.05, **P < 3

0.01. (G) Schematic procedures for in vivo therapy. CAL62/effluc tumor-bearing mice were 4

divided into four groups as follows, group 1, vehicle-treated group; group 2, 131I-treated group; 5

group 3, DN200434-treated group; and group 4, DN200434 + 131I-treated group. In vivo BLI 6

imaging was conducted to assess therapeutic response over time. DN200434 was orally 7

administered once a day for 6 days. (H) In vivo BLI imaging depicting therapeutic outcomes 8

of respective treatments on CAL62 tumor-bearing mice (left panel) and the relative increase in 9

BLI signals of CAL62 tumors between day 0 and day 27 (right panel). ***P < 0.001. 10




28

Figure 1




29

Figure 2




30

Figure 3




31

Figure 4




Published OnlineFirst April 22, 2019.Clin Cancer Res Thoudam Debraj Singh, Jaeyoung Song, Jina Kim, et al. anaplastic thyroid cancer

), DN200434, a booster of NIS inγreceptor gamma (ERRA novel orally active inverse agonist of estrogen-related

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