Helix12-Stabilization Antagonist of Vitamin D Receptor

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Article

Helix12-stabilization antagonist of vitamin D receptor Akira Kato, Toshimasa Itoh, Yasuaki Anami, Daichi Egawa, and Keiko Yamamoto Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00246 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Bioconjugate Chemistry

Helix12-stabilization antagonist of vitamin D receptor

Akira Kato,1 Toshimasa Itoh,1 Yasuaki Anami,1 Daichi Egawa,1 Keiko Yamamoto1,*

1

Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165

Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan

*Corresponding author

Keiko Yamamoto: [email protected]; Phone, +81 42 721 1580; Fax, +81 42 721 1580

1

ACS Paragon Plus Environment


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Abstract To develop strong vitamin D receptor (VDR) antagonists and reveal their antagonistic mechanism, we designed and

synthesized vitamin D analogues with bulky side chains based on the “active antagonist” concept in which

antagonist prevents helix 12 (H12) folding. Of the synthesized analogues, compounds 3a and 3b showed strong antagonistic activity. Dynamic hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) and

static X-ray crystal structure analyses indicated that compound 3a stabilizes H11-H12 but displaces H6-H7 so that

3a is a novel rather than “active” or “passive” type of antagonist. We classified 3a as a third type of antagonist and

called it “H11-H12 stabilization antagonist”. HDX-MS analysis indicated that antagonist 3b is an “active” antagonist. To date there are no reports relating to nuclear receptor antagonist that strongly stabilizes H12. In this

study, we found first VDR antagonist that stabilizes H12 and we showed that antagonistic mechanism is diverse

depending on each antagonist structure. Additionally, HDX-MS was proved to be very useful for investigations of

protein structure alterations resulting from ligand binding.

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Introduction

1α,25-Dihydroxyvitamin D3 (1,25D3) is the active form of vitamin D3 and plays important roles in calcium metabolism, immunomodulation, and cellular differentiation and proliferation.1 1,25D3 exerts these actions by modulating gene transcription through binding to the vitamin D receptor (VDR), a member of the nuclear receptor (NR) superfamily.2 Helix 12 (H12) of the NR ligand-binding domain (LBD) plays a significant role in the

formation of the active form of NRs. The conformation of VDR changes from the inactive to active form by

binding 1,25D3 and then forms a heterodimer with retinoid X receptor (RXR). The VDR/RXR heterodimer binds to vitamin D response elements (VDREs) of the target gene and recruits the coactivator, resulting in transcription of the target gene.3 In order to recruit the coactivator, H12 of VDR adopts a stable active conformation by binding the

VDR agonist.

Various VDR ligands have been developed by many groups, and several vitamin D analogues are used in clinical applications, such as the treatment of metabolic bone diseases and skin diseases such as psoriasis.4,5 All of

the clinically used vitamin D analogues are VDR agonists. However, antagonists are needed to treat diseases of VDR hyperfunction, such as Paget’s disease of bone and osteopetrosis,6,7 but no VDR antagonists are currently

available for use in medicine.

In 2002, Greene et al. proposed that steroid hormone receptor antagonists could be categorized as either “active” or “passive”.8 Active antagonists have a bulky structure and inhibit the agonistic behavior of NRs in any

situation because a bulky substituent actively prevents H12 folding, which is a significant aspect of NR activation. 4-Hydroxytamoxifen,9 a selective estrogen receptor modulator, is a representative active antagonist. Indeed, X-ray 3



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crystal structure analyses showed that 4-hydroxytamoxifen blocks H12 folding in estrogen receptor α (ERα).10 By contrast, a passive antagonist is comparatively small and fits into the ligand-binding pocket (LBP), permitting H12

folding but with an antagonistic effect. As the crystal structure of the complex of ERβ and its antagonist, 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC), adopts an agonist-binding form, THC is classified as a passive antagonist.8 Bulky side chain–containing VDR antagonists such as carboxylic ester (ZK series) compounds,11,12 adamantane compounds,13 and 1α,25-dihydroxyvitamin D3-26,23-lactams,14 are classified as active antagonists, and their antagonistic activity is believed to be caused by prevention of H12 folding by the bulky side chain.

However, this hypothesis has not been proven in structural biological studies. VDR analogues lacking a bulky side chain, such as TEI964715 and its derivatives15,16 and 22S-butyl vitamin D analogues17,18 (e.g., compound 1) (Figure

1), are classified as passive antagonists. The X-ray crystal structure showed that antagonist 1 fits into the LBP and permits H12 folding.17 In some situations, a passive antagonist may exhibit agonist activity due to the absence of

steric repulsion between H12 and the ligand.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is often used to investigate protein-ligand interactions, ligand-binding modes, protein-protein interactions, and protein folding.19–22 HDX-MS

enables the analysis of protein dynamics in solution, unlike static X-ray crystal structure analysis. HDX-MS

utilizing deuterium labeling can be used to investigate the rate at which protein backbone amide hydrogens undergo exchange.19 The rate of hydrogen/deuterium exchange of backbone amide hydrogens reflects the conformational mobility, hydrogen bonding strength, and solvent accessibility in the protein structure.20 In fact, the in-solution 4


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dynamics of several NRs, including VDR, have been investigated using HDX-MS.23–33

To develop strong antagonists and to reveal the mechanism of active antagonism of the VDR, we

designed and synthesized vitamin D analogues with bulky substituents at the side chain terminus and used them to

probe changes in the VDR-LBD structure upon binding to synthetic active antagonists. Here, we report the

synthesis and biological evaluation of compounds 2a,b-6a,b (Figure 1) and the results of static X-ray crystallographic and dynamic HDX-MS analyses of the VDR-LBD/antagonist complex.

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OH

H

HO

OH

1α,25-(OH)2D3 (1,25D3)

ZK168281

22

TEI9647

25

2a : R=H 2b : R=Bu

1

3a : R=H 3b : R=Bu

R OH

H

HO

4a : R=H 4b : R=Bu

OH

5a : R=H 5b : R=Bu

Figure 1. Structures of 1,25-(OH)2D3 (1,25D3) and its analogues.

6


6a : R=H 6b : R=Bu

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Results

Design and Synthesis Vitamin D analogues 2a,b-6a,b were designed with a bulky substituent at the 25 position (Figure 1). These bulky substituents were expected to prevent H12 folding due to steric repulsion. In addition, a 22S-butyl group was introduced in 2b-6b because it was found to be an effective VDR antagonist.18

The synthesis of target compounds 2a,b-6a,b is shown in Scheme 1. Alcohol 7, which was synthesized in our previous study,18 was tosylated with TsCl to give 8. Treatment of tosylate 8 with KCN afforded cyanate 9,

which was then reduced to aldehyde 10. Aldehyde 10 was converted to compound 11a using a mild iodine oxidation method developed by our group previously.34 Compound 11b was prepared according to a previously reported procedure.18

Key compounds 11a,b were treated with n-BuLi to give dibutyl compounds 12a,b, which were then

deprotected with 10-camphorsulfonic acid (CSA) to afford target compounds 2a and 2b, respectively. Compounds

11a,b were treated with BnMgCl and then CSA to give compounds 3a and 3b, respectively. Diphenyl compounds

4a,b, dinaphthylmethyl compounds 5a,b, and dinaphthyl compounds 6a,b were obtained by similar synthetic procedures using appropriate Grignard reagents.

Scheme 1. Synthesis of compounds 2a,b-6a,b.

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X

X

H

TBSO

H KCN

I2, KOH

DMSO

MeOH

OTBS

TBSO

OTBS

COOMe

H

TBSO

OTBS 11a (94%)

9 : X = CN (92%) 10 : X = CHO (63%) R1

7 : X = OH 8 : X = OTs (78%) R1

Bu OH Bu

CO2Me

H

TBSO

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H n - BuLi

CSA

THF

MeOH

OTBS

TBSO

11a : R1 = H 11b : R1 = Bu

2a : R1 = H (96%) 2b : R1 = Bu (88%)

OTBS

12a : R1 = H (89%) 12b : R1 = Bu (75%) R1 Bn OH Bn H

11a 11b

BnMgCl

CSA

THF

MeOH TBSO

3a : R1 = H (84%) 3b : R1 = Bu (92%)

OTBS 13a : R1 = H (64%) 13b : R1 = Bu (64%) R1 R2 OH R2 H

11a 11b

R2MgBr

CSA

THF

MeOH TBSO

OTBS

4a : R1 = H, R2 = Ph (89%) 4b : R1 = Bu, R2 = Ph (79%) 5a : R1 = H, R2 = 2-Np-CH 2- (75%) 5b : R1 = Bu, R2 = 2-Np-CH 2- (94%) 6a : R1 = H, R2 = 2-Np (92%) 6b : R1 = Bu, R2 = 2-Np (96%)

14a : R1 = H, R2 = Ph (59%) 14b : R1 = Bu, R2 = Ph (81%) 15a : R1 = H, R2 = 2-Np-CH 2- (50%) 15b : R1 = Bu, R2 = 2-Np-CH 2- (57%) 16a : R1 = H, R2 = 2-Np (61%) 8 16b : R1 = Bu, R2 = 2-Np (65%)


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Biological Activities VDR binding affinity was evaluated using a competitive binding assay according to a previously reported procedure.35 This assay was performed using [3H]-1,25D3 and recombinant human VDR-LBD expressed as the N-terminal GST-tagged protein using pGEX-VDR vector in Escherichia coli BL21. The results are summarized in

Table S1. All synthetic compounds showed significant binding to the VDR (IC50: 0.14-130 nM), indicating that they are VDR ligands. The 25-dibenzyl analogues 3a (IC50: 0.43 nM), 3b (IC50: 0.14 nM) and 25-dinaphthylmethyl analogues 5a (IC50: 7.4 nM), 5b (IC50: 8.0 nM) showed stronger affinity than the 25-diphenyl analogues 4a (IC50: 24 nM), 4b (IC50: 12 nM) and 25-naphthyl analogues 6a (IC50: 130 nM), 6b (IC50: 34 nM), respectively. These results indicate that compounds with flexible substituents at C-25 show stronger affinity than those with rigid

substituents. The 22S-butyl compounds 2b-4b and 6b (except 5b) showed stronger affinity than the corresponding

22-H compounds 2a-4a and 6a, respectively. That is, the 22S-butyl group increased VDR affinity.

The ability of synthetic compounds 2a,b-6a,b to induce transcription of vitamin D–responsive genes was examined using the mouse osteopontin luciferase reporter gene assay system in Cos7 and HEK293 cells, according to a previously reported procedure.35 The results are shown in Figure 2. Transcriptional activities in HEK293 cells were stronger than in Cos7 cells, which is the same as previous studies.17,36 Except for compounds 2a, 5b, and 6b, most compounds did not show transactivation activity in Cos7 cells but instead exhibited concentration-dependent

inhibition of transactivation induced by 1,25D3. In HEK293 cells, only compounds 3a,b did not exhibit transactivation activity and inhibited the transactivation induced by 1,25D3. From these results, we concluded that 9



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compounds 3a and 3b are potent VDR antagonists.

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Cos7

3b

4b

5b

6b

2b

3b

-log [M]

2a

3a

4a

5a

6a

1,25D3 10-8 M

4b

10 9 8 7 6 5

0

-log [M]

10 9 8 7 6 5

0.5

10 9 8 7 6 5

10 9 8 7 6 5

10 9 8 7 6 5

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

0.5

1

10 9 8 7 6 5

1

1.5

EtOH 1,25D3

relative luciferase activity

1.5

0

2b

d

EtOH 1,25D3


c

12 11 10 9 8 7 6 5

6a

12 11 10 9 8 7 6 5

5a

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

4a

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

3a

0

-log [M]

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

2a

0.5

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

0

12 11 10 9 8 7 6 5

0.5

1

EtOH 1,25D3

1

12 11 10 9 8 7 6 5


b

EtOH 1,25D3


a

-log [M]

5b

6b

1,25D3 10-8 M

HEK293

12 11 10 9 8 7 6 5

3b

4b

5b

6b

2b

3b

-log [M]

2a

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

EtOH 1,25D3

0.5

3a

1

0.5

0

-log [M]

5a

EtOH 1,25D3

1

0

2b

h relative luciferase activity

g

12 11 10 9 8 7 6 5

6a

12 11 10 9 8 7 6 5

5a

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

4a

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

3a

0

-log [M]

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

2a

0.5

12 11 10 9 8 7 6 5

12 11 10 9 8 7 6 5

0

12 11 10 9 8 7 6 5

0.5

1

EtOH 1,25D3

1

12 11 10 9 8 7 6 5


f

EtOH 1,25D3


e


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1,25D3 10-9 M

4b 1,25D3 10-9 M

Figure 2. Transactivation of synthetic compounds. Transactivation of compounds 2a,b-6a,b was evaluated using Cos7 (a-d) and HEK293 (e-h) cells. Transcriptional activity was evaluated using a dual luciferase assay with a full-length human VDR expression plasmid (pCMX-hVDR), a reporter plasmid containing three copies of mouse osteopontin VDRE (SPPx3-TK-Luc), and an internal control plasmid containing sea pansy luciferase expression constructs (pRL-CMV), as described previously.35 In experiments in Cos7 cells, luciferase activity equal to 10−8 M 1,25D3 was defined as 1, and in experiments in HEK293 cells, activity equal to 10−9 M 1,25D3 was defined as 1. Inhibitory effects on transactivation were evaluated in the presence of 1,25D3 (c, d, g, h).

11


-log [M]


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

X-ray Crystal Structure Analysis To understand the mechanism of antagonism of compounds 3a and 3b, we attempted to analyze the X-ray

crystal structure of rat VDR-LBD complexed with 3a or 3b. In the absence of coactivator peptide, we could not obtain crystals. As all crystal structures of rat VDR-LBD/ligand complex reported so far were ternary complex with

DRIP205, we also added DRIP205. In the presence of coactivator peptide DRIP205, co-crystals of 3a were

obtained but not co-crystals of 3b. X-ray diffraction images of the 3a/VDR-LBD complex were obtained at 2.0-Å resolution. The data collection and refinement statistics are summarized in Table S2.

Crystals of the 3a/VDR-LBD complex were an asymmetric homodimer (Figure 3), whereas all crystals of ligand/VDR-LBD complexes reported to date are monomers. We termed the monomers “chain A” (red) and “chain

B” (yellow), respectively (Figure 3a). Chain A could be analyzed to Ser114 based on the clear 2Fo − Fc map at the N-terminus, whereas chain B was analyzed to Lys123 at the N-terminus (Figure 3a). In chain A, we found a new

1.5-pitch α-helix at the N-terminus and designated it “helix 0” (H0). H0 formed significant interactions with chain B and neighboring chain A (chain A’), as shown in Figure 3b, c. H0 appears to have been detected due to

stabilization caused by these interactions. N-terminus helix similar to H0 was observed in a complex of 1,25D3/VDR-LBD (R270L) (PDB code 3VT3),37 but the interactions differed from those of the 3a/VDR-LBD complex. This difference was considered reasonable because the crystal packing of the complexes differ.

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Figure 3. X-ray crystal structure of the 3a/VDR-LBD complex. (a) Overall view of the 3a/VDR-LBD asymmetric homodimer. Chains A and B are shown as red and yellow ribbons, respectively. Only chain A contained a novel 1.5-pitch α-helix, “helix 0” (H0), at the N terminus. (b,c) Interactions between H0 of chain A and surrounding amino acid residues (pale blue shows neighboring chain A, chain A’). Hydrogen bonding and hydrophobic interactions are shown with red and green dashed lines, respectively.

As shown in Figure 3a, except for the N-terminus, almost no differences were observed between chain A

and chain B including the 3a conformation. Indeed, the Cα RMSD value between chain A and chain B was 0.24 Å. We therefore discuss chain A as being representative for this study. Figure 4a shows chain A (3a/VDR-LBD), and 13



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Figure 4b shows the superimposition of chain A on the 1,25D3/VDR-LBD complex (PDB code 2ZLC).38 There are two significant differences between chain A and the 1,25D3/VDR-LBD complex. First, H0 was observed in chain A but not in the 1,25D3/VDR-LBD complex (Figure 4b). Second, because of steric repulsion between the benzyl group of 3a and loop 6-7, the main chain between Asp292 of H6 and Leu305 of H7 was markedly shifted from the canonical positon, and consequently, one pitch of the H7 N-terminus unraveled (Figure 4c). The shifted H6/loop

6-7/H7 and the unraveled H7 N-terminus were clearly observed in the electron density map (Figure 4d).

The A and CD rings of ligand 3a adopted a similar position to those of 1,25D3 (Figure 4e). The hydroxyl groups of 3a formed hydrogen bonds with five amino acid residues (Ser233, Arg270, Tyr143, Ser274, and His393),

just like 1,25D3, but interestingly, the 25-OH group of 3a formed a hydrogen bond with Gln396 of H10 instead of H301 of loop 6-7 (Figure 4e, f). Because of steric repulsion between 3a and loop 6-7, His301 shifted dramatically

from its original position, and Gln396 was rotated at Cβ to occupy empty space (Figure 4e). Figure 4g shows the electron density map of 3a and the benzyl group of 3a fits the electron density map well. The H12 of chain A adopted an almost identical position to that of the 1,25D3/VDR-LBD complex (Figure 4b). This result indicates that H12 of 3a/VDR-LBD assumes an active conformation similar to that of agonist/VDR-LBD.

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Figure 4. X-ray crystal structure of chain A of 3a/VDR-LBD. (a) Overall view of chain A. (b) Superposition of 1,25D3/VDR-LBD (green) and chain A (red). (c) Conformational change in H6/loop 6-7/H7 of chain A. Compared with 1,25D3/VDR-LBD, the main chain was shifted from a canonical positon, and one pitch of H7 unraveled because the benzyl group of 3a pushes against loop 6-7. (d) The electron density map of H6/loop 6-7/H7 in chain A. (e) His301 was shifted and Gln396 was flipped because the benzyl group of 3a pushes against loop 6-7. The 25-OH group forms a hydrogen bond with Gln396 on H10 instead of with His301. Yellow dashed line shows the hydrogen bond between 1,25D3 and VDR-LBD; orange dashed line shows the hydrogen bond between 3a and VDR-LBD. (f) Hydrogen bonds between VDR-LBD and 3a. (g) The electron density map with refined 3a bound to VDR-LBD.

HDX-MS Analysis To investigate protein dynamics in solution, we performed HDX-MS analyses in the presence and

absence of synthetic antagonists. Rat (rather than human) VDR-LBD was used to enable direct comparison with the

crystal structure of rat VDR-LBD. HDX-MS experiments were performed in D2O buffer (10 mM Tris-HCl [pH 8.0], 200 mM NaCl, 2mM DTT) at six time points (10, 30, 60, 300, 900, and 3600 sec). Deuterated VDR-LBD was

digested with pepsin, and the resulting peptides were analyzed by HPLC-MS/MS. In these experiments, duplicates

were analyzed for each time point. The rate of hydrogen/deuterium exchange was calculated using the following

equation: deuterium level (%) = [(mt – m0%)/(m100% – m0%)] × 100, where mt, m0%, and m100% represent the centroid value of the partially deuterated, nondeuterated, and fully deuterated peptides, respectively.21 A total of 23 peptic

peptides covering 95% of the VDR-LBD sequence (residues 125-423) were analyzed.

Apo VDR-LBD First, we performed HDX-MS analysis of apo VDR-LBD (Figure 5 and Figure S1) to characterize the

protein’s stability. In addition, the resulting data enabled us to calculate the average difference in deuterium level 16


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between VDR-LBD in the presence of ligand and apoVDR-LBD (differential HDX analysis). Results of the

HDX-MS analysis of apoVDR-LBD are shown in Figure 5. Peptic peptides are depicted according to color-coded

deuterium level for each exchange time point. The most unstable regions were H1 (residues 117-133), loop 2-3

(residues 151-215), H3 (residues 216-229), loop 11-12 (residues 408-415), and H12 (residues 416-423); >80% of

these regions were deuterated with a 10-sec exposure to D2O (Figure 5). The most stable region was a portion of the N-terminus of H10 (residues 375-381), which underwent only about 20% exchange to deuterium after 3600 sec (Figure 5). Similar results were observed in HDX-MS analyses of human VDR-LBD and some NR-LBDs.21,29

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Hinge

loop 1-2

H1

Page 18 of 35

loop 2-3

H2

106 116 120 130 140 150 160 215 GS HMGS P NS P L KD S L RP K L S E EQQH I I A I L L D A HHK T YD P T YAD F RD F RP P V RMDGS TGS V T L 10 sec 30 sec 60 sec 300 sec 900 sec 3600 sec 10 sec 30 sec 60 sec 300 sec 900 sec 3600 sec 10 sec 30 sec 60 sec 300 sec 900 sec 3600 sec

H3

H3’

H4

H5

S1

D (%) ≥ 90 < 90 < 80 < 70 < 60 < 50 < 40 < 30 < 20 < 10

216 220 230 240 250 260 270 280 D L S P L SM L P H L AD L V S YS I QK V I GF A KM I PGF RD L T SDDQ I V L L K S S A I E V I M L RS NQS F TMDD M

S2

S3

H6

loop 6-7

H7

loop 7-8

loop 8-9

H8

H9

281 290 300 310 320 330 340 347 SWDCGSQD YK YD V TD V S K AGH T L E L I E P L I K F QVG L K K L N L HE E E HV L L MA I C I V S PD R PGVQD A K L

H9

loop 9-10

H10

H11

loop 11-12

H12

348 360 370 380 390 400 410 420 423 V E A I QD R L S N T L Q T Y I RC RHP P PGS HQ L YA KM I QK L AD L RS L NE E HS KQ YR S L S F QP E N SMK L T P L V L E V F GN E I S

Figure 5. Amino acid sequences and deuterium levels of apoVDR-LBD peptic peptides. In this study, 23 VDR-LBD peptic peptides were analyzed, covering 95% of the sequence of VDR-LBD (residues 125-423). Horizontal blocks represent peptic peptides for which the deuterium levels (D) are shown in color code for the six different time points. Amino acid residues that form hydrogen bonds with 1,25D3 are identified by solid red circles.

VDR-LBD/Ligand Complex 18


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Next, we analyzed VDR-LBD by HDX-MS in the presence of ligand (1,25D3, 3a, and 3b) and calculated the deuterium levels (Figures S2 and S3). To investigate the ligand-induced conformational dynamics of VDR-LBD,

we then conducted differential HDX analyses of VDR-LBDs in the presence and absence of ligand. The average

differences in VDR-LBD deuterium levels are shown in Table 1. A positive value indicates an increase in the

deuterium level of a peptide region destabilized by ligand-binding. In contrast, a negative value represents a

decrease in deuterium exchange due to stabilization brought about by ligand-binding. The HDX profiles of

VDR-LBD in presence of 1,25D3, 3a, and 3b were then mapped onto the crystal structure of the 1,25D3/VDR-LBD complex (PDB code 2ZLC) (Figure 6).

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Table 1. Change in HDX profile for each peptide in the presence of ligands. Region

z

Sequence

Structure

1,25D3

3a

3b

117-133 128-136 134-147 134-150 151-215 216-229 230-240 245-255 251-275 288-299 300-323 305-312 305-321 316-329 330-347 348-359 360-384 375-381 376-400 385-405 403-413 408-415 416-423

2 1 2 2 2 1 1 1 3 1 3 1 2 1 2 1 3 1 3 2 1 1 1

KDSLRPKLSEEQQHIIA QQHIIAILL ILLDAHHKTYDPTY ILLDAHHKTYDPTYADF RDFRPPVRMDGSTGSVTL DLSPLSMLPHLADL VSYSIQKVIGF PGFRDLTSDDQ TSDDQIVLLKSSAIEVIMLRSNQSF DYKYDVTDVSKA GHTLELIEPLIKFQVGLKKLNLHE LIEPLIKF LIEPLIKFQVGLKKLNL KKLNLHEEEHVLL MAICIVSPDRPGVQDAKL VEAIQDRLSNTL QTYIRCRHPPPGSHQLYAKMIQKLA LYAKMIQ YAKMIQKLADLRSLNEEHSKQYRSL DLRSLNEEHSKQYRSLSFQPE QPENSMKLTPL MKLTPLVL EVFGNEIS

Hinge/H1 H1 H1/Loop H1/Loop/H2 Loop Loop/H3 H3/H3’ H3’/H4 H4/H5/S1 S3/H6 Loop/H7/Loop H7 H7/Loop Loop/H8 H8/Loop/H9 H9 H9/Loop/H10 H10 H10/H11 H10/H11 Loop/H12 H12 H12

4 1 -4 -31 3 -52 -22 3 -33 -13 -22 -29 -42 -1 -1 -2 -22 6 -27 -20 -39 -25 -31

-2 1 -3 -29 -5 -56 -30 4 -27 5 13 8 -26 -12 -2 -1 -18 16 -25 -27 -34 -38 -57

-1 -2 4 -17 -4 -48 -24 2 -47 -2 7 1 -41 -14 4 0 -12 16 -23 7 6 0 -11

∆D (%) ≤ 20 ≤ 10 n.s. ≥ -10 ≥ -20 ≥ -30 ≥ -40 < -40

The values represent difference in percentage of deuterium level of apo VDR-LBD and VDR-LBD in the presence of ligand. Deuterium level is the average of six H/D exchange time experiments: 10, 30, 60, 300, 900 and 3600 sec. A positive value (red) indicates a region increased in deuterium exchange in that region of VDR-LBD in the presence of ligand. A negative value (blue) represents a decrease in exchange. z value is the charge state of the peptide ion.

20


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Figure 6. Differences in HDX profile for each ligand. (a) 1,25D3, (b) 3a, (c) 3b. The changes in HDX profile presented in Table 1 were mapped onto VDR-LBD (PDB: 2ZLC). Undetected regions are shown in black.

We would like to emphasize that, in the presence of 1,25D3, both sets of HDX data of ours and Grrifin's show good agreement, including the values of protection and deprotection, across the entire LBD.21 In the presence

of 1,25D3, deuterium exchange decreased in peptide regions constituting the VDR-LBP (H1-2, H3, H5, H6-7, and H10-12) (Table 1 and Figure 6a). These regions include amino acid residues that form hydrogen bonds with 1,25D3 [Tyr143 (H1), Ser233 (H3), Arg270 (H5), Ser274 (H5), His301 (loop 6-7), and His393 (H10)] and hydrophobic

residues (H12) that form hydrophobic interactions. These data indicate that the above-mentioned regions are

stabilized by 1,25D3 binding. The region encompassing residues 375-381 (H10), which is considerably resistant to exchange in apo VDR-LBD, nevertheless exhibited a slight increase in exchange upon 1,25D3 binding (Table 1). A similar observation was reported for human VDR-LBD.21 As this region is a part of the dimerization interface with

RXR, an increase in flexibility may be advantageous for heterodimerization with RXR.

Compound 3a caused a decrease in deuterium exchange similar to that associated with 1,25D3, except for H6-H7 (Table 1 and Figure 6b). H1-2, H3, H5, and H10-H12 were stabilized by hydrogen bonding and

hydrophobic interaction between 3a and VDR-LBD (Figure 4a, 4f). Interestingly, we found that the deuterium

levels in H6, loop 6-7, and H7 increased markedly in the presence of ligand 3a (Table 1), indicating that the

flexibility of these regions increases upon ligand 3a binding. This increased flexibility is consistent with the results

of analyses of the crystal structure of 3a/VDR-LBD, which showed that this region shifted substantially from the 22


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canonical position because the benzyl group of 3a makes contact with loop 6-7 in the canonical position (Figure 4c).

Surprisingly, 3a decreased deuterium levels of H12 and its vicinity (-38% and -57%) and the decrease rate was

bigger than that by 1,25D3 binding, indicating that 3a strongly stabilizes H12 though it is VDR antagonist (Table 1 and Figure S2). Moreover, exchange in the region encompassing residues 375-381 (H10) was higher with 3a (16%) than 1,25D3 (6%). The HDX profile of compound 3b was similar to that of 3a except for H11, loop 11-12, and H12 (Table 1

and Figure 6c). In contrast to the results obtained with 3a, the regions encompassing residues 385-405 (H10-H11) and 403-413 (loop 11-12) exhibited a marked increase in deuterium level; that is, H11 and loop 11-12 became

unstable upon ligand 3b binding. In addition, the regions encompassing residues 408-415 (H12) and 416-423 (H12) exhibited an increase in deuterium level compared with 1,25D3 (Figures S2 and S3). Interestingly, the deuterium level of H12 was quite similar to that of apo VDR-LBD (Figure S3). These results indicate that ligand 3b does not contribute to the stabilization of H12 but instead destabilizes H11 and loop 11-12.

Docking Study Because we were unable to obtain crystals of the 3b/VDR-LBD complex, we conducted a docking study. As ligand 3b has a 22S-butyl group, VDR must form a butyl pocket.17,39,40 Therefore, we selected a VDR-LBD (PDB code 3WTQ), for which 22S-butyl-2-methylidene-19-nor-1α,25-dihydroxyvitamin D3 is a ligand.40 Crystals of this complex (3WTQ) exhibit the best resolution among crystals forming the butyl pocket. We performed a

normal docking study; however, 3b could not be docked in VDR-LBD. We hypothesized that 3b is too large to

dock in the LBP and therefore removed H12 (residues 412-420) and used this VDR-LBD (∆412-420) for the 23



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docking study. The results of this analysis are shown in Figure S4a. Ligand 3b was then extracted from the data shown in Figure S4a and superimposed upon the original VDR-LBD, 3WTQ (Figure S4b), which revealed that the

22S-butyl group was accommodated in the butyl pocket (Figure S4c) while 25-benzyl group came into contact with

Val414 and Phe418 of H12 (Figure S4d). Repulsion between the benzyl group and H12 must therefore prevent the

proper folding of H12.

Recruitment of the Coactivator Peptide and RXRα α To elucidate the mechanism of antagonism of 3a and 3b, we examined ligand-dependent recruitment of a

coactivator peptide, SRC-1, and RXRα to the VDR using a mammalian two-hybrid assay in HEK293 cells according to a previously reported procedure.35 As shown in Figure S5, 3a exhibited moderate SRC-1 recruitment

and weak RXRα, whereas 3b did not recruit SRC-1 and exhibited only marginal recruitment of RXRα.

Discussion Synthetic compounds 2a,b-6a,b showed specific binding affinity for hVDR (Table S1). Bulky

compounds 5 and 6 exhibited less affinity for the VDR compared with 3 and 4, respectively, indicating that the

bulkiness of analogues is important for high VDR affinity. The 25-dibenzyl analogues 3a,b inhibited the activation

induced by 1,25D3 in Cos7 and HEK293 cells (Figure 2). The naphthyl analogues 5a,b and 6a,b, which had more bulkier substituents than 3a,b, showed agonistic activity, albeit weak, indicating that the appropriate bulkiness like

3a,b are critical for antagonistic activity. At the present time, we cannot explain why naphthyl analogues 5a,b and

6a,b show weak agonistic activity. 24


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Extensive HDX-MS analyses of NRs have been conducted by Griffin et al. and other groups. Using the cutting-edge technology of HDX-MS, the behavior and mechanism of NRs such as RXRα,24,25 peroxisome proliferator-activated receptor γ (PPARγ),26-28 ERα,29,30 receptor-related orphan receptor γ (RORγ),31 liver receptor homolog-1 (LRH-1),32 and the progesterone receptor (PR)33 have been described. The VDR has also been studied,21-23 and those studies demonstrated that the stability of each region in apo hVDR-LBD differs and the

mobility of each region in hVDR-LBD or VDR/RXR heterodimer changes upon binding of agonists such as 1,25D3, ED-71, or alfacalcidol.21 Moreover, the stability of hVDR changes upon 1,25D3 binding, heterodimerization, VDRE binding, and coactivator recruitment.22

Using HDX-MS, we examined the effects of antagonists instead of agonists and found that three regions,

H6-H7, the N-terminus of H10, and H11/loop 11-12, become flexible upon ligand binding. 1,25D3 was shown to destabilize only the N-terminus of H10, similar to the results observed in NR-LBDs including hVDR-LBD.21,29

Antagonist 3a destabilized two regions, H6-H7 and the N-terminus of H10, and antagonist 3b destabilized three regions, loop 6-7/H7, H11/loop 11-12, and the N-terminus of H10 (Table 1 and Figure 6).

Antagonist 3a caused a dynamic conformational change in the main chain of H6-H7, as shown in the crystal structure (Figure 4c), and destabilized the same region, as shown by differential HDX analysis (Figure 6b).

Unexpectedly, H11-H12 of 3a/VDR-LBD adopted an almost canonical position (Figure 4b) and was stabilized more than that of 1,25D3/VDR-LBD (Table 1, Figure 6b). Thus, both crystal structure and HDX-MS analyses demonstrated that although 3a is a VDR antagonist, H11-H12 is stabilized. Surprisingly, H12 was strongly

stabilized by binding of antagonist 3a than 1,25D3. These structural analyses indicate 3a exhibits antagonistic 25



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activity dependent on destabilization of the structure of H6-H7 but not H12. All VDR antagonists reported so far

are thought to destabilize H12 or be intact to H12, and no antagonist is known that stabilize H12. The same is

applicable for other NR antagonists. Therefore, compound 3a is a first antagonist against not only VDR but also

NRs that stabilizes H12. Thus, compound 3a stabilizes H11-H12 but crashes H6-H7 so that 3a is a novel rather than

“active” or “passive” type of antagonist. We classified 3a as a third type of antagonist and called it “H11-H12 stabilization antagonist”.

Antagonist 3b destabilized H11 and loop 11-12 and induced flexibility in H12 similar to apoVDR-LBD

(Table 1 and Figure 6c). Therefore, 3b is thought to inhibit VDR activity by preventing H12 folding. The docking study also demonstrated that it is reasonable that the 22-butyl group is accommodated in the butyl pocket despite

3b-induced prevention of H12 folding (Figure S4). This characteristic behavior (prevention of H12 folding)

explains why co-crystals of 3b were not obtained. As all VDR-LBD crystals reported to date have shown an H12-folding structure, H12 folding seems to be necessary for crystallization in terms of crystal packing. Antagonist

3b actively prevents H12 folding, thus preventing the formation of co-crystals of 3b. Binding of 3b also increased the deuterium levels in loop 6-7/H7. As reported in our previous studies, 22S-butyl compounds change the

side-chain conformation of residues in loop 6-7/H7 to induce butyl pocket formation but do not change the

conformation of the main chain. Therefore, 3b seems to destabilize loop 6-7/H7 without altering the conformation of the main chain. Taken together, these data indicate that the mechanism underlying destabilization of this region

upon 3b binding is distinct from that associated with 3a binding.

This study demonstrated that 3a is neither an “active” nor “passive” antagonist. Antagonist 3a is a third 26


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type of antagonist, namely “H11-H12 stabilization antagonist”. In contrast, 3b is an active antagonist that prevents

H12 folding. It seems that 3a binds to the VDR-LBP, forming the activation function 2 (AF-2) surface upon H12

folding (Figures 3 and 4). Therefore, 3a binding might result in low-level coactivator recruitment (Figure S5). However, destabilization of regions H6-H7 and H10, which form part of the dimer interface, may inhibit

heterodimerization with RXR (Figure S5). Considering that the N-terminus of H10 becomes more unstable in

3a/VDR-LBD (16%) than 1,25D3/VDR-LBD (6%), H10 might adopt an unsuitable conformation for heterodimerization. In the case of antagonist 3b, 3b is thought to bind to the VDR-LBP but prevent H12 folding.

Therefore, the AF-2 surface is not formed and 3b inhibits coactivator recruitment (Figure S5). Moreover, 3b must

inhibit heterodimerization because, in addition to loop 6-7/H7, H10 becomes unstable, as in the case with 3a binding (Table 1, Figure S5).

The mechanism of the antagonistic activity of 3a differs from that of 3b. All VDR antagonists reported to date are side-chain analogues of 1,25D3. Those analogues must destabilize all or some of three regions via a similar or different mechanism than that described in this study. For example, binding of antagonist 1, which induces formation of the butyl pocket,17 must destabilize loop 6-7/H7 and the N-terminus of H10 by a mechanism

similar to that of 3b. However, unlike 3a and 3b, neither stabilization nor destabilization of H12 would occur

because there are no interactions between H12 and antagonist 1, as shown in the crystal structure of the 1/VDR-LBD complex.17 Thus, the flexibility of H12 in 1/VDR-LBD may be similar to that in the apo-form. These considerations are consistent with the observation that 1 does not recruit either RXR or the SRC-1 peptide.39 Based on these results, we conclude that the mechanism of VDR antagonistic behavior varies. 27



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In conclusion, we designed and synthesized vitamin D analogues 2a,b-6a,b and evaluated their biological

activities. The 25-dibenzyl analogues 3a and 3b showed strong antagonistic activity. HDX-MS and X-ray crystal

structure analyses indicated that 3a is a “H11-H12 stabilization antagonist”. HDX-MS analysis also clearly showed

that 3b is an active antagonist. In this study we found first VDR antagonist 3a that strongly stabilizes H12. This finding must be applicable for other NRs and greatly affect drug design to treat NR related diseases. In addition, we

demonstrated that VDR antagonists have diverse mechanisms depending on each antagonist structure. Our results

also demonstrate that HDX-MS can provide information about dynamic changes in protein structure induced by

ligand binding that could prove useful in drug discovery research.

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Experimental Section HDX-MS. A mixture of rVDR-LBD in buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 2mM DTT) and ligand

(10 equiv.) was incubated for 1 h on ice. Each exchange reaction was initiated by incubating 8 µL of VDR/ligand

complex with 32 µL of D2O protein buffer for 10, 30, 60, 300, 900, or 3600 sec at 4°C. The exchange reaction was quenched by mixing with 60 µL of quench buffer (3 M urea, 1% TFA) on ice. Protein digestion was carried out with pepsin at an enzyme to substrate ratio of 1:1 for 5 min on ice. The digested samples were immediately frozen

in liquid N2. Peptide separation and mass spectrometry were carried out using an Agilent 6550 iFunnel Q-TOF LC/MS and MassHunter Workstation Qualitative Analysis Software (Agilent Technologies, CA, U.S.A.). Peptides

were separated using a JUPITER 4 µm Proteo 90 Å LC Column 50 × 4.6 mm (Phenomenex, CA, U.S.A.) with a linear gradient of 4–40% CH3CN/0.3% formic acid over 5 min at a flow rate of 1 mL/min. Peptide separation was performed within a thermal chamber held at 1°C to reduce D/H back exchange. The operating conditions for MS

analysis were as follows: positive ion mode, capillary voltage (VCap) 5000 V, gas temperature 220°C, drying gas

20 L/min, nebulizer gas 50 psig, fragmentor 400 V. Duplicates were performed for each on-exchange time point.

The intensity weighted average m/z value (centroid) of each deuterated peptide was calculated using MassHunter

Workstation Qualitative Analysis Software (Agilent Technologies) and Microsoft Excel. The deuterium level was

calculated according to the following equation: deuterium (%) = [(mt – m0%)/(m100% – m0%)] x 100, where mt, m0%, and m100% represent the centroid value of the partially deuterated, nondeuterated, and fully deuterated peptide, respectively.21

29



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Supporting Information The Supporting Information is available.

List of contents: Supplementary Figures and Tables, Supplementary Methods (Synthesis, Competitive Binding

Assay, Transfection and Transactivation Assay, Protein Expression and Purification, X-ray Crystallographic

Analysis, Docking Study, RXR and SRC-1 Recruitment), Supplementary References

Acknowledgement This work was financially supported by the Platform for Drug Discovery, Informatics, and Structural Life

Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Agency

for Medical Research and Development (AMED), a Grant-in-Aid for Scientific Research (No. 26460155) from

MEXT, and the Takeda Science Foundation, Japan, to K.Y. Synchrotron-radiation experiments were performed at

the Photon Factory (Proposal No. 2013G656), and we are grateful for the assistance provided by the beamline

scientists at the Photon Factory.

AUTHOR CONTRIBUTIONS A.K. and K.Y. designed the research. K.Y. supervised the overall project. A.K. performed synthesis and biological

evaluation. A.K., T.I. and Y.A. performed x-ray crystal structure experiment and analyzed the data. A.K. and D.E.

performed the HDX-MS experiments and A.K. analyzed the data. A.K. and K.Y. wrote the paper. All authors

discussed the results and contributed to the final version of the manuscript.

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Helix12-Stabilization Antagonist of Vitamin D Receptor

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