Structural insights for producing CK2α1-specific inhibitors

Journal Pre-proofs Str uctur al insights for pr oducing CK2 α 1-specific inhibitor s Masato Tsuyuguchi, Tetsuko Nakaniwa, Akira Hirasawa, Isao Nakanishi, Takayoshi Kinoshita PII: DOI: Reference:

S0960-894X(19)30809-1 https://doi.org/10.1016/j.bmcl.2019.126837 BMCL 126837

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Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

2 October 2019 6 November 2019 15 November 2019

Please cite this article as: Tsuyuguchi, M., Nakaniwa, T., Hirasawa, A., Nakanishi, I., Kinoshita, T., Str uctur al insights for pr oducing CK2 α 1-specific inhibitor s, Bioorganic & Medicinal Chemistry Letters (2019), doi: https:// doi.org/10.1016/j.bmcl.2019.126837

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Structural insights for producing CK2α1-specific inhibitors Masato Tsuyuguchia*, Tetsuko Nakaniwab, Akira Hirasawac, Isao Nakanishid and Takayoshi Kinoshitaa Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 c Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan d Faculty of Pharmacy, Department of Pharmaceutical Sciences, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan a

b

ARTICLE INFO

ABSTRACT

Article history: Received Revised Accepted Available online

Casein kinase 2 catalytic subunit (CK2α) is classified into two subtypes CK2α1 and CK2α2. CK2α1 is a drug discovery target, whereas CK2α2 is an off-target of CK2α1 inhibitors. High amino acid sequence homology between these subtypes hampers efforts to produce ATP competitive inhibitors that are highly selective to CK2α1. Hematein was identified previously as a non-ATP-competitive inhibitor for CK2α1, whereas this compound acts as an ATP competitive CK2α2 inhibitor. Crystal structures of CK2α1 and CK2α2 in complex with hematein revealed distinct binding features that provide structural insights for producing CK2α1-selective inhibitors.

Keywords: CK2α1 Selectivity Binding mode Crystal structure Conformational change

Casein kinase 2 (CK2) is a serine/threonine kinase that regulates miscellaneous cell signaling events through the phosphorylation of various substrates.1 The holoenzyme of CK2 consists of two catalytic subunits (CK2α1 and/or CK2α2) and two regulatory subunits (CK2β).2 CK2α1 is an important drug discovery target for serious diseases including nephritis and cancer.3 CK2α2 knock-out mice exhibit pathological conditions, such as globozoospermia in humans,4 which suggests that inhibition of CK2α2 likely causes testicular toxicity. High amino acid sequence homology between CK2α1 and CK2α2 (identities: 85%, positives: 92%) renders it difficult to produce ATP competitive inhibitors that are highly selective to CK2α1. In particular, the ATP binding site is almost conserved. Only two residues in the hinge region of the ATP binding site are different between CK2α1 and CK2α2; however, these residues interact with the ligand using only main chain atoms. Various types of CK2 inhibitors have been reported, including emodine,5 4,5,6,7tetrabromo-1H-benzotriazole (TBB),6 (5-oxo-5,6dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid (IQA)7 and CX4945.8,9 CX-4945 has been clinically studied as an anti-cancer agent.8 However, our experiments11 showed that existing inhibitors have insufficient selectivity for CK2α1 vs. CK2α2. Hematein (Fig. 1) is a natural compound that is a non-ATPcompetitive inhibitor of CK2α1.10 Binding characteristics of hematein for CK2α1 were confirmed by our experiments.11,12 Surprisingly, hematein inhibited CK2α2 in an ATP-competitive manner.11,12 These distinct behaviors of hematein against CK2α1

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and CK2α2 presumably give rise to different binding modes of hematein to the two isozymes, although the inhibitory activities are nearly identical (IC50: 0.2 M and 0.4 M against CK2α1 and CK2α2, respectively). 6

5

7 6a

8 9

4 3

2

1

11

10

Fig. 1. Chemical structure of hematein.

The similar inhibitory activities can be accounted for by the following two possibilities. For the first possibility, hematein binds to allosteric binding sites of CK2α1. The regions in the vicinity of the αD-helix and the CK2β binding surface are defined as allosteric sites of CK2α1. CAM4066, a CK2α1 inhibitor, binds to a novel pocket that emerges by inhibitorinduced structural rearrangement in which the αD-helix moves outside compared with that of the apo state.13 CAM4066 binds not only to an allosteric pocket but also to the ATP binding site; although, the mechanisms of inhibition has yet to be verified. A cyclic peptide binds to the CK2β binding site and exerts inhibitory activity.14 This peptide competes with CK2β but not ATP. For the second possibility, hematein binds to the ATP site with an inhibitor-induced conformational change. There are no

reports describing hematein binding to the ATP site of CK2α1; however, there are reports for other kinases. For example, The proto-oncogene Src inhibitor PP1 is a non-ATP-competitive inhibitor that binds to the ATP binding site.15 In the crystal structure of Src complexed with PP1, Src adopts an autoinhibition form that is induced by PP1. In this structure, Lys295 does not form a salt bridge with Glu310, an essential interaction for the active form, but with Asp404.

accompanied with the structural change of His160 between the up and down forms. This behavior may explain the non-ATPcompetitive manner of hematein binding to CK2α1. In contrast, the down form of His161 of CK2α2 does not disrupt ATP binding. Therefore, hematein can compete with ATP without structural changes in binding to CK2α2. This feature likely explains the ATP-competitive manner inhibition of hematein against CK2α2.

In this report, the crystal structures of CK2α1 and CK2α2 complexed with hematein at 1.9 Å and 3.1 Å resolutions, respectively,16,17 were determined to reveal the distinct features of hematein binding to CK2α1 and CK2α2. The overall folds of CK2α1 and CK2α2 do not differ significantly (Fig. 2). In both CK2α1 and CK2α2 complexes, hematein binds to the ATP binding site and was not found at any other region including the CK2β binding site and αD-helix site.

σ＝1.0

σ＝1.0

Fig. 3. Binding modes of hematein to CK2α1 (blue and red) and CK2α2 (green and red), and electron density maps with hematein.

K68 Aq1 D175 W176 Fig. 2. Superposed structures of CK2α1 (blue) and CK2α2 (green).

However, the electron density maps suggested that hematein bound to CK2α1 and CK2α2 in unique manners even though the hematein binding site structure is highly conserved between these subtypes (Fig. 3). In the CK2α1 complex, hematein binds to the ATP back pocket at Lys68 and Asp175 directly, and Trp176 via a water molecule (Fig. 4a). This water molecule is observed frequently in crystal structures of CK2α1 and CK2α2.18,19 In the CK2α2 complex, hematein binds more deeply within the ATP back pocket when compared with that of CK2α1, which eliminates the water molecule observed in the CK2α1 complex (Fig. 4b). Thus, hematein directly binds to Lys69, Asp176 and additionally to Glu82 in the CK2α2 complex (Fig. 4b). Hematein interacts with the main chain atoms of Glu114 and Val116 in the hinge region via a water molecule in the CK2α1 complex (Fig. 5a). In the CK2α2 complex, hematein directly forms hydrogen bonds with the main chain atoms of Ile117 in the hinge region (Fig. 5b). In both structures the side chains of these residues in the hinge region are not involved in hematein binding. In the ribose binding site, His160 faces the ATP binding site (up form) and make a CH- π interaction with hematein in the CK2α1 complex (Fig. 6a). His161 of CK2α2 faces in the opposite direction (down form) and makes no interaction with hematein (Fig. 6b). In the several structures of CK2α1 in the apo form or in complex with other compounds, His160 adopts the down form.18,20 The up form of His160 is likely induced by hematein and causes the non-ATP-competitive inhibition of CK2α1. In the crystal structure of CK2α1 complexed with AMPPNP, an ATP analogue, His160 adopts the down form.20 The up form of His160 would sterically clash with the ribose group of AMPPNP. This structural insight suggests that ATP competes with hematein for binding to CK2α1, which is

(a)

K69

E82

D176 (b) Fig. 4. Close-up views of the ATP back pocket. (a) CK2α1; (b) CK2α2.

(a)

(b)

Fig. 5. Close-up views of the hinge region. (a) CK2α1; (b) CK2α2.

(a)

(b) Fig. 6. Close-up views of the ribose binding site including His160 and His161. (a) CK2α1; (b) CK2α2.

The crystal structure16 of a His160Ala mutant (H160A) of CK2α1 showed that the binding mode of hematein completely changed to the CK2α2-type (Table 1). This result suggests that His160 of CK2α1 is critical for forming the CK2α1-type binding mode of hematein. As mentioned above, the amino acid residues within 8 Å of His160 including the αD helix are well-conserved between CK2α1 and CK2α2. Thus, we focused on the two nonconserved residues in the hinge region, which are not less than 13 Å away from His160 and recognize all kinds of inhibitors using only main chain atoms. To evaluate the effect of these two residues on the binding modes of hematein, we solved the crystal structures16 of other mutants in which the hinge-region residues of CK2α1 corresponded to those of CK2α2 (H115Y, V116I, H115Y/V116I). These mutations conferred a decrease in the rate of the CK2α1binding mode. The ratio of the CK2α1-binding mode in H115Y and V116Y decreased and that of the double mutant H115Y/V116I decreased even further (Table 1). These results revealed that the non-conserved residues in the hinge region between CK2α1 and CK2α2 critically correlate with the conformational alternation in His160/His161 of CK2α1 and CK2α2, and the binding mode; although, hematein did not form any interaction with the side chains of these non-conserved residues. However, these residues presumably cause a significant difference in structural flexibility, resulting in increasing the propensity of the His160-up configuration of CK2α1. The hinge region of CK2α1 forms two van der Waals contacts with the neighborhood lysine residue (Fig. 7a). In case of CK2α2, the hinge region forms the two van der Waals contacts and two hydrogen bonds in addition to the interactions observed in CK2α1 (Fig. 7b). Furthermore, the neutron crystal structure of CK2α1 presented that His115 in the hinge region was in the protonated state and thus possessed a positive charge.21 These differences likely form a conformation in the αD helix, that supports the His160-up configuration of CK2α1. Table 1. Ratio of the hematein binding modes. 7

CK2α1

CK2α2

CK2α1

CK2α1

CK2α1

CK2α1

(wild)

(wild)

H115Y

V116I

H115Y/

H160A

V116I CK2α1-

100

0

40

30

20

0

type CK2α2type

0

100

60

70

80

100

In summary, hematein binds to the ATP binding site of CK2α1 and CK2α2 with distinct binding modes (Fig. 3). This distinction was caused by a conformational difference of His160 (up and down forms) (Fig. 6). The crystal structures showed that the conformation of His160 is allosterically regulated by two residues in the hinge region, which are non-conserved between CK2α1 and CK2α2. These structural insights into hematein binding to CK2α1 and CK2α2 likely account for the inhibition mechanisms, and provide a crucial clue for designing CK2α1 selective inhibitors. For example, the replacement of the 3position hydroxyl group of hematein with a methoxy group may confer higher selectivity towards CK2α1. The crystal structures suggest that this modification does not affect the binding of hematein to CK2α1 (Fig. 6a) but causes steric clashes with the hinge region when hematein binds to CK2α2 (Fig. 5b). Synthesizing hematein derivatives as described above are very difficult at present. Therefore, we next plan to understand the mechanism of the alternative binding modes in detail by further structural studies and kinetics analysis. Nonetheless, two residues in the hinge region are the most important residues in the regulation mechanism. Unraveling the mechanism should facilitate production of CK2α1 specific inhibitors with other scaffolds.

(a)

(b) Fig. 7. Interaction network around the hinge region. (a) CK2α1 (b) CK2α2. The dotted lines show the hydrogen bond (purple) or van der Waals contact (yellow).

Acknowledgments Preliminary experiments and diffraction data collection were carried out at the beam lines of the Photon Factory (2007G016 and 2016G665) and at the Osaka University beam line BL44XU of SPring-8 (2018A6814 and 2018B6814). We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Appendix A. Supplementary data

15. 16.

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Masato Tsuyuguchi, Tetsuko Nakaniwa, Akira Hirasawa, Isao Nakanishi, Takayoshi Kinoshita

CK2α1-hematein CK2α2-hematein