Discovery of a Highly Selective STK16 Kinase ... - ACS Publications

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Discovery of a Highly Selective STK16 Kinase Inhibitor Feiyang Liu, Jinhua Wang, Xingxing Yang, Binhua Li, Hong Wu, Shuang Qi, Cheng Chen, Xiaochuan Liu, Kailin Yu, Wenchao Wang, Zheng Zhao, Aoli Wang, YongFei Chen, Li Wang, Nathanael S Gray, Jing Liu, Xin Zhang, and Qingsong Liu ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00250 • Publication Date (Web): 15 Apr 2016 Downloaded from http://pubs.acs.org on April 16, 2016

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ACS Chemical Biology Discovery of a Highly Selective STK16 Kinase Inhibitor Feiyang Liu1,2,6, Jinhua Wang3,6, Xingxing Yang1,6, Binhua Li1,4,6, Hong Wu1,2, Shuang Qi1,4, Cheng Chen1,4, Xiaochuan Liu1,2, Kailin Yu1,2, Wenchao Wang1,4, Zheng Zhao1,4, Aoli Wang1,2,Yongfei Chen1,4, Li Wang1,4, Nathanael S.Gray3, Jing Liu1,4*, Xin Zhang1*, Qingsong Liu1,2,4,5* 1. High Magnetic Field Laboratory, Chinese Academy of Sciences, 350 Shushanhu Road, P.O. Box 1110, Hefei, Anhui 230031, P. R. China 2. University of Science and Technology of China, Anhui, Hefei, 230036, P. R. China 3. Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave, SGM 628, Boston, MA 02115, USA 4. CHMFL-HCMTC target therapy Joint Laboratory, Shushanhu Road, Hefei 230031, Anhui, P. R. China 5. Hefei Science Center, Chinese Academy of Sciences, Shushanhu Road, Hefei 230031, Anhui, P. R. China 6. These authors contributed equally *

Correspondence:

[email protected] (JL), [email protected] (XZ), [email protected] (QL)


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ACS Chemical Biology Abstract STK16, a serine/threonine protein kinase, is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion but the pathways through which these effects are mediated remain to be elucidated. Through screening of our focused library of kinase inhibitors we discovered a highly selective ATP competitive inhibitor, STK16-IN1, which exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 µM) with excellent selective across the kinome as assessed using the KinomeScanTM profiling assay (S score (1)=0.0). In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine and paclitaxel results in a slight potentiation of the anti-proliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16.

Keywords: STK16 kinase, Kinase inhibitor, Binucleated cell, Drug combination



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ACS Chemical Biology Text STK16 (serine/threonine kinase 16, also known as Krct, PKL12, MPSK1 and TSF-1) is a unique member of the NAK (Numb associated Kinase) subfamily of serine/threonine protein kinase discovered in the late 1990s by several labs using degenerate PCR screens.1-4 It is ubiquitously expressed with highest expression observed in the liver, testis and kidney. STK16 is conserved among all of the eukaryotes.1 Despite being discovered 17 years ago, the biological functions of STK16 are not well understood yet. STK16 appears to be associated with the Golgi and is believed to be involved in the regulation of VEGF expression and soluble cargo secretion as well as end-bud morphogenesis during murine mammary gland development.5-7 STK16 has been demonstrated to interact with MAL2 protein, GlcNAcK kinase, and DRG1 protein and is involved in the TGF-β signaling pathway.6, 8-10 As an active serine/threonine kinase, STK16 has been reported to phosphorylate DRG1 and 4EBP1 in vitro.10 To the best of our knowledge, there are no reported selective STK16 inhibitors. In order to further elucidate the biological functions of STK16, we sought to identify a selective STK16 inhibitor that resulted in the identification of STK16-IN-1. During kinase profiling of our in house focused kinase inhibitor library, we identified a pyrrolonaphthyridinone STK16-IN-1 (Structure shown in Fig. 1A). Selectivity profiling using the DiscoveRx’s KinomeScanTM technology demonstrated that the compound is highly selective: among the 442 kinases tested, STK16-IN-1 only potently inhibited STK16 (relative activity remaining 0.65%) and mTOR (relative activity remaining 0.4%) at a concentration of 10 µM.11 This corresponds to a selectivity score (S score (1)=0.0) which ranks STK16-IN-1 as a highly selective inhibitor (Fig. 1B and Supplemental Table 1). To corroborate these findings using enzymatic assays (LifeTechnology SelectScreen®) STK16-IN-1 was demonstrated to inhibit STK16 with an IC50 of 0.295 µM and mTOR kinase with an IC50 of 5.56 µM (Table 1). STK16-IN-1 was evaluated for its ability to inhibit several PI3Ks due to the fact that the known mTOR inhibitors can also inhibit PI3K. STK16-IN-1 inhibited PI3Kδ with an IC50 of 0.856 µM, PI3Kγ with an IC50 of 0.867 µM while inhibited other isoforms with IC50 greater than 1 µM. In addition, in the cell-based assays, STK16-IN-1 did not exhibit any apparent inhibitory activity


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ACS Chemical Biology against all of those four class I isoforms of PI3K up to 10 µM (Supplemental Fig. 1). We next evaluated the ability of STK-16-IN-1 to inhibit STK16 using an in vitro IP kinase assay monitoring phosphorylation of T37/46 of 4EBP1 as readout, where it displayed an IC50 of 0.91 µM (Fig. 1C). In addition, it exhibited IC50 of 1.2 µM when using DRG1 as substrate by detection of the general threonine phosphorylation (Fig. 1D). Enzymatic studies demonstrated that STK16-IN-1 is an ATP competitive inhibitor against STK16 kinase (Fig. 1E). A docking study using the experimentally determined STK16 crystal structure (PDB ID: 2BUJ) suggests that the pyrrolopyridine moiety forms two hydrogen bonds with Phe101 the kinase hinge binding segment and that the fluorinated benzene group is orientated beneath the P-loop roof (Fig. 1F) .10 We next tested STK16-IN-1 against a small panel of cancer cell lines including the cervical cancer, B-cell leukemia, breast cancer, lung cancer and colon cancers to see if sensitivity correlated with any cancer types or genetic factors. STK16-IN-1 only weakly inhibited growth of MCF-7 (GI50 about 10 µM) but not any other cancer cell lines (GI50 over 10 µM) and seems there is no correlation between proliferation and the expression level of STK16 kinases in these cells (Fig. 2A and Supplemental Fig. 2). However, there was dose-dependent induction of apoptosis in both MCF-7 and HeLa cells but not in HCT-116 cells (Fig. 2B and Supplemental Fig. 3). We next evaluated the ability of STK16-IN-1 to inhibit STK16 and putative downstream substrates in cells. Due to the lack of STK16 phosphorylation specific antibodies, we transfected a GFP-FLAG tag labeled STK16 kinase into HeLa cells and did the IP/IB blotting of the general serine, tyrosine and threonine phosphorylation of STK16. The results demonstrated that at a concentration of 10 µM, the phosphorylation of serine was inhibited by approximately 70% while the phosphorylation of tyrosine and threonine were not affected by STK16IN-1 (Fig. 2C). In the cellular context, STK16-IN-1 only weakly affected 4EBP1’s phosphorylation at about 5 µM in MCF-7 cells but not in HeLa or HCT-116 cells (Fig. 2D). In addition, due to lacking of antibodies specific for the phosphorylation of DRG1, we blotted general threonine phosphorylation of DRG1in HCT-116 cells but did not observe any inhibitory activity (Supplemental Fig. 4).10 These observations seem to contradict the in vitro biochemical assay results. However, this implied that either in



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ACS Chemical Biology cellular context 4EBP1 and DRG1 were not the direct the downstream substrates of STK16 or due to the ATP concentration difference between the in vitro assay (20 µM) and in cellular assays (about 5 mM), so that the inhibitor could not efficiently compete with ATP. Interestingly, neither of the AKT phosphorylation site (T308 and S473) was affected by STK16-IN-1 even at 10 µM concentration. This suggests that STK16 did not affect PI3K isoforms, at least for those expressed in these cell lines such as PI3Kα, which further supported the idea that STK16-IN-1 is a selective STK16 inhibitor. The difference between the cellular signal transduction pathway and invitrogen’s biochemical data (listed in Table 1) might come from the ATP concentration difference as mentioned above. In addition, since there has been some preliminary evidence indicating that knockdown of STK16 kinase may enhance the anti-proliferative efficacy of chemotherapy, we tested STK16-IN-1’s combinatorial effect with doxorubicin, cisplatin, paclitaxel and colchicine.12 Combinatorial anti-proliferation efficacy was observed for these drugs in MCF-7 cells but no synergistic effect was observed (Fig. 2E and Supplemental Fig. 5). Furthermore, the drug combination enhanced cell apoptosis by examination of the cleavage PARP with the cytokinesis inhibitors colchicine and paclitaxel and chemotherapy drug doxorubicin (Fig. 2F). In order to further investigate the cellular pharmacology of the inhibitor we screened cell lines for STK16 expression using western blot. We identified several cell lines that express STK16 at relative higher levels including the breast cancer cell line MCF-7, the colon cancer cell line HCT-116 and the cervical cancer cell line HeLa (Supplemental Fig. 6). Interestingly, we observed dose dependent alive cell number decrease and meanwhile a dose-dependent binucleated cells number increase in MCF-7 cells, which usually result from a failure to complete cytokinesis (Fig. 3A).13-15 Similar results were observed in HeLa cells (Supplemental Fig. 8). Quantification of DNA content in all three cell lines following 72 hours treatment with STK16-IN-1 using flow cytometry demonstrated an increase in the number of cells in G2/M phase of the cell cycle, which is consistent with the phenotype of increased binucleated cells (Fig. 3C and Supplemental Fig. 7).

In addition, although STK16 RNAi only achieved approximately 47%

knockdown, we observed an approximate 50% reduction of cell number for MCF-7 cells


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ACS Chemical Biology (Fig. 3D, F). Meanwhile the binucleated cell number was also increased about 2-fold (Fig. 3E, F). Similar results were observed for HeLa cells (Supplemental Fig. 8). These results suggest that the cell proliferation and induction of binucleated cells could be seen using both pharmacological and RNAi-mediated depletion of STK16, which suggest that STK16 may have functions during mitosis and cytokinesis. In order to further confirm the STK16-IN-1’s on target effect, we generated a STK16 inducible knockdown cell line of HCT-116 cell, HCT116-shSTK16, in which the STK16 can be conditionally knockdown by doxycycline addition, as well as the STK16-GFPFlag HCT116 cells, which overexpress a GFP-Flag tagged STK16, for the comparison purpose (Fig. 4B). Although the inducible knockdown is not very efficient, we could still see that after STK16 kinase was knockdown, the STK16-IN-1’s efficacy to reduce cell numbers was significantly decreased compare to the non-knockdown cells (Fig. 4A, C). In addition, STK16-IN-1’s efficacy to arrest cell cycle into G2/M phase was decreased too (Fig. 4D). This indicated that STK16-IN-1 exerted its effect through STK16 kinase. We then screened a panel of mutations16 in the STK16 kinase ATP binding pocket including the L98F, L98Q, G104 (N, D, E) and F100C and found that STK16 F100C mutation could significantly abolish STK16-IN-1’s inhibitory effect (Supplemental Fig. 9). Transformation of this mutant into HeLa cells with a GFP-FLAG labeled STK16 kinase showed that the G2/M-phase cell cycle arrest effect of STK16-IN-1 was rescued (Fig. 4E, F). All of these results confirmed the specificity of STK16-IN-1 on STK16 in cells.

STK16 kinase has been discovered for almost two decades but the detailed biological function is still poorly understood. One of the reasons might be the lack of selective STK16 inhibitors that can be used as a complementary tool to the genetic methods such as RNAi knockdown or gene knockout. Here we report the discovery and characterization of a highly selective small molecule inhibitor of STK16, STK16-IN-1. Our initial characterization of this compound suggests that inhibition of STK16 results in reduced cell proliferation, accumulation of cells in the G2/M-phase of the cell cycle and appearance of binucleated cells and these efficacies could be rescued by the drug resistant mutation of F100C in the drug binding pocket of STK16 kinase. These phenomena were



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ACS Chemical Biology also observed upon RNAi-mediated STK16 knock down. Furthermore, the previously identified protein substrates such as 4EBP1 and DRG1 seem not the direct downstream targets in the cellular context and may be not be related to the observed mitosis regulation function. In summary we believe STK16-IN-1 could serve as a useful chemical probe to elucidate the biological function of STK16. Conflict of Interest The authors declare no conflict of interest. Supporting information Supplemental information includes Experimental Procedures, supplemental figure 1-9 and supplemental table 1 can be found with this article via Internet at http://pubs.acs.org

Acknowledgements W. Wang, J. Liu and Q. Liu are supported by the grant of “Cross-disciplinary Collaborative Teams Program for Science, Technology and Innovation (2014-2016)" from Chinese Academy of Sciences. Z. Zhao is supported by Anhui Province Natural Science Foundation Annual Key Program (grant number: 1301023011). We want to thank China “Thousand Talents Program” support for Prof. Q. Liu and “Hundred Talents Program” of The Chinese Academy of Sciences support for Prof. J. Liu and W. Wang. We also want to thank National Natural Science Foundation of China (Project No.31301112) to X. Zhang and Scientific Research Grant of Hefei Science Center of CAS (SRG-HSC # 2015SRG-HSC022) for Q. Liu. Q. Liu was also supported by the CAS/SAFEA international partnership program for creative research teams. References: (1) Ligos, J. M., Gerwin, N., Fernandez, P., Gutierrez-Ramos, J. C., and Bernad, A. (1998) Cloning, expression analysis, and functional characterization of PKL12, a member of a new subfamily of ser/thr kinases, Biochem. Biophys. Res. Commun. 249, 380-384. (2) Stairs, D. B., Perry Gardner, H., Ha, S. I., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Chodosh, L. A. (1998) Cloning and characterization of Krct, a member of a novel subfamily of serine/threonine kinases, Hum. Mol. Genet. 7, 2157-2166.


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ACS Chemical Biology (3) Kurioka, K., Nakagawa, K., Denda, K., Miyazawa, K., and Kitamura, N. (1998) Molecular cloning and characterization of a novel protein serine/threonine kinase highly expressed in mouse embryo, Biochim. Biophys. Acta. 1443, 275-284. (4) Berson, A. E., Young, C., Morrison, S. L., Fujii, G. H., Sheung, J., Wu, B., Bolen, J. B., and Burkhardt, A. L. (1999) Identification and characterization of a myristylated and palmitylated serine/threonine protein kinase, Biochem. Biophys. Res. Commun. 259, 533538. (5) Guinea, B., Ligos, J. M., Lain de Lera, T., Martin-Caballero, J., Flores, J., Gonzalez de la Pena, M., Garcia-Castro, J., and Bernad, A. (2006) Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/threonine kinase involved in VEGF expression regulation, Exp. Cell Res. 312, 135-144. (6) In, J. G., Striz, A. C., Bernad, A., and Tuma, P. L. (2014) Serine/threonine kinase 16 and MAL2 regulate constitutive secretion of soluble cargo in hepatic cells, Biochem. J. 463, 201-213. (7) Stairs, D. B., Notarfrancesco, K. L., and Chodosh, L. A. (2005) The serine/threonine kinase, Krct, affects endbud morphogenesis during murine mammary gland development, Transgenic Res. 14, 919-940. (8) Ligos, J. M., de Lera, T. L., Hinderlich, S., Guinea, B., Sanchez, L., Roca, R., Valencia, A., and Bernad, A. (2002) Functional interaction between the Ser/Thr kinase PKL12 and N-acetylglucosamine kinase, a prominent enzyme implicated in the salvage pathway for GlcNAc recycling, J. Biol. Chem. 277, 6333-6343. (9) Ohta, S., Takeuchi, M., Deguchi, M., Tsuji, T., Gahara, Y., and Nagata, K. (2000) A novel transcriptional factor with Ser/Thr kinase activity involved in the transforming growth factor (TGF)-beta signalling pathway, Biochem. J. 350 Pt 2, 395-404. (10) Eswaran, J., Bernad, A., Ligos, J. M., Guinea, B., Debreczeni, J. E., Sobott, F., Parker, S. A., Najmanovich, R., Turk, B. E., and Knapp, S. (2008) Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture, Structure 16, 115124. (11) Fabian, M. A., Biggs, W. H., 3rd, Treiber, D. K., Atteridge, C. E., Azimioara, M. D., Benedetti, M. G., Carter, T. A., Ciceri, P., Edeen, P. T., Floyd, M., Ford, J. M., Galvin, M., Gerlach, J. L., Grotzfeld, R. M., Herrgard, S., Insko, D. E., Insko, M. A., Lai, A. G., Lelias, J. M., Mehta, S. A., Milanov, Z. V., Velasco, A. M., Wodicka, L. M., Patel, H. K., Zarrinkar, P. P., and Lockhart, D. J. (2005) A small molecule-kinase interaction map for clinical kinase inhibitors, Nat. Biotechnol. 23, 329-336. (12) Balzano, D., Santaguida, S., Musacchio, A., and Villa, F. (2011) A General Framework for Inhibitor Resistance in Protein Kinases, Chem Biol 18, 966-975. (13) Swanton, C., Marani, M., Pardo, O., Warne, P. H., Kelly, G., Sahai, E., Elustondo, F., Chang, J., Temple, J., Ahmed, A. A., Brenton, J. D., Downward, J., and Nicke, B. (2007) Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs, Cancer cell 11, 498-512. (14) Normand, G., and King, R. W. (2010) Understanding cytokinesis failure, Adv. Exp. Med. Biol. 676, 27-55. (15) Shi, Q., and King, R. W. (2005) Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines, Nature 437, 1038-1042.



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ACS Chemical Biology (16) Nishimura, K., Watanabe, S., Hayashida, R., Sugishima, S., Iwasaka, T., and Kaku, T. (2015) Binucleated HeLa cells are formed by cytokinesis failure in starvation and keep the potential of proliferation, Cytotechnology.

Figure Legends: Fig. 1. Characterization of STK16-IN-1: A. Chemical structure of STK16-IN-1; B. DiscoveRx KinomeScanTM profiling of STK16-IN-1; C. IP kinase assay of STK16-IN-1 with 4EBP1 as substrate; D. IP kinase assay of STK16-IN-1 with DRG1 as substrate; E. Kinetic study of STK16-IN-1 against STK16 kinase with ATP concentration variation; F. Molecule modeling study of STK16-IN-1 in STK16 kinase (PDB ID: 2BUJ) Fig. 2. STK16-IN-1 anti-proliferation and signaling pathway effect on cancer cells: A. Examination of anti-proliferation efficacy of STK16-IN-1 against a panel of cancer cell lines with 1, 3 and 10 µM by Cell Titer-Glo assay; B. FACS analysis of STK16-IN-1 apoptosis induction effect in MCF-7, HCT-116 and Hela cells; C. IP/IB assay of STK16IN-1 effect on pSTK16(total phosphorylation sites). D. STK16-IN-1’s effect on signaling pathways in MCF-7, HeLa and HCT-116 cells. E. Combination study of STK16-IN-1 with colchicine, paclitaxel, doxorubicin and cisplatin in MCF-7 cells after 72 hours of drug treatment; F. Western blotting detection of apoptotic effect of STK16-IN-1 with colchicine, paclitaxel, doxorubicin and cisplatin combination in MCF-7 cells after 72 hours of drug treatment. Fig. 3. STK16 affects cell division: A. MCF-7 cells were treated with 0, 0.1 µM, 0.5 µM and 10 µM STK16-IN-1 for 72 hours before they were stained with DAPI (blue) and antitubulin (green); B. Quantification of control or 10 µM STK16-IN-1 treated MCF-7 cells in (A) for total cell numbers or binucleated cells from three independent experiments; C. FACS analysis of cell cycle progression in MCF-7, HCT-116 and HeLa cells after 72 hours of STK16-IN-1 treatment; D. MCF-7 cells were treated with 40 nM negative control or STK16 siRNA for 72 hours and blotted for STK16, tubulin and β-actin; E. Immunofluorecence of MCF-7 cells that were treated with control or STK16 RNAi 72 hours and stained with DAPI (blue) and anti-tubulin (green); F. Quantification of (E) for relative cell number (left) and percentage of binucleated cells (right) from two independent experiments. * p

Discovery of a Highly Selective STK16 Kinase ... - ACS Publications

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