Letter pubs.acs.org/jcim
In Search of Novel CDK8 Inhibitors by Virtual Screening Malika Kumarasiri,†,‡ Theodosia Teo,†,‡ Mingfeng Yu,‡ Stephen Philip,‡ Sunita K. C. Basnet,‡ Hugo Albrecht,‡ Matthew J. Sykes,‡ Peng Wang,§ and Shudong Wang*,‡ ‡
Centre for Drug Discovery and Development, Sansom Institute for Health Research, Centre for Cancer Biology, and School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5001, Australia § Yabao Pharmaceutical Group Co., Ltd., Suzhou Industrial Park, Suzhou, China, 215123 S Supporting Information *
conformation.2 This conformation allows access to a deep hydrophobic pocket where the 4-chloro-3-trifluoromethylphenyl moiety of sorafenib binds. In contrast, this pocket is inaccessible in the active conformation (DMG-in), where the Met174 side-chain is reoriented to make the site available to ATP or a competitive inhibitor. Typically, the inhibitors that bind to DMG-in and DMG-out conformations are termed type I and type II, respectively. The structural difference of CDK8 between the active and inactive states explains the structural diversity of its inhibitors. Among the few known selective CDK8 inhibitors, several are sorafenib analogs that contain an aryl urea core and are type II.3,4 The Senexin-type and the newer CCT-type compounds, which possess 4-aminoquinazoline and 3,4,5-trisubstituted pyridine scaffolds, respectively, are type I.3,5,6 Additionally, Cortistatin A, a natural product with high selectivity, is worth mentioning.7 The high structural diversity of selective CDK8 inhibitors raises the possibility of the existence of different scaffolds, even among the drug-like molecules from commercially available compound databases, which may further be structurally optimized into novel and selective inhibitors. We report herein the identification of CDK8 inhibitor scaffolds using two complementary virtual screening cascades, followed by a series of biological and cellular assessments of top ranking compounds. From 127 virtual screening hits, 33 were confirmed as moderate to potent CDK8/CycC inhibitors by enzymatic testing. Subsequently, 16 molecules were shown to exert antiproliferative effects on colorectal cancer cell lines. Finally, Western blotting analysis was used to demonstrate that three hits target CDK8 in HCT 116 colorectal cancer cells. The results showcase the successful application of virtual screening cascades to identify CDK8-targeted scaffolds that can be developed into a drug discovery program.
ABSTRACT: Aberrant activity of cyclin-dependent kinase (CDK) 8 is implicated in various cancers. While CDK8targeting anticancer drugs are highly sought-after, no CDK8 inhibitor has yet reached clinical trials. Herein a large library of drug-like molecules was computationally screened using two complementary cascades to identify potential CDK8 inhibitors. Thirty-three hits were identified to inhibit CDK8 and seven of them were active against colorectal cancer cell lines. Finally, the primary target was confirmed using three promising hits.
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INTRODUCTION Cyclin-dependent kinases (CDKs) are a family of key regulatory proteins that oversee diverse cellular events, and their main involvement is in the cell cycle and transcription. CDK function generally depends on partnering with various cyclins (Cycs), and currently about 20 CDKs and 29 Cycs are known.1 Given the fundamental biological roles CDKs perform, it is not surprising that their aberrant activities are a common feature of many diseases, especially cancer. CDK8, an atypical, primarily transcriptional CDK family member, has recently attracted considerable attention due to recognition of its key roles in oncogenesis (see CDK8 Biology in the Supporting Information). Thus, its selective pharmacological inhibition offers a novel therapeutic strategy for the treatment of various solid and blood cancers. The hunt for selective CDK8 inhibitors has commenced only recently, and to date none have progressed to clinical trials. However, several multikinase inhibitors, including the FDAapproved drug sorafenib, are known to inhibit CDK8. A CDK8 X-ray crystal structure with bound sorafenib has been resolved, with the kinase in an inactive state, where a common feature is the rotation of the D173M174G175 motif. This rotation positions the Met174 side-chain to a location that assists in obstructing the ATP binding pocket; hence called the DMG-out © 2017 American Chemical Society
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RESULTS AND DISCUSSION Detailed computational and biological methods are provided in the Supporting Information. Virtual Screening. The objectives behind the design of two virtual screening cascades were to capture compounds with scaffolds similar to those of known type I and type II CDK8 inhibitors, and to explore novel and diverse scaffolds using the OpenEye and Schrödinger suites. We have previously utilized similar virtual screening protocols successfully to filter large compound libraries.8,9 Here we observed that the Glide XP Published: February 13, 2017 413
DOI: 10.1021/acs.jcim.6b00711 J. Chem. Inf. Model. 2017, 57, 413−416
Letter
Journal of Chemical Information and Modeling
Figure 1. Binding modes of typical type I (hit 1, A) and type II (hit 29, B) hits to CDK8. Backbones of the CDK8 homology models are depicted as white cartoons with the hinge region in yellow. The inhibitor carbon atoms are colored green. Hydrogen bonds are in black dashed-lines, and hydrophobic and cation−π interactions are depicted in orange. The interactions d1 and d2 are common to both types while d3−5 are unique.
good agreement with reported values of 0.280 μM for Senexin A and 0.199 μM for sorafenib.5,6 The above identified 9 hits were further evaluated using the ADP-Glo CDK8/CycC assay. The obtained kinase activity data at 1 μM were comparable to the previous 33PanQinase data (Table S1). The only exception was hit 1, which was found to be the most potent CDK8 inhibitor using the radiometric assay (−6.0% at 1 μM), while only moderately inhibited CDK8/ CycC with a residual kinase activity of 25.3% at 1 μM in the ADP-Glo assay. We note that the distinct technologies used in the two assays, luminescence and radiometry, significantly limit the possibility of having pan assay interference compounds (PAINS).10 Nonetheless, the hits in Table S1 were screened for PAINS, using 480 filters, and were cleared. The subsequent 10point dose−response study further confirmed that hit 1 has moderate inhibitory potency against CDK8/CycC (IC50 = 0.386 μM). In contrast, the other eight candidates exhibited much higher potency toward CDK8/CycC with IC50 values between 0.018−0.184 μM. We also compared these nine hits with known CDK8 inhibitors listed in ChEMBL. The 422 ChEMBL entries we encountered exhibited 0.148−0.377 similarity scores to our hits (Table S1). Cell Viability Assay. To investigate the sensitivity of our hits to CDK8-overexpressing colon cancer cells, the 127 hits were tested against three colorectal cell lines, Colo 205, HCT 116 and HT-29. These cell lines were previously reported to express high levels of CDK8 in addition to other mutations involved in the stimulation of the β-catenin signaling pathway (Table S2).3,11,12 Cells were treated with the compounds at a range of concentrations for a period of 72 h followed by cell viability determination. The compounds with GI50 < 15 μM are summarized in Table S3, and data for nine CDK8 inhibitors and two comparators (Senexin A and sorafenib) are presented in Table S1. Senexin A displayed little antiproliferative activity against all three cell lines with GI50 values greater than 15 μM, while sorafenib inhibited cell growth at low micromolar concentrations (GI50 = 4.80−5.65 μM). Among the 127 hits, 16 showed good antiproliferative activity against at least two colon cancer cells (GI50 = 0.75−11.3 μM) (Tables S2), and 7 of them were potent CDK8 inhibitors (Table S1). Cellular CDK8 Inhibition. CDK8 phosphorylates interferon-γ (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) at the transactivation domain, specifically at Ser727.13 To confirm the cellular CDK8 inhibitory activity of the active hits, hits 1, 29, and 47 (three
refinement scores for type I inhibitors were significantly lower than those for type II. Therefore, a cutoff of −14.00 was set for the top-ranking type I hits and −9.5 for type II. This resulted in ∼5000 type I and II hits from the biased screening cascade and ∼3000 from the unbiased. The lower scores of type I inhibitors however did not translate into more potent CDK8 inhibitory activities, compared to type II, as reported by the CDK8 kinase assay (vide inf ra). A significant overlap was observed between the top scoring compounds from each cascade. This was expected as both routes used the same two homology models for docking. Nonetheless, upon removal of duplicates, 103 type I and 373 type II inhibitors were obtained. Several classes of scaffolds dominated the results. Upon visual inspection of compounds and scaffold classes, we procured 127 for biological testing. Binding Mode Analysis of Type I and Type II Hits. Figure 1 depicts the typical binding modes of active type I and type II hits. Compound 6-fluoro-N-(2-fluorophenyl)quinolin-4amine (hit 1) demonstrates a common binding mode that is often observed by ATP-competitive kinase inhibitors. It forms a 2.1 Å hydrogen bond with the hinge region at the carbonyl of Ala100 (d2) and a π−π stacking interaction (d1, 4.0 Å) with the gatekeeper Phe97 of CDK8. In addition, a weak cation−π interaction can be seen between the guanidinium of Arg356 and the phenyl π-electron cloud of hit 1 (d3, 4.4 Å). In contrast, 1-(3,4-dimethylphenyl)-3-(4-(pyridin-4-ylmethyl)phenyl)urea (hit 29) binds as a type II inhibitor, forming two additional hydrogen bonds through the urea moiety: one with Asp173 of the DMG motif (d5, 2.1 Å) and a bifurcated one with Glu66 (d4, 1.9/2.1 Å). It also maintains similar interactions to hit 1 with Ala100 (1.9 Å) and Phe97 (4.4 Å). The hydrophobic 3,4dimethylphenyl moiety of hit 29 projects into the deep hydrophobic pocket in a similar manner to sorafenib. Biochemical Assay. For this study, 127 compounds from the screening cascades were purchased and initially assessed for inhibitory activities using 33PanQinase radiometric CDK8/ CycC assay. Here, 33 hits caused moderate to potent CDK8/ CycC kinase inhibition (Figure S1) at 10 μM, whereas 9 were potent inhibitors at 1 μM. To confirm the primary hits, a CDK8/CycC kinase assay was established using the ADP-Glo luminescence-based technology. Its performance was validated by dose−response experiments, which produced IC50 values of 0.183 and 0.133 μM for the reference compounds, Senexin A and sorafenib,2,5 respectively (Table S1). These results are in 414
DOI: 10.1021/acs.jcim.6b00711 J. Chem. Inf. Model. 2017, 57, 413−416
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Journal of Chemical Information and Modeling ORCID
representative scaffolds) were chosen, while sorafenib served as a positive control. Each compound was incubated with HCT 116 colorectal cancer cells at 7 or 28 μM in the presence of IFN-γ for 1 h. Western blotting analysis demonstrated that all four compounds significantly reduced the level of STAT1S727 phosphorylation at GI50 concentration (i.e., 7 μM), (Figure 2), thus confirming the inhibition of CDK8 in HCT 116 cells.
Hugo Albrecht: 0000-0002-3951-1866 Matthew J. Sykes: 0000-0002-2509-5531 Shudong Wang: 0000-0001-6225-5525 Author Contributions †
M.K. and T.T. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Yabao Pharmaceutical Group Co., Ltd., China. M.J.S. acknowledges the receipt of a no-cost academic licence from OpenEye Scientific Software Incorporated.
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Figure 2. Effects of hit compounds and sorafenib on STAT1 S727 phosphorylation in HCT 116 cells by Western blotting analysis. Cells were treated with 7 or 28 μM compound for 1 h. The protein molecular weight marker is indicated on the right side of the panel. GAPDH was used as an internal loading control. A representative blot was selected from at least four independent repeats (top). A graphical representation is shown of the ratio of p-STAT1S727 to its parental protein after normalization with GAPDH (bottom, * indicates p < 0.05).
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CONCLUSIONS In this work, several CDK8 inhibitors containing a variety of scaffolds have been identified from a library of more than 2 million drug-like compounds, using two complementary virtual screening cascades. The antiproliferative activities of the compounds against colorectal cancer cell lines were observed. Inhibition of CDK8 by key compounds was further confirmed in HCT 116 cells. The chemical scaffolds of the CDK8 inhibitor hits can further be optimized for potency, selectivity, and pharmaceutical properties, and offer unique opportunities to develop CDK8-targeted anticancer therapeutics.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00711. Detailed experimental methods. Figure S1 and Tables S1−S3 showing biological results. Additional references (PDF)
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REFERENCES
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