Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to

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Discovery of novel polo-like kinase 1 polo-box domain inhibitors to induce mitotic arrest in tumor cells Tan Qin, Fangjin Chen, Xiaolong Zhuo, Xiao Guo, Taikangxiang Yun, Ying Liu, Chuanmao Zhang, and Luhua Lai J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00261 • Publication Date (Web): 16 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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Journal of Medicinal Chemistry

Discovery of novel polo-like kinase 1 polo-box domain inhibitors to induce mitotic arrest in tumor cells Tan Qin, 1 Fangjin Chen, 1, 2 Xiaolong Zhuo, 3 Xiao Guo, 3 Taikangxiang Yun 2, Ying Liu1, Chuanmao Zhang, 3,* Luhua Lai.1, 2,*

1

BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College

of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China 2

Center for Quantitative Biology, Peking University, Beijing, 100871, China

3

The Ministry of Education Key Laboratory of Cell Proliferation and Differentiation and the State

Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing, 100871, China

*Correspondence: [email protected]; [email protected]

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ACS Paragon Plus Environment


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Abstract Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anti-cancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Though several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 µM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

Key Words: Polo-like kinase 1, polo-box domain, anti-cancer, virtual screening, microscale thermophoresis.

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Introduction Polo-like kinase 1(Plk1) is an evolutionarily conserved serine/threonine kinase which plays essential role in cell mitosis.1-4 Plk1 participates in all phases of mitosis and regulates cell events such as mitotic entry, centrosome maturation, spindle assembly and cytokinesis.2, 5 Dozens of proteins including Cdc25, TCTP, and Myt1 are known substrates of Plk1 phosphorylation.6 Plk1 is a validated target for anti-cancer therapy.7, 8 Over-expression of Plk1is observed in various human tumors.7 Inhibition of Plk1 using antisense oligonucleotides9 or chemical compounds10,

11

causes the cultured cancer cell mitosis arrest and subsequent apoptosis. Plk1 is also verified as a selective anti-cancer target by induced-knockdown mouse model.12 To date there are several Plk1 ATP competitive inhibitors in clinical trial.13-16

Full length Plk1 consists of two domains, the N-terminus kinase domain (KD)17 and the C-terminus polo-box domain (PBD).18 PBD, which is featured for Plk family, binds to specific phosphoepitope and determines the subcellular localization of Plk1.19, 20

Crystallography studies reveal that the substrate phosphopeptide binds to the cleft

in PBD formed by two small polo box domains.21-23 Chemical compounds targeting PBD cause mislocalization of Plk1 in cell and induce mitotic arrest of the cultured cancer cells.24-26 Distinct from the mono-polarity phenotype caused by Plk1 ATP competitive inhibitors in cell,10 Plk1 PBD inhibitors induce multi-polarity and chromosome misalignment of the cultured cells.24 The abnormal cell cycle progression caused by Plk1 PBD inhibitor leads to the death of the treated cancer cells,25 indicating that Plk1 PBD inhibitors can be developed into anti-cancer drugs. 3



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Furthermore, targeting Plk1 PBD helps avoid the selectivity issue of ATP competitive inhibitors resulting from the conservative structure of kinase ATP binding site, since the PBD is featured only for Plk family.

In previous studies, several low molecular weight compounds including thymoquinone (TQ) 26, Poloxin25 and green tea catechins27 were identified as Plk1 PBD inhibitors (Figure 1). However, the structural diversity was still limited for Plk1 PBD small molecular inhibitor development. Moreover, certain chemical groups in TQ, Poloxin and green tea catechins made them pan assay interference compounds (PAINS), compromising their use as lead compounds for Plk1 drug design.28 Furthermore, most of the known Plk1 PBD inhibitors suffered from the loss of activity when applied in cell, especially for the PBD peptide substrate derivative inhibitors. In some cases, large amount of modifications were made to improve the bioactivities of Plk1 PBD inhibitors.11, 29, 30

In the present study, we aimed to develop Plk1 PBD inhibitors with new chemical scaffolds and improved bioactivity in cell. Through a combined virtual and experimental screening approach, we identified 10 small molecules with diverse scaffolds to induce mitotic arrest of HeLa cells and bind to Plk1 PBD in vitro. These inhibitors represent new scaffolds for future development of drugs against Plk1 PBD.

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Figure 1. Structures of thymoquinone, Poloxin, (-)-epigallocatechin (EGC), D84 and D116. Compound D84 and D116 are inhibitors discovered in the present study.

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Results Virtual screening In order to develop Plk1 PBD inhibitors, we first used virtual screening to search for compounds that potentially bind to PBD from the SPECS compound library (November 2011 version of Specs_SC_10mg, ~200,000 compounds). The X-ray crystallography structure of Plk1 PBD with the minimal phosphopeptide PLHSpT (PDB ID: 3HIK) as the ligand was used for docking. The structure was chosen for that PLHSpT is part of the natural substrate of Plk1 yet with a relatively low molecular weight like small molecules. Meanwhile the PBD structures with different ligands were compared. The shapes of pocket and the rotamers of key residues are very similar in multiple Plk1 PBD-ligand complexes. The binding pocket of PBD substrate was delimitated with Cavity 2.031 and used for docking studies. Scheme 1 shows the virtual screen scheme used in this study. After screening the SPECS library using DOCK632 and then AutoDock4,33 five thousands compounds were kept and further evaluated with PSCORE.34 Then binding conformations of 562 compounds with the lowest estimated Kd were exported and manually evaluated according to the following criteria: (1) forming H-bonds or electrostatic interactions with PBD key residues His538, Lys540 or Trp414, side-chain or main-chain; (2) forming good hydrophobic interactions within the binding site consists of residues Trp414, Leu491 or Phe535. After this step, 81 compounds were selected and purchased for flow cytometry and immunofluorescence microscopy testing.

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Scheme 1. Steps of virtual and cellular screening for Plk1 PBD inhibitors. PBD binding site was shown in grey shadow. Key residues for Plk1 PBD substrate binding His538, Lys540 and Trp414 were shown in orange sticks.

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Table 1. Inhibition strength of the active compounds. The half effective constants (EC50) for HeLa cell proliferation measured by FACS and dissociation constants of the compounds from Plk1/Plk2/Plk3 PBD given by MST assay were listed. Compounds

Chemical Structure

O2N

D84

EC50 of cell assay (µM)

Kd to Plk1 PBD (µM)a

Kd to Plk2 PBD (µM)

Kd to Plk3 PBD (µM)

21.1 ± 1.5

57.8 ± 6.4

159 ± 65

n.d.

25.6 ± 14.1

44.2 ± 4.8

197 ± 79

n.d.

n.d.b

34.7 ± 2.5

n.d.c

n.d.

S N H

N H

O S

N O

O O

O

D88

O

N H

N

Br O N

D96 HN N

N H

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Cl O N

D109

N

N H

Cl

Cl N H

Cl

N

O

210 ± 96

n.d.

4.4 ± 1.7

46.7 ± 4.3

n.d.

n.d.

8.6 ± 1.4

83.9 ± 11.5

n.d.

n.d.

S N H

D208

68.1 ± 3.9

N

O

D116

20.4 ± 33.0

N

Cl

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N

NO2 O

D222

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N H

N

5.3 ± 3.6

66.1 ± 5.3

n.d.

n.d.

n.d.

26.8 ± 4.1

n.d.

n.d.

18.7 ± 1.6

65.9 ± 10.1

n.d.

n.d.

N O N H

D225

Cl O S

D254

N

N N H

10


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O

O S

D278

S N H

O

O O

63.8 ± 11.3

n.d.

n.d.

35.7 ± 2.0

87.3 ± 11.5

296 ± 103

23.0 ± 3.6

n.d.

n.d.

36 ± 2 % of 4N

O

Poloxin

44.8 ± 2.9

O

N

DNA cells at O

100µM

PLHSpT

n.d.

a

MST data shown represent the mean ± SEM (n = 3).

b

n.d., for cell assay, not determined for data did not fit Hill1 equation.

c

n.d., for MST assay, not determined because of weak binding.

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Cellular assay testing Before testing the inhibition of the purchased SPECS compounds to Plk1 PBD in

vitro, we first investigated the anti-proliferation effect of the compounds in HeLa cells using flow cytometry, based on the fact that inhibition of Plk1 PBD causes mitotic arrest during the cell cycle progression. Through quantitative analysis of the percentage of cells in G0/G1, S and G2/M phases by fluorescence-activated cell sorting (FACS), we identified 10 (out of 81) compounds that arrested HeLa cells in G2/M phase (Figure 2A) in the first round screening. These compounds increased the proportion of 4N DNA HeLa cells (refer to G2/M cells) to more than 50% at screening concentrations. While the 4N DNA cells proportion of DMSO treated HeLa cells was less than 25%. Some of the active compounds, including D96, D116 and D222, significantly increased the proportion of 4N DNA HeLa cells at low concentrations of 5 µM or 10 µM. Further analysis showed that most of the active compounds induced the accumulation of mitotic HeLa cells in a dose-dependent manner (Figure 2B, Supplementary Figure S1, S2), which was a primary phenotype expected for Plk1 inhibition in cell. The EC50 values of the active compounds to induce HeLa cell mitotic arrest were listed in Table 1. The most potent compound D116 induced the accumulation of 4N DNA HeLa cells with an EC50 of 4.4 ± 1.7 µM. These data indicate that we have obtained compounds through first round of virtual and cell screening for Plk1 PBD inhibitors that strongly block HeLa cells in mitosis.

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Figure 2. Cellular assay testing. (A) Quantification analysis of the cell-cycle distribution induced by screened compounds and DMSO. (B) Dose-dependent FACS analysis of D84.

Binding test To characterize the cellular active compounds targeting Plk1 PBD in vitro, we used the microscale thermophoresis (MST) method35, 36 to measure the binding affinity of the compounds to Plk1 PBD. The previously reported Plk1 PBD ligand Poloxin and the phosphopeptide PLHSpT were used as positive controls. The dissociation constants (Kd) of Poloxin and PLHSpT to Plk1 PBD measured by MST were 35.7 ± 13



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2.0 µM and 23.0 ± 3.6 µM, respectively. These data were comparable with the results of fluorescence polarization assay24 or enzyme linked immunosorbent assay (ELISA)37 experiments before. The dissociation constants (Kd) of the cellular active compounds with Plk1 PBD measured by MST are shown in Table 1. Kds of these compounds binding to Plk1 PBD varied from ~20 to ~100 µM. To further investigate the selectivity of these Plk1 PBD inhibitors, we tested the binding affinity of these compounds to Plk2/Plk3 PBD. Plk2 and Plk3 are Plk family members with sequence similarity of 67.9% and 64.8% in PBD region with Plk1, respectively. A recent study revealed that Plk2 promotes the survival of human tumor cells,38 whereas Plk3 has been proposed as a tumor suppressor.39, 40 MST experiments showed that most of the tested compounds showed no or weak binding to Plk2/Plk3 PBD with Kd much weaker or undeterminable under working condition, indicating the selectivity of these Plk1 inhibitors over Plk2 and Plk3 (Table 1, Supplementary Figure S3). These data also suggest that the newly discovered Plk1 PBD inhibitors (except for D254 and D278) exhibit very low inhibition effect on the tumor suppressor Plk3. We further tested the binding of the compounds to Plk1 using surface plasmon resonance (SPR) technique. The SPR data showed that key compounds including D84, D116 and D222 bind to Plk1 with Kds of ~ 10 µM (Supplementary Figure S4). We also tested the inhibition of the compounds in fluorescent polarization (FP) assay. Most of the compounds (except for D84) do not show significant activity in FP (data not shown). This is similar to the small phosphopeptide PLHSpT, which showed much weaker binding in FP assay (IC50 > 100 µM and could not be determined due to weak binding) 41 compared to other methods including ELISA37 and MST, ITC21. These compounds may not occupy the entire binding space of the probe peptide in FP, therefore causing limited reduction of the polarization. Binding kinetics is another 14


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issue, as compounds with slow binding and dissociating behavior like Poloxin and TQ26, are more potent in FP assay. All of the inhibitors were subjected to the pan assay interference compounds (PAINS) online filter (http://cbligand.org/PAINS/).28 PAINS analysis showed that nine of the ten active compounds passed the filter, the exception being compound D225.

Binding analysis To further characterize the binding modes of the inhibitors, we designed competitive experiments to examine the binding behavior of testing compounds in presence of the phosphopeptide PLHSpT. If the compound and PLHSpT competitively binds to the substrate site of Plk1 PBD, the presence of PLHSpT would impair the binding ability of the PBD inhibitor. By adding 100 µM PLHSpT to the PBD-inhibitor incubation solution, the MST binding amplitude of all testing compounds significantly decreased (Figure 3A, 4A, Supplementary Figure S5). For most of the Plk1 PBD inhibitors, the binding amplitude in the presence of PLHSpT dropped to less than half of the non-competitive signals. These results suggested that these inhibitors competed with the PBD ligand PLHSpT. Furthermore, we conducted mutation studies to testify the key residues predicted for PBD inhibitor binding. According to the docking analysis, PBD residues His538, Lys540 and Trp414, which were also key residues for PBD substrate binding, formed polar contacts or hydrophobic interactions with most inhibitors we identified. We then measured the binding affinity of the inhibitors to PBD mutants H538A, K540A and W414A. Circular dichroism (CD) studies showed that these PBD variants maintained good secondary structures after mutation (Supplementary Figure S6). In MST assay, the binding signals of tested compounds to these PBD variants significantly decreased 15



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compared to the wild type PBD (Table 2, Figure 3B, 4B, Supplementary Figure S5). These decrements in binding affinity supported the conformers predicted by docking analysis. Based on the docking study and experimental results, we proposed binding conformations of the tested compounds (Figure 3C, 4C, Supplementary Figure S7). The docking analysis gives two major predicted binding modes of the inhibitors. In the first mode, at least one main-chain H-bond is formed between PBD and the inhibitor, which is very similar to the phosphopeptide PLHSpT.21 In the other mode, no main-chain H-bonds were formed (D116 and D222). These predictions were supported by the binding testing the the compounds with the binding site mutants.

Table 2. Mutation effects on the binding strength of PBD inhibitors.

a

WT PBD (µM)

H538A (µM)

K540A (µM)

W414A (µM)

Poloxin

35.7 ± 2.0

n.d. a

n.d.

408 ± 16

D84

57.8 ± 6.4

n.d.

n.d.

n.d.

D88

44.2 ± 4.8

n.d.

n.d.

412 ± 27

D96

34.7 ± 2.5

n.d.

n.d.

n.d.

D109

68.1 ± 3.9

n.d.

339 ± 16

n.d.

D116

46.7 ± 4.3

n.d.

n.d.

n.d.

D208

83.9 ± 11.5

n.d.

251 ± 14

n.d.

D222

66.1 ± 5.3

n.d.

n.d.

n.d.

D225

26.8 ± 4.1

n.d.

n.d.

n.d.

D254

65.9 ± 10.1

418 ± 11

n.d.

n.d.

D278

63.8 ± 11.3

n.d.

n.d.

n.d.

n.d.: not determined because of weak binding. 16


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Figure 3. Binding analysis of D84. (A) Competitive binding of D84 and PLHSpT to Plk1 PBD in MST assay. (B) Mutational effects of Plk1 PBD to binding strength of D84 in MST experiments. (C) Predicted binding mode of compound D84 to Plk1 17



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PBD. Key residues from PBD were shown in green sticks and the predicted H-bonds were shown in blue dashes.

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Figure 4. Binding analysis of D116. (A) Competitive binding of D116 and PLHSpT to Plk1 PBD in MST assay. (B) Mutational effects of Plk1 PBD to binding strength of D116 in MST experiments. (C) Predicted binding mode of compound D116 to Plk1 PBD. Key residues from PBD were shown in green sticks and the predicted H-bonds were shown in blue dashes.

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Cancer cell line inhibition To evaluate the anti-cancer activity of the Plk1 PBD inhibitors, we used MTT assay to test the inhibition of these compounds to the cancer cell lines including HeLa, RKO and U2OS cells. These cell lines were chosen because they have over-expression of Plk1. Results showed that most compounds induce dose-dependent inhibition on cell growth (Supplementary Figure S8). The EC50 values of the Plk1 PBD inhibitors against cancer cell lines were listed in Table 3. The most potent compound D96 inhibits the viability of HeLa cells with EC50 of 2.6 µM. These evidences showed the anti-cancer potency of the Plk1 inhibitors.

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Table 3. Inhibition of the active compounds to cultured cancer cell lines. The half effective constants (EC50) for HeLa, RKO and U2OS cell survival rates measured by MTT assay were listed.

a

HeLa cell (µM)

RKO cell (µM)

U2OS cell (µM)

D84

37.3 ± 2.6 a

24.1. ± 5.5

30.4. ± 2.7

D88

40.2 ± 1.6 % inhibition at 100 µM


37.8. ± 10.6

D96

2.6 ± 0.3


59.8 ± 2.4 % inhibition at 20 µM.

D109


19.2 ± 1.0


D116

15.6 ± 4.2

10.0 ± 1.5


D208

18.8 ± 3.3

37.8 ± 11.9


D222

15.0 ± 3.3



D225



18.1 ± 1.0

D254




D278




Data shown represent the mean ± SEM (n = 3).

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Discussion Plk1 is a validated target for cancer therapy. Targeting the PBD of Plk1 as an approach to inhibit cancer cell proliferation has been investigated. However, most of the known Plk1 PBD inhibitors showed low or no activity in cell assays. Peptide analogs as Plk1 PBD inhibitors possessed high binding strength in vitro, yet suffered from substantial activity loss in cell.37, 41, 42 In previous reports, the most potent small molecular Plk1 PBD inhibitor to induce cell mitosis arrest was Poloxin-2, an inhibitor modified from Poloxin, with an EC50 of ~ 15µM in HeLa cells.11 In the present study, we used cellular assays to find active compounds after the virtual screening step, which were then studied by in vitro binding experiments. Using this approach, we identified 10 inhibitors that induced mitosis arrest of HeLa cells in single digit micromolar concentrations, among which the strongest inhibitor (D116) has an EC50 of 4.4 µM. The cellular active inhibitors contain novel chemical scaffolds. The inhibitors include derivatives of benzimidamide (D84), acrylamide (D225), carboxylate (D278) and hydrazide (D116 and other compounds). Among these inhibitors, the derivative of benzimidamide D84 was the least cytotoxic, while the derivatives of hydrazide (D116 and other compounds) were most potent in cell. Furthermore, these newly discovered inhibitors (except for D225) passed the PAINS filter.28 The previously reported Plk1 PBD inhibitors Poloxin and thymoquinone (TQ) contain the PAINS structure quinone, which is considered problematic because of the protein reactivity,11 signaling assay interference and cytotoxicity issue.28 The green tea catechins27 were also identified as PAINS PBD inhibitors for being epoxides. The newly discovered non-PAINS inhibitors therefore serve as more suitable leads for tool compounds development.

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In the present study, MST was used to validate the binding abilities of Plk1 PBD inhibitors in vitro. The MST signal altitude reveals the difference between thermophoresis of bound and unbound protein, which mostly affected by molecule size, charge and hydration shell.35, 36 Fitting of the unsaturated MST curves was based on the hypothesis that thermophoresis signals are close between similar PBD-ligand complexes. MST method applies to direct and competitive binding measurements of the compounds to PBD, also the mutational analysis. In previous studies, fluorescent polarization (FP) assay was the most frequently used assay for PBD inhibitors. FP assay can only give the competitive strength of PBD inhibitors. It was suggested that FP assay suffered from high false positive rate for Plk1 PBD inhibitor screening.43, 44 In conclusion, we have identified 10 small molecular inhibitors with diverse scaffolds to induce mitotic arrest of HeLa cells in the present study. The EC50 of the strongest inhibitor to induce mitotic arrest of HeLa cell was 4.4 µM. These cellular active inhibitors serve as both lead compounds for anti-cancer development and tools to delineate Plk1 PBD function in cell.

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Materials and Methods Molecular docking targeting Plk1 PBD The crystal structure of Plk1 PBD in complex with PLHSpT (PDB code: 3HIK) was used to identify potential inhibitors of polo-box domain of Plk1. The resolution of the structure is 1.77 Å, the R value free and R value work were 0.237 and 0.205 respectively. Before docking analysis, PBD structure was prepared using Schrödinger software with procedures including remove of the ligand, waters and addition of the polar hydrogens. The phosphopeptide ligand binding pocket of Plk1 PBD was used for the docking studies. The pocket was delimitated with Cavity 2.031 using default settings. The 3D structures of the SPECS library compounds were built with the OMEGA program45 from the Openeye Scientific Software (using default settings). Each compound had 120 conformations generated in average. Rigid body docking was done using DOCK6 with default parameters followed by semi-flexible docking using Autodock 4. For the Autodock 4 docking step, flexible ligand and rigid receptor docking was performed using empirical free-energy function and the GALS algorithm (genetic algorithm with local search); The number of runs was 30; The number of individuals was set as 300; The maximum number of energy evaluations was set as 15,000,000; The maximum number of generations was set as 270,000; The grid box was set as 70 × 70 × 70 points.

Small molecular conformations with the lowest

energies in the largest clusters were selected as final docking poses.

After docking,

the top 562 compounds with predicted KD lower than 600 nM were evaluated manually according to the following criteria: (1) forming H-bonds with PBD-ligand binding key residues His538, Lys540, Trp414, side-chain or main-chain; (2) forming good hydrophobic interaction within the binding site consists of residues Trp414, Leu491 or Phe535. After this step, 81 compounds were selected for further 24


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experimental tests. Ranks of identified hits in virtual screen are given in Supplementary Table S1.

Chemicals Structural information of all identified hit compounds was confirmed using 1H NMR. The purities of most tested compounds were above 95%. The 1H NMR spectrums of all hits were listed in supplementary materials. Reports from the suppliers for all tested compounds such as LC-MS data are available at website http://www.specs.net. Compound Poloxin was purchased from MedChem ExpressTM with purity above 98%. Phosphopeptide PLHSpT was synthesized by GL Biochem Ltd. with purity above 95%.

Plasmids and antibodies Human Plk1 DNA was cloned from a cDNA library by RT-PCR and confirmed by sequencing (Plk1, NM_005030). cDNA encoding Plk1was first subcloned into pEGFPC1 vectors. cDNA of Plk1 PBD (Plk1 327-603), Plk2 PBD (Plk2 355-685) and Plk3 PBD (Plk3 335-646) were subcloned into pGEX-6p-1vector respectively for expression. All Plk1 PBD mutations were introduced into wild type constructs by PCR and the mutation sites were confirmed by sequencing. Both mouse anti-α-tubulin and anti-γ-tubulin were purchased from Sigma-Aldrich. Anti-PARP antibody was purchased from BD Biosciences.

Expression and purification of PBD GST-PBD plasmid was transformed into Escherichia coli strain Rosseta (DE3). The GST-tagged-PBD protein variants were expressed as previously described.21 Cells 25



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were collected and stored at –80℃prior to use. Cell pellets were first resuspended and then lysed by homogenization in solution contained 20mM HEPES, pH7.4, 300mM NaCl, 0.1 mM phenylmethylsulfonylfluoride (PMSF) and 0.1% (v/v) β-ME. The cell lysates were clarified by centrifugation at 35000g for 40min. Then the supernatants were applied to glutathione-sepharose column (GE Healthcare), washed with equilibration buffer containing20mM HEPES, pH7.4, 150mM NaCl and 0.1% (v/v)β-ME. The GST-tagged PBD variants were cleaved on column using PreScission protease (1:50) at 4 ℃overnight. The cleaved protein was eluted in equilibration buffer and applied to S200 gel-filtration column. The PBD variants were then eluted and concentrated in gel-filtration buffer containing 20mM HEPES, pH7.4,150mM NaCl and 0.1% (v/v) β-ME, and stored at –20℃ with 50% (v/v) glycerol prior to MST experiments.

Cell culture HeLa cells were grown at 37℃ in a 5% (v/v) CO2 atmosphere in DMEM (Gibco), supplemented with 10% (v/v) fetal bovine serum (HyClone Laboratories Inc.), 100 U mL-1 penicillin and 100 µg mL-1 streptomycin (Invitrogen). Cells were synchronized at G1/S phase by a 250 µM thymidine(Sigma) block. Testing compounds was dissolved in DMSO at a concentration of 10mM and added to the culture media. In the control population, DMSO was added instead.

Analysis of cell cycle progression For the cell-cycle analysis, cells were seeded onto 6-well plates (3×104cells/well) and incubated at 37℃ in a 5% (v/v) CO2 atmosphere overnight. After being incubated with DMSO or various concentrations of testing compounds for 24 hr, all cells were 26


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detached with trypsin and collected by centrifugation at 600×g for 5 min. The final concentration of DMSO was 0.1% (v/v). The cells were washed twice with PBS, fixed in 70% (v/v) ethanol-PBS for 2 h at 4℃, then washed with PBS and collected by centrifugation. Cells were resuspended with 400 µl PBS containing 100 mg mL-1 RNase, 1% (v/v) Triton X-100, and 0.5mg mL-1 propidium iodide (PI) and incubated for 30 min at 37℃. The suspensions were then analyzed by Becton Dichinson FACScan (BD Biosciences, San Jose, CA). For the determination of mitotic indexes, the number of mitotic cells treated as described below under “Immunofluorescence microscopy” within a population of 200–300 cells was counted.

Immunofluorescence microscopy HeLa cells were grown on glass coverslips and fixed with –20℃methanol for 5 minutes. Fixed cells were incubated with the appropriate primary antibodies overnight at 4℃, washed with PBS, and then incubated with the fluorescently labeled secondary antibodies for 1 hour at room temperature. The coverslips were mounted with mowiol (Sigma-Aldrich) containing 1 µg mL-1 DAPI (Sigma-Aldrich) for DNA staining. Cells were observed using a×63 oil objective on an Axiovert 200M microscope (Zeiss) and the Axiovert software was used for acquisition and analysis.

Microscale thermophoresis (MST) analysis The MST experiments were conducted with the Nanolith NT1.05 instrument (NanoTemper Technologies, Munich) in buffer containing 20 mM Tris (pH 8.0), 200mMNaCl, 0.1% (v/v) TWEEN-20, 1 mM EDTA and 0.1 mg mL-1 BSA. Plk1 PBD was labeled using the Lys labeling kit for detection in the MST experiments with the red fluorescent dye NT-647. For tested compounds, a serial dilution was set up, 27



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starting with a concentration of 100–500µM (according to the solubility of each compound), with a 2-fold dilution down to 0.05– 0.25 µM. The concentration of PBD was adjusted based on the fluorescent counts reported from the instrument with a typical range of 1200–1500 counts at 20% LED power. The tested compounds dilution series and PBD solution (incubated with PLHSpT or not) were mixed 1:1 to obtain the final measurement samples. The final concentration of DMSO was 2.5% (v/v) for each sample. Each sample was centrifuged at 13000 rpm for 5 min before filled into capillaries. The measurements were conducted using the Temperature Jump analysis mode with 20% of the instrument MST power. The dissociation constant (Kd) of the binding was calculated using Kd Formula (law of mass action): f(c) = unbound + (bound-unbound) / 2 * (FluoConc + c + Kd – Sqrt ((FluoConc + c + Kd)2 4*FluoConc*c), which provided by NT Analysis 1.5.41 software, Nanolith.

Surface plasmon resonance (SPR) analysis The binding affinity of tested compounds to Plk1 was analyzed using SPR Biacore T200 instrument. Plk1 was immobilized on a CM5 sensor chip through standard amine-coupling procedure at 25℃ in running buffer contains 10 mM HEPES, pH 7.2, 150 mM NaCl, 0.05% TWEEN-20 and 1 mM EDTA.

~1200 Response Units (RU)

of Plk1 was immobilized on the chip to measure the binding affinity of compounds. Series of compounds dilutions with 5% DMSO in running buffer were injected into the channels. Regeneration was achieved by 1 min washing with the running buffer after injection. The equilibrium dissociation constant Kds of compounds were determined with Hill equation. The SPR dose-response curves of the compounds were shown in Supplementary Figure S4.

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MTT assay For the MTT assay, cells were seeded onto 96-well plates (2×103 cells per well) and incubated at 37℃ in a 5% (v/v) CO2 atmosphere overnight. The cells were incubated with DMSO or various dilutions of tested compounds for 72 h. Discarded the culture medium and then MTT (0.5 mg mL-1 in DMEM) was added to every well at 37℃ in a 5% (v/v) CO2 atmosphere for 4 h. Cells were lysed in lysis buffer (10% (m/v) SDS, 0.01 M HCl) for 2 h at 37℃. The amount of survival cells was determined by measuring the absorbance at the wavelength of 490 nm.

Acknowledgements This work was supported in part by grants from the Ministry of Science and Technology of China (2015CB910300) and the National Natural Science Foundation of China (31430051, 91313302).

Supporting Information Available Table S1 and Figures S1-S7, including calculated docking score and ranks of identified hits in virtual screening, dose−response behavior of compounds, predicted binding modes, circular dichroism spectra of Plk1 PBD variants and NMR spectra of identified hits. Molecular formula strings.

Corresponding Author Telephone: (+86)10-62757486. Fax: (+86)10-62751725. E-mail: [email protected] . 29



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Abbreviations used Polo-like kinase 1 (Plk1); kinase domain (KD); polo-box domain (PBD); microscale thermophoresis (MST); fluorescent polarization (FP); pan assay interference compounds (PAINS); fluorescence-activated cell sorting (FACS); circular dichroism (CD).

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Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to

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