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Optimized Plk1 PBD inhibitors based on Poloxin induce mitotic arrest and apoptosis in tumor cells Andrej Scharow, Monika Raab, Krishna Saxena, Sridhar Sreeramulu, Denis Kudlinzki, Santosh Gande, Christina Dötsch, Elisabeth Kurunci-Csacsko, Susan Kläger, Bernhard Kuster, Harald Schwalbe, Klaus Strebhardt, and Thorsten Berg ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00565 • Publication Date (Web): 17 Aug 2015 Downloaded from http://pubs.acs.org on August 18, 2015

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ACS Chemical Biology

Optimized Plk1 PBD inhibitors based on Poloxin induce mitotic arrest and apoptosis in tumor cells

Andrej Scharow‡1, Monika Raab‡2, Krishna Saxena3,5, Sridhar Sreeramulu3, Denis Kudlinzki3,5, Santosh Gande3,5, Christina Dötsch2, Elisabeth Kurunci-Csacsko2, Susan Kläger4,5, Bernhard Küster4,5, Harald Schwalbe3,5, Klaus Strebhardt*‡2,5, and Thorsten Berg*‡1

1

Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany

2

Johann Wolfgang Goethe-University, Medical School, Department of Gynecology and Obstetrics, Theodor-Stern-Kai 7-9, 60596 Frankfurt, Germany

3

Johann Wolfgang Goethe-University Frankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Max-von-Laue-Str. 7, 60438 Frankfurt

4

Technische Universität München, Emil Erlenmeyer Forum 5, 85354 Freising, Germany

5

German Cancer Consortium (DKTK), Heidelberg, Germany

*Corresponding Authors. E-mail: [email protected], [email protected]

‡

These authors contributed equally

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Abstract Polo-like kinase 1 (Plk1) is a central regulator of mitosis, and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity, and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, whilst avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure-activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells.


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Polo-like kinases are a family of serine/threonine kinases with critical cellular functions.1 Plk1 is the most well-studied member of the family, and has been shown to be a crucial mediator of cell cycle progression and a valid target for tumor therapy.2 As a result of promising clinical results, the FDA has recently granted Breakthrough Therapy designation to volasertib, an ATPcompetitive inhibitor of Plk1. The treatment of patients suffering from previously untreated acute myeloid leukemia (AML) with volasertib in combination with low-dose cytarabine (LDAC) prolonged their survival significantly compared to patients receiving LDAC alone.3 However, activity profiling of the volasertib-related inhibitor BI 2536 in a chemical proteomics approach identified off-target effects of this compound.4 These novel targets of BI 2536 include Death Associated Protein Kinases (DAPK), which are activated and contribute to cell death upon prolonged mitotic arrest. The simultaneous inhibition of DAPK and Plk1 by the ATP-competitive inhibitor BI 2536 was shown to counteract, at least partially, the pro-apoptotic effect of Plk1 inhibition and led to an improved survival of cancer cells. This highlights the need for more specific Plk1 inhibitors which are less prone to off-target effects.

In addition to its enzymatic domain, Plk1 harbors a protein-protein interaction domain known as the polo-box domain (PBD).5,

6

The PBD comprises two conserved motifs referred to as polo-

boxes, and has been reported to bind to peptide motifs bearing phosphorylated side chains of serine or threonine. Early evidence for the important role of the polo-box 1 in cell division came from a study using a hybrid protein encompassing the Plk1 polo-box 1 fused to an Antennapedia peptide for the treatment of cancer cells.7 The PBD is unique to the family of polo-like kinases, with phosphoserine/phosphothreonine binding PBDs having been reported for Plk1, Plk2 and Plk3. Consequently, targeting of the Plk1 PBD has been proposed as a promising strategy for functional inhibition of Plk1.8 This approach circumvents the specificity issues typically associated with targeting the conserved ATP binding pocket of protein kinases.9 ATP-binding domains are found in approximately 500 protein kinases and many other ATP hydrolases, and 3 ACS Paragon Plus Environment


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off-target effects of small molecules are most likely to arise from binding to protein domains with structural or functional similarity to their target protein domain. Therefore, the likelihood that an ATP-competitive kinase inhibitor recognizes any of the other ATP binding domains in the proteome is relatively high. In contrast, an inhibitor of the Plk1 PBD inhibitor has a higher chance of being specific on a proteome-wide level, because there are only two other domains in the proteome with structural and functional similarity to its target domain.

Importantly, small-molecule inhibitors of the Plk1 PBD are also valuable molecular research tools to analyze the role of the PBD for Plk1-mediated processes.1 We recently presented Poloxin as the first small organic molecule shown to functionally inhibit the Plk1 PBD both in vitro and in human tumor cells (Figure 1A).10 Treatment of tumor cells with Poloxin results in mitotic arrest associated with chromosome congression defects, erroneous localization of Plk1, and the induction of apoptosis. Subsequently, purpurogallin,11 Poloxipan,12 and green tea catechins13 were reported as inhibitors of the Plk1 PBD. Several peptide-based agents have been reported,14 the most potent of which gain nanomolar affinities by exploiting a hydrophobic channel in the Plk1 PBD15-20 that had been occluded in the first-reported X-ray structure of a Plk1 PBD-phosphopeptide complex.6 However, their use as Plk1 PBD inhibitors in cells has either not been reported,16-19 or requires high micromolar concentrations.15,

20

Monoanionic

peptide-based reagents have been developed that retain low nanomolar activities against the Plk1 PBD in vitro, yet are more active in live cells than the dianionic species.21 Of all the Plk1 PBD inhibitors reported to date, Poloxin is the only one for which anti-tumor effects could be shown in vivo. Poloxin was demonstrated to lower the proliferation rate and increase the apoptotic rate of tumors in mouse xenograph models.22


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RESULTS AND DISCUSSION In order to develop second-generation tool compounds for the functional inhibition of the Plk1 PBD with higher activities and selectivities, we explored the influence of systematic changes in the structure of Poloxin (1) on the activity of its derivatives. Synthesis was carried out by converting the quinones into the respective oximes, which were acylated to yield the Poloxin derivatives (Figure 1B). Quinones that were not commercially available were synthesized by Co(II)(salen)-catalyzed oxidation of the corresponding phenols.23 The activities of the test compounds against the PBDs of Plk1, Plk2, and Plk3 were determined in binding assays based on fluorescence polarization (Figure 2A).24, 25 A

B O

O N

O

O R R

O

O

N R NH2OH x HCl

R

R

R

OH R

HO R DCC DMAP

R O

O

O or

Cl

R NEt3

N

R O

R

R

R

R O

Poloxin (1)

Figure 1: A) Structure of Poloxin (1). B) General scheme for the synthesis of Poloxin analogs.

As a first step, we explored various substitution patterns on the iminoquinone moiety. Replacement of the 2-isopropyl group of 1 by a methyl group as represented by 2 resulted in a 4-fold increase in activity against the Plk1 PBD, with 10-fold and more than 70-fold selectivity over the PBDs of Plk2 and Plk3, respectively (Table 1 and Figure 2B, C, D). Placement of the two methyl groups in the 2,5-positions (compound 2) was more effective than in the 2,6-positions (compound 3). Deletion of the 5-methyl group as in compound 4 increased activity by more than twofold compared to 1; however, increasing the size of the substituent in the 2-position slightly by changing the isopropyl group for a tert-butyl group (compound 5) led to a drastic decrease in activity. The 2,3,5,6-tetramethyl derivative 6 had a similar activity to 5, and placement of two tertbutyl groups in the 2,6-positions as in compound 7 led to virtually complete loss of activity. These data suggest the existence of a defined protein pocket in the Plk1 PBD which interacts with the iminoquinone moiety. 5 ACS Paragon Plus Environment


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1

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2

General compound structure: R –R

app. IC50 [µM] Plk1 PBD

app. IC50 [µM] [a] Plk2 PBD

app. IC50 [µM] [a] Plk3 PBD

app. IC50 [µM] Plk1 PBD w/ [a] 1 mM DTT

Poloxin (1)

6.4 ± 1.2

19.4 ± 0.8

69.3 ± 3.6

58.2 ± 11.3

2

1.36 ± 0.13

14.2 ± 1.0

49 ± 1% inhibition at 100 µM

42.7 ± 1.9

3

4.63 ± 0.89

n.d.

n.d.

n.d.

4

2.54 ± 0.28

10.7 ± 0.7

41 ± 2 % inhibition at 100 µM

65.8 ± 4.5

5

22.0 ± 1.8

n.d.

n.d.

n.d.

6

23.2 ± 2.1

n.d.

n.d.

n.d.

7

-4 ± 13% inhibition at 80 µM

n.d.

n.d.

n.d.

8

0.74 ± 0.07

8.4 ± 0.8


72 ± 3

9

2.26 ± 0.44

n.d.

n.d.

n.d.

10

1.24 ± 0.38

n.d.

n.d.

n.d.

11

0.81 ± 0.19

34.8 ± 1.4

45.7 ± 3.6

n.d.

12

11.4 ± 1.3

n.d.

n.d.

n.d.

13

0.53 ± 0.04

3.52 ± 0.47

20.5 ± 5.3

23.4 ± 0.3 % inhibition at 100 µM

14

0.60 ± 0.08

2.09 ± 0.10

15.4 ± 8.7

n.d.

15

1.02 ± 0.14

n.d.

n.d.

n.d.

16

3.25 ± 1.17

n.d.

n.d.

n.d.

17

0.47 ± 0.12

1.47 ± 0.15

4.21 ± 0.47

n.d.

18

4.95 ± 0.55

n.d.

n.d.

n.d.

19

70.1 ± 23.1

n.d.

n.d.


Compound

[a]

1

R

2

R

n.d.: not determined

Table 1. Activities of test compounds in fluorescence polarization assays against the PBDs of Plk1, Plk2, and Plk3.


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Figure 2: Activities of Poloxin (1) and new analogs synthesized in this study as analyzed in fluorescence polarization assays. (A) Principle of the assay. A Plk1 PBD-binding peptide (shown in red) which is labeled with a fluorophore (depicted as a green star) is incubated with the Plk1 PBD (shown in light gray), and the fluorophore is excited with linearly polarized light. The emitted fluorescence of the protein/peptide complex has a high degree of polarization owing to its large molecular weight. A functional inhibitor of protein/peptide binding liberates the fluorophore-labeled 6 peptide, leading to a reduction of fluorescence polarization. The structures are based on PDB entry 1UMW. B) Activities of Poloxin (1) and analogs 2, 19–23 against the Plk1 PBD. C, D, E) Activity profiles of compounds 1, 2, and 22 against the PBDs of Plk1, Plk2, and Plk3.

In the second step of the process, we explored variations in the substitution pattern on the aromatic ring in the acyl part of 1. Shifting the methyl group in the phenyl ring from the orthoposition as in 1 to the meta- (8) or para-position (9) increased the potency of the compounds significantly, as did the deletion of the methyl group (10) (Table 1). An ortho-methoxy substituent (11) improved not only activity, but also selectivity over the PBDs of Plk2 and Plk3. In contrast, 7 ACS Paragon Plus Environment


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the trifluoromethyl group (12) did not improve activity as compared to the methyl group contained in 1. Introduction of a fluorine atom was more beneficial in the ortho-position (13) than in the meta-(14) and para-(15) position, although the selectivity of 13 did not reach that of the ortho-methoxy compound 11. Simultaneous placement of a methoxy group and a fluorine in both ortho-positions as in compound 16 was not superior to either monosubstituted compound 11 or 13. The trifluorosubstituted compound 17 displayed good activities, but its selectivity did not match that of the ortho-monofluorinated compound 13.

The structure-activity relationships obtained for substitutions on the aromatic ring indicated that changes which increase the reactivity of the ester group, either by introducing electronwithdrawing substituents, or by altering the ortho-methyl group, increase the activity against the Plk1 PBD. Thus, the main function of the aromatic group might not be to provide binding interactions with the Plk1 PBD itself, but rather to allow the attachment of substituents, which tailor the reactivity of the activated ester group of Poloxin derivatives. To test this hypothesis, we synthesized compound 18 bearing an aliphatic residue (Table 1). 18 displays similar potency to that of 1, indicating that the aromatic ring itself is not strictly required for activity. Finally, we substituted the activated ester group by an acyl hydrazone, which cannot react via protein acylation (compound 19). The strong loss of activity observed for 19 is consistent with the notion that a reactive ester group is required for Poloxin’s activity against the Plk1 PBD (Table 1 and Figure 2B). This suggests protein acylation as a possible mechanism of action of Poloxin and analogs.

To improve the activity of the compounds further, we aimed to combine the most favorable structural variations of the iminoquinone and acyl moiety of the analogs. To this end, we combined the 2,5-dimethyl substitution pattern on the iminoquinone moiety of 2 with the substitutions on the benzoyl group found to lead to the highest activities and selectivities (Table 8 ACS Paragon Plus Environment

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1). Merging the iminoquinone moiety of 2 with the ortho-methoxy benzoyl group of 11, the metamethyl benzoyl group of 8, and the ortho-fluoro benzoyl group of 13, respectively, resulted in compounds 20, 21, and 22 (Table 2 and Figure 2B). Compound 20 is the only compound under investigation that displays higher selectivity against the Plk2 PBD (30-fold) than against the Plk3 PBD (9-fold). Compound 21 is the most selective compound, with 18-fold selectivity over the Plk2 PBD and more than 238-fold selectivity over the Plk3 PBD. Compound 22 is the most active compound identified under the assay conditions (app. IC50 (Plk1 PBD) = 0.31 ± 0.02 µM). It is approximately 20-fold more active than 1 (app. IC50 (Plk1 PBD) = 6.4 ± 1.2 µM) (Figure 2B) and displays more than 7-fold and 59-fold selectivity against the PBDs of Plk2 and Plk3, respectively (Table 2 and Figure 2E). Introduction of a second methyl group which reduces the reactivity of the ester group as exemplified by 23 resulted in 8-fold weaker activity compared to 2 (Figure 2B). Cmpd

Structure




app. IC50 [µM] Plk1 PBD with 1 mM DTT

Loss of activity with 1 mM DTT

Poloxin (1)

6.4 ± 1.2

19.4 ± 0.8

69.3 ± 3.6

58.2 ± 11.3

9-fold

2

1.36 ± 0.13

14.2 ± 1.0


42.7 ± 1.9

31-fold

20

0.59 ± 0.31

17.8 ± 1.1

5.3 ± 0.6

74.9 ± 8.0

127-fold

21

0.42 ± 0.24

7.7 ± 3.1



> 238-fold

22

0.31 ± 0.02

2.32 ± 0.44

18.3 ± 1.8

8±1% inhibition at 100 µM

> 238-fold

23

11.0 ± 0.8

15.8 ± 0.7

63.3 ± 12.4

22.8 ± 2.4

2-fold

Table 2. Activities of test compounds combining the most favorable substitutions of the iminoquinone and the benzoyl moiety in fluorescence polarization assays against the PBDs of Plk1, Plk2, and Plk3.



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The activity data of Poloxin and its analogs were analyzed up to this point in the absence of nucleophiles in the assay buffer. Since the structure-activity relationships described in Table 1 had suggested cleavage of the activated ester to play an important role for compound activity, we investigated the inhibitory potential of Poloxin and selected derivatives in the presence of the nucleophilic reducing agent dithiothreitol (DTT). From the first series of derivatives harboring different substitution patterns on the iminoquinone ring, we chose the most active derivative 2 and derivative 4, as representatives of compounds in which the substituents at the 2,5 positions have been altered (Table 1). From the second series harboring different acyl groups, we chose compound 8 as the most active derivative amongst the methyl-substituted regioisomers 1, 8, and 9, and compound 13, as the most active derivative amongst the monofluorinated compounds 1315 (Table 1). In addition, compounds 20–22 displaying the most favorable structural variations of both parts of the molecule were tested (Table 2). The activity of Poloxin decreased by a factor of 9 in the presence of 1 mM DTT (Figure 3A, B, Tables 1 and 2). Derivatives 2 and 4 suffered greater relative losses in activity than 1, but 2 was still more potent than 1 in the presence of 1 mM DTT (Table 1 and Figure 3C). Analogs 8 and 13 also displayed significant loss of activity, and were less active than 1 in the presence of DTT (Table 1). Similarly, analogs 20, 21, and 22 were less active than 1 in the presence of 1 mM DTT, despite their nanomolar potencies in the absence of DTT. The largest drop in activity was observed for 22, the most active compound in the absence of DTT. These data strongly suggest that the ortho-methyl group protects the ester group of Poloxin analogs from non-specific attack by nucleophiles. Activity gains arising from removal of steric hindrance (by shifting the methyl group) or by increasing the activity of the ester (by introducing electron-withdrawing substituents) may thus not be sustainable in the presence of nucleophiles. Consistent with this mechanistic model, introduction of a second methyl group in the para-position in order to reduce the susceptibility of the ester against nonspecific attack, as exemplified in compound 23, led to the highest activity of all test compounds in the presence of DTT (Figure 3A, D and Table 2). The weakly active control compound 19, 10 ACS Paragon Plus Environment

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which lacks the reactive ester group, lost its remaining activity in the presence of DTT (Table 1), suggesting that the hypothesized addition of thiols to the iminoquinone moiety may also, in part, be responsible for the activity of Poloxin.26 However, overall analysis of the structure-activity conducted in this study is more consistent with protein acylation as the main mechanism of action of Poloxin and derivatives.

Figure 3: Activities of Poloxin (1) and new analogs in the presence of 1 mM DTT in the assay buffer analyzed in fluorescence polarization assays. A) Activities of Poloxin (1) and analogs 2, 20-23 against the Plk1 PBD. B, C, D) Activity profiles of compounds 1, 2, and 23 against the PBDs of Plk1, Plk2, and Plk3.

Analysis of the activities of the compounds against the Plk2 PBD and the Plk3 PBD revealed that the presence of 1 mM DTT in the assay buffer also affected the selectivity profiles of the test compounds. Compound 2 displayed superior selectivity in the presence of 1 mM DTT compared to Poloxin (1), with a selectivity factor of 5 for the Plk1 PBD over the Plk2 PBD, and a selectivity > 5 over the Plk3 PBD (Figure 3B, C, Supplementary Table 1). Compound 23, as the most active compound in the presence of DTT, displayed a selectivity of approximately 2-fold over the 11 ACS Paragon Plus Environment


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Plk2 PBD, and a selectivity of approximately 9-fold over the Plk3 PBD (Figure 3D, Supplementary Table 1).

Recent X-ray structural investigations showed that thymoquinone blocks the phosphoserine / phosphothreonine (pSer/pThr) recognition site of the PBD as phosphate mimetic.27 Soaking experiments attempted with Poloxin found that the oxime resulting from hydrolysis, dubbed Poloxime, also binds to the phosphate recognition site between the two polo boxes.27 To gain further structural insight into the interaction of Poloxin with Plk1, we aimed to investigate this complex by solution NMR. The suitability of the PBD construct used for biophysical studies was investigated by recording 1H,15N-TROSY NMR spectra. The PBD shows a highly resolved spectrum, which indicates that this domain is properly folded (Supplementary Figure 1). Furthermore, addition of the PBD binding peptide MQSpTPL,5 dubbed PBD-tide, induces significant chemical shifts of a large proportion of the amide groups, indicative of binding (Supplementary Figure 1). However, at the high protein concentrations required for the NMR studies, the addition of Poloxin (300 µM) to the PBD (250 µM) induces its protein oligomerization or multimerization (data not shown), rendering the PBD-Poloxin complex inaccessible to structural biology in solution. Size exclusion chromatography (SEC) in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP) confirmed the multimerizing effect of Poloxin on the PBD, as monitored by the time-dependent decrease of the peak for the monomeric PBD, and concomitant increase of the multimer peak (Supplementary Figure 2A, B, C). Of note, Poloxin does not lead to multimerization of the Plk1 kinase domain (Supplementary Figure 2D, E, F) or the N-terminal domain of the protein Cdc37 (Supplementary Figure 2G, H, I).

Analysis of most of the new Poloxin analogs by SEC demonstrated their tendency to induce protein multimerization under the high concentrations of test compounds (300 µM) and protein (50 µM) required for the SEC assays, also preventing their use in solution-based structural 12 ACS Paragon Plus Environment

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biology studies. Comparison of the compounds’ tendencies to induce protein oligomers or multimers as analyzed by SEC, and their activities in fluorescence polarization assays in the absence of DTT revealed no significant correlation between the two assay types (Supplementary Table 2), indicating that protein multimerization is not the underlying reason for the biochemical activities of the compounds in the absence of DTT. This is illustrated by the observation that a low degree of multimerization is displayed by the inactive compound 7 and the poorly active compound 19, but also by the highly active compound 4. Another example is that compounds 2, 4, 10, and 11 are more active than 1, yet induce multimerization either to a lower (compounds 2, 4) or a higher (compounds 10, 11) degree than 1. The biochemical activities of the test compounds in the presence of DTT also do not correlate with the SEC data, as both 2 and 23 are more active than 1, yet exert either a weaker (compound 2) or a slightly stronger (compound 23) effect in the SEC assay (Supplementary Table 2). Therefore, it appears likely that the use of high protein concentrations required for the SEC and NMR studies (50–300 µM) induces unspecific non-covalent and/or covalent binding events, which can alter the multimerization tendencies of the Plk1 PBD. Non-specific protein binding and/or modification is far less likely to occur under the more physiological conditions of the fluorescence polarization assay (carried out at a Plk1 PBD concentration of 15–20 nM), owing to the ~3,000–15,000 fold lower protein concentrations used compared to the SEC and the NMR experiments.

Binding of phosphopeptide ligands to the Plk1 PBD is mediated by three key amino acids: Trp414, His538, and Lys540.6 Mutation of these three amino acids to alanine has a detrimental effect on binding of the PBD to its interaction partners.28, 29 In order to analyze whether the same amino acids are directly involved in binding to Poloxin, we developed a direct binding assay based on fluorescence polarization. To this end, we designed Poloxin derivate 24 (Figure 4A), which is labeled with the fluorophore BODIPY-FL30 in the para-position of the benzoyl group, and synthesized it in a seven-step procedure (Supplementary Figure 3). Incubation of 24 with GST13 ACS Paragon Plus Environment


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tagged, wild-type Plk1 PBD resulted in a time- and dose-dependent increase in fluorescence polarization, which is indicative of protein binding of either the entire molecule, or the fluorophore-containing part, via an irreversible mechanism (apparent Kd after 6 h of incubation = 1.04 ± 0.04 µM) (Figure 4B and Supplementary Figure 4A).

Figure 4: Activities of the BODIPY-FL-labeled Poloxin derivative 24 in direct binding assays based on fluorescence polarization. A) Structure of 24. B) 24 was incubated with wild-type GST-tagged Plk1 PBD (WT), the triple mutant W414A, H538A, K540A (3M), or GST alone for 6 h, followed by analysis of fluorescence polarization. C) 24 was incubated with the MBP-tagged PBDs of Plk1, Plk2, and Plk3, or with MBP alone, for 6 h, followed by analysis of fluorescence polarization.


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Under otherwise identical assay conditions, incubation with the GST-tagged triple mutant Plk1 PBD W414A, H538A, K540A (Plk1 PBD-GST 3M) showed a slight decrease in fluorescence polarization as compared to wild-type Plk1 PBD, but essentially the same apparent Kd of 1.06 ± 0.18 µM (Figure 4B and Supplementary Figure 4B). In contrast, incubation with GST alone showed only a very minor increase in fluorescence polarization. These data indicate that binding of Poloxin to the Plk1 PBD is not mediated by the same amino acids involved in interactions with the phosphate group of phosphoserine/phosphothreonine-containing peptides. Binding of 24 to the MBP-tagged PBDs of Plk1, Plk2, and Plk3 indicated preferential inhibition of Plk1 (apparent Kd after 6 h of incubation: 0.87 ± 0.02 µM) over Plk2 and Plk3 (Figure 4C), and thus followed the same trend as was observed for most Poloxin derivatives in the competition-based assays. Incubation of 24 with MBP alone did not result in a significant increase in fluorescence polarization, indicating the specificity of 24 for the Plk1 PBD.

Plk1 is a key protein for progression through mitosis. Consequently, any efficient inhibitor of Plk1 must induce mitotic arrest in mammalian cells. Mitotic arrest is associated with increased concentrations of Plk1, Cyclin B1, phospho histone H3, and hyperphosphorylation of Cdc25C. In order to investigate whether the most potent inhibitors in the absence of DTT (compounds 20– 22), or the most active compounds in the presence of DTT (compounds 2 and 23) in the fluorescence polarization assay are most potent in inducing mitotic arrest in HeLa cells, we investigated both groups of compounds. Poloxin (1) and the poorly active acyl hydrazone 19 served as control compounds. Treatment of HeLa cells, which were synchronized at the G1/S transition by release from thymidine block, with 10 µM of the test compounds revealed that compounds 20–22, the most potent compounds in the absence of DTT, were only approximately as active as Poloxin (1) (Figure 5A). In contrast, compounds 2 and 23, as the most potent compounds in the presence of DTT, were superior to Poloxin in their ability to induce mitotic arrest. Quantitative analysis of the cell cycle distribution by flow cytometry confirmed the data 15 ACS Paragon Plus Environment


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obtained by Western blot analysis (Figure 5B and Supplementary Figure 5A). Of note, phosphorylation of Cdc25C in cells treated with 2 and 23 was only slightly increased as compared to untreated cells (Figure 5A), with stronger relative increases of the other mitotic markers Plk1, Cyclin B1, and phospho histone H3 being observed. This can be explained by the facts that Cdc25C is directly phosphorylated by Plk1 on Ser198,31 and that Cdc25C contains a binding site for the Plk1 PBD.5 Thus, the relatively low degree of Cdc25C phosphorylation in cells arrested in mitosis by 2 and 23 is consistent with the assumed mechanism of action of the compounds. Taken together, the data demonstrate the improved potency of 2 and 23 as compared to Poloxin, and indicate that low susceptibility of compound activity to the presence of DTT in a fluorescence polarization assay is a suitable criterion by which to pre-select promising compounds for use in cell-based assays. However, it is not possible to formulate a quantitative correlation between in vitro activities in the absence or presence of DTT and activity in cellbased assays. The complexity of cellular events cannot be mimicked by the addition of any single nucleophile in a biochemical assay, regardless of whether the nucleophile is physiological or non-physiological.


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Figure 5: Induction of mitotic arrest by Poloxin and analogs in HeLa cells. A) HeLa cells synchronized in G1/S by release from thymidine block were treated with test compounds at 10 µM for 14 h. Changes in the fraction of cells arrested in mitosis are analyzed by Western Blots against the mitotic markers Plk1, Cyclin B1, phospho histone H3, and Cdc25C. B) Cells treated like in A) were analyzed by flow cytometry. C) Dose-dependent analysis of the activities of Poloxin (1), 2, 19, and 23 on induction of mitotic arrest. D) Cells treated like in C) were analyzed by flow cytometry. E) Representative primary data of the flow cytometry data quantified in D).

The activities of the most potent new Poloxin derivatives 2 and 23 were subsequently analyzed in more detail. Treatment of HeLa cells released from G1/S arrest with compounds 2 and 23 increased the proportion of mitotic cells in a dose-dependent manner, as analyzed by Western blot (Figure 5C) and flow cytometry (Figure 5D, E, and Supplementary Figure 5B-D). At 10 µM, both 2 and 23 induced a 2.5–fold higher mitotic arrest than Poloxin (1) (30% vs 12% of the cells in G2/M). In the presence of 20 µM 2 and 23, respectively, approximately 70% of the cells were 17 ACS Paragon Plus Environment


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in G2/M, compared to 44% in the presence of 20 µM Poloxin (1). The negative control compound 19 did not induce a significant degree of mitotic arrest at concentrations of up to 20 µM.

Tumor cells arrested in mitosis typically undergo apoptosis. Consistently, Poloxin (1) increased the apoptotic rate of HeLa cells in a dose-dependent manner (Figure 6A). Consistent with their relative abilities to induce mitotic arrest, compounds 2 and 23 caused a stronger increase in the apoptotic rate than 1. The inactive compound 19 did not induce apoptosis. In line with the results of the cell cycle assays (Figure 5) and the apoptosis assay (Figure 6A), compounds 2 and 23 were also significantly more potent than Poloxin (1) in a cell viability assay, while 19 only had a minor effect on cell viability (Figure 6B).

Figure 6: Poloxin and active derivatives induce apoptosis in HeLa cells and reduce cell viability. A) Derivatives 2 and 23 are more potent than Poloxin (1) in inducing apoptosis in unsynchronized HeLa cells. Error bars depict standard error of the mean (SEM). B) Derivatives 2 and 23 inhibit cell viability of unsynchronized HeLa cells to a larger extent than Poloxin (1) and the negative control 19. Test compounds were used at a concentration of 20 µM. Error bars depict standard deviation (SD). a.u.: arbitrary units.


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Conclusions Small-molecule inhibitors of the protein-protein interaction domain of Plk1, the polo-box domain, can be powerful inhibitors of the anti-cancer target Plk1, circumventing specificity problems associated with ATP-competitive kinase inhibitors. In this study, we have analyzed structureactivity relationships and carried out mechanistic studies for the first reported small-molecule inhibitor of the PBD, Poloxin. These studies have determined that a 2,5-dimethyl substitution pattern on the iminoquinone ring is associated with improved inhibitory activities of Poloxin analogs. Moreover, the activated ester group of Poloxin and analogs was found to be essential for inhibitory activity. Shifting, deletion, or substitution of the ortho-methyl group on the benzoyl moiety results in compounds which mostly display improved in vitro activities compared to Poloxin in the absence of DTT in the assay buffer. The most potent molecules 20–22 under these assay conditions display nanomolar activities against the Plk1 PBD, along with exquisite selectivities over the PBDs of Plk2 and Plk3. However, the compounds’ high in vitro activities are not maintained in the presence of DTT in the assay buffer, and the compounds’ cellular activities are similar to that Poloxin. In contrast, although analogs 2 and 23 bearing an ortho-methyl group at the benzoyl moiety were less active than 20–22 in the absence of DTT, their activities were more stable in the presence of DTT, suggesting that the ortho-methyl group protects the activated ester functionality from non-specific attack by nucleophiles. Importantly, compounds 2 and 23 are significantly more potent than Poloxin in inducing cell cycle arrest and apoptosis in cultured human tumor cells, placing them amongst the most active small-molecule inhibitors of the Plk1 PBD reported to date. Owing to its superior specificity profile, we propose compound 2, dubbed Poloxin-2, for use in future research studies aimed at exploring the function of the Plk1 PBD in living cells.

Acknowledgements 19 ACS Paragon Plus Environment


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Work in the laboratory of T.B. was supported by the Deutsche Forschungsgemeinschaft (BE4572/1-1). Work in the laboratory of K.S. was supported by grants from the German Cancer Consortium (DKTK, Heidelberg), Carls-Stiftung, Deutsche Krebshilfe, and BANSS Stiftung. We thank T. Holak and H. Cavga for initial experiments, C. Birkemeyer, S. Billig and L. Hennig for analytical support, and A. Berg for critical reading of the manuscript.

METHODS Experimental details are given in the Supporting Information.

Supporting Information. Synthesis of compounds, their spectroscopic characterization, plasmid construction, protein expression, details and results from cell-based assays, and biophysical assays are summarized in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

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