A New Class of Peptidomimetics Targeting the Polo-Box Domain of

Oct 27, 2014 - ABSTRACT: Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the...
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A New Class of Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1 Mija Ahn,†,# Young-Hyun Han,‡,# Jung-Eun Park,§,# Sungmin Kim,† Woo Cheol Lee,† Soo Jae Lee,∥ Pethaiah Gunasekaran,‡ Chaejoon Cheong,† Song Yub Shin, Sr.,⊥ Hye-Yeon Kim,† Eun Kyung Ryu,† Ravichandran N. Murugan,† Nam-Hyung Kim,‡ and Jeong Kyu Bang*,† †

Division of Magnetic Resonance, Korea Basic Science Institute, 804-1, Yangcheong Ri, Ochang, Chungbuk, Cheongwon 363-883, Republic of Korea ‡ Molecular Embryology Laboratory, Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea § Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States ∥ College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea ⊥ Department of Bio-Materials, Graduate School and Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Republic of Korea S Supporting Information *

ABSTRACT: Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.

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INTRODUCTION Inhibition of certain protein kinases that are overexpressed in human tumor tissues was established as a therapeutic approach for identifying potent inhibitors during the past decade.1 However, protein kinases have high similarities in the ATPbinding pockets, and the available kinase inhibitors compete with ATP at the ATP-binding site of the enzymes.2 An alternative approach with potential for identifying potent and highly selective kinase inhibitors is to target the interfaces of protein−protein complexes of interest.3 Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that acts as key regulator of cell cycle progression, particularly mitosis.4 Plk1 © XXXX American Chemical Society

modulates the transition through the G2/M checkpoint by influencing the activation of the CDC25C phosphatase and cyclin B1. A high level of Plk1 is required for the viability of cancer cells bearing oncogenic Ras or an inactivating p53 mutation(s) but not for isogenic wild-type (WT) cells, and therefore Plk1 has been considered as a primary target for anticancer drug development.5 Five Plks have been identified in Special Issue: New Frontiers in Kinases Received: July 28, 2014

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mammals (Plk1−5), and Plk1−4 consists of a N-terminal catalytic domain and a C-terminal domain having 1 or 2 highly conserved sequences, termed polo-box domains (PBDs).6 A large body of evidence suggests that PBD directs the Nterminal catalytic domain for specific subcellular localization through interacting with phosphoserine/phosphothreonine (pS/pT)-containing motifs. Moreover, the subcellular targeting binding site in PBD forms a compact and druggable interface.7 Recently, a minimal peptide (PLHSpT) was identified with high affinity toward the Plk1 PBD that blocked Plk1 binding to polo-box-interacting protein 1 (PBIP1), a centromere/ kinetochore-associated target. The N-terminal Pro residue of PLHSpT confers crucial specificity by docking its side chain into a unique hydrophobic core (hereafter designated the pyrrolidine-binding pocket), which is surrounded by the Trp414, Phe535, and Arg516 residues (Figure 1).8 The same

with the peptide 2 (PL-2), which has only three amino acids. Moreover, our structure−activity relationship study with peptide 2, involving the replacement of the N-terminal ProLeu residues with nonpeptide variants, dramatically increased the binding affinity in comparison with the parent peptide, PLHSpT. Furthermore, crystallographic analyses of the Plk1 PBD in complex with candidate ligands showed that peptidomimetics 7 (AB-102), 8 (AB-103), and 22 (AB-1035) recognize well-reported key binding sites such as the Tyrrich channel and the phosphate-binding sites.10 However, unlike peptidomimetic 7, the amide nitrogen atoms of peptidomimetics 8 and 22 mediate hydrogen-bonding interactions with the backbone carbonyl on D416 for increased affinity. Finally, direct incubation of cultured HeLa cells with PEGylated 22 and 24 showed significant antiproliferative activities due to increase in intracellular bioavailability.

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RESULT AND DISCUSSION Design Strategy of Peptidomimetic Ligands. Lowmolecular-weight compounds that disrupt protein−protein interactions (PPIs) have tremendous potential applications as clinical agents or as chemical probes.10 However, designing drug-like small inhibitors of PPIs remains challenging, and in fact, only a few small molecule PPIs have been developed to date.11,12 Our approach in designing enhanced peptidomimetic ligands began with the systematic deletion of amino acid residues in the most potent 4j peptide analogues (Figure 2) and identifying key residues in the peptide responsible for biological effects. N-Terminal capping of the minimal peptide with small organic moieties was also investigated to generate potent, cellpermeable peptidomimetic ligands. Preliminary Evaluation of Minimal Phosphopeptides for Plk1 PBD Binding. Beyond the various peptide derivatives that have been found to interact with the Plk1 PBD, only a few systematic analysis studies have been conducted to ascertain key residues and their contributions to Plk1 inhibition.13−15 To identify the minimal set of key residues required for a high binding affinity, we examined the previously reported cocrystal structure of Plk1 PBD in complex with the 4j peptide9 that shows a ∼1000-fold increase in binding affinity compared to the parent PLHSpT peptide. According to the reported results, the C-terminal pT and the alkylated (C6H5(CH2)8−) imidazole ring at the π nitrogen of the His residue are essential for high binding affinity. Remarkably, the C6H5(CH2)8− moiety of 4j was well embedded into a Tyr-rich hydrophobic channel formed by multiple PBD residues, such as Val415, Tyr417, Tyr421, Leu478, Tyr481, Phe482, and Tyr485. Hence, a long alkyl side chain is required for effective binding with the Tyrrich hydrophobic channel. In addition, the side chain of the Nterminal Pro residue was docked into a hydrophobic core surrounded by the Trp414, Phe535, and Arg516 residues (Figure 1).9 These observations suggest that the Pro, Leu, or Ser residues do not mediate direct interactions with the PBD residues and that the systematic deletion of above residues from the Nterminal region of 4j may be an effective way to derive a minimal peptide with moderate inhibitory activity. Consistent with this interpretation, we found that peptide analogues with the N-terminal Pro and Leu deleted in 4j (2) or with the pT-1 Ser substituted with Ala on peptide 2 (5) had either potent inhibitory activity or retained the binding affinity of the parent PLHSpT peptide (Figure 2). The peptides used in this study were synthesized using a 9-fluorenylmethoxycarbonyl (Fmoc)-

Figure 1. Crystal structure of the previously reported Plk1 PBD in complex with 4j (PDB ID 3RQ7), which shows the presence of both the unique pyrrolidine-binding pocket and the conserved Tyr-rich hydrophobic channel.9

group of investigators also synthesized PLHSpT derivatives, which led to the identification of a narrow, Tyr-rich hydrophobic channel that recognizes the most potent peptide analogue 4j through hydrophobic interactions with four aromatic residues (Tyr417, Tyr421, Tyr481, and Phe482; PDB ID 3RQ7, Figure 1).9 Although PLHSpT and its derivatives including 4j have advanced as drug candidates for targeting the Plk1 PBD, they have several drawbacks in terms of limited bioavailability, poor protease stability, and high production cost. To overcome these drawbacks, the amino acids of the parental peptide should be substituted with unnatural amino acids as much as possible while maintaining high affinity and specificity against Plk1 vs the closely related Plk2 and Plk3 kinases. In this study, we performed systematic deletions in the 4j peptide to synthesize shortened and more stable peptidomimetics through deconstruction and reassembly processes and study critical residues and nonpeptide variants on a modified scaffold. This approach takes advantage of the good drug properties of the parent compound, such as stability and bioavailability, while enabling low manufacturing costs. We observed an unexpected preservation of Plk1 inhibitory activity B

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Figure 2. Minimization of the 4j peptide and quantification of the Plk1 PBD inhibitory activities of the resulting analogues. (A) Peptides of variable length were generated using solid-phase peptide synthesis (SPPS). (B) ELISA-based Plk1 PBD-binding assay results using minimal peptides derived from systematic N-terminal deletion of 4j. Representative graphs from three independent experiments are shown (OD, optical density).

Figure 3. Crystal structure of the Plk1 PBD in complex with peptide 2 (PDB ID: 4RCP). (A) The crystal structure of the Plk1 PBD−peptide 2 complex shows that the C6H5(CH2)8-group on the imidazole ring was directed toward the previously occluded Tyr-rich hydrophobic channel, whereas the negatively charged pThr phosphoryl group mediated strong electrostatic interactions with the H538 and K540 residues. The complex structure of peptide 2 is shown in a gray-colored ribbon model. The 4j structure in a cyan-colored ribbon model (PDB ID: 3RQ7) is overlaid on the peptide 2. (B) The omit map for the peptide 2 complex structure (PDB ID: 4RCP). The map was calculated with the final coordinates of peptide 2 and contoured at 2.5 σ.

position, showed decreased binding affinity compared to peptide 2, peptide 2 was chosen as promising lead compound to generate a new class of peptidomimetics. Because the fact “SpT” dipeptide motif is critical for highaffinity Plk1 PBD binding,8 we also synthesized and evaluated nonphospho derivatives of peptide 2 and 5 (3 and 6) to rule out promiscuous modes of binding. As expected, we found that the Ser-to-Ala substituted peptide 5 or the corresponding nonphospho derivative peptides 3 or 6 exhibited either reduced

based solid-phase method on a Rink amide resin, and Plk1 PBD binding activities were evaluated using an enzyme-linked immunosorbent assay (ELISA)-based Plk1 PBD-binding assay.8 Synthesis was accomplished by the sequential coupling of amino acids on the Rink amide resin in the presence of 1-Obenzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), and N,Ndiisopropylethylamine (DIEA) as coupling agents. Because peptide 5, which has an Ala instead of a Ser at the pT-1 C

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Figure 4. First-phase diversification of peptides and quantification of their inhibitory activities against Plk1 PBD. (A) First-phase N-terminally capped peptide 2 derivatives generated using SPPS. (B) ELISA-based Plk1 PBD-binding assay results using first-phase N-terminally capped peptide derivatives. Representative graphs from three independent experiments are shown (OD, optical density).

phase derivatization, a series of HSpT-derived peptide analogues, with the N-terminal His-2 acylated by bulky, short, or long aromatic or nonaromatic ring systems, were synthesized and evaluated for Plk1 inhibition activity (Figure 4 and Supporting Information, Figure S1). From our initial screen of derivatives 7 to 19, we obtained six confirmed hits when compared to the tripeptide 2. Surprisingly, peptidomimetic 8 having an N-terminal ethylbenzamide substituent showed the highest binding affinity of any compound in the series and was comparable to that of the p-13mer. In contrast, the lack of an amide functional group at the N-terminal peptidomimetic 7 dramatically decreased the binding affinity when compared to peptidomimetic 8. Among the substituted benzene analogues 12−16, peptidomimetics 14 and 15 having either dimethoxy-substituted ethenyl benzene or 1-naphthoic acid showed the next highest binding affinities. In addition, the higher binding affinity of 16 compared to 9 and 19 suggested that substituted bicyclic aromatic systems 16 performed better than either anthracene 9 or linear functional groups 19. Two double-ring heterocyclic ring systems 10 and 11, however, showed no indication of improved binding affinity. All the results described above showed that the best candidates for N-terminal capping to derive potent peptidomimetic ligands were tethered, hydrogenbond mediating substituted aryl derivatives rather than either short and bulky aromatic 17 or nonaromatic 18. To rationalize the differences in binding affinities between 7 and 8, a crystallographic study was performed with Plk1 PBD in complex with 7 and 8 (Figure 5 and Supporting Information, Table S1). Interestingly, the crystal structures of Plk1 PBD in complex with either peptidomimetic 7 or 8 showed different binding modes of their N-terminal acylated fragments. For instance, the crystal structure of peptidomimetic 8 in complex with Plk1

or nearly abolished inhibitory affinity. Thus, we concluded that the binding of peptide 2 was specific. A fluorescein isothiocyantate (FITC)-labeled version of peptide 2 was prepared for use in fluorescence polarization-based determinations of binding affinities to Plk1 PBD. We replaced the acyl group of peptide 2 with the FITC group to yield the corresponding peptide 1. ELISA results showed that the moderate increase in affinity and binding was still specific, as the mutant peptide 4 (S4A) variant showed a dramatic loss in affinity (Figure 2). To unambiguously identify the peptide 2 mode of binding, the X-ray cocrystal structure of peptide 2 (PDB ID: 4RCP) with the Plk1 PBD was solved (Figure 3 and Supporting Information, Table S1). We observed that both the protein backbone and the peptide ligand in the Plk1 PBD−peptide 2 complex were superimposable with the Plk1 PBD−4j structure. The cocrystal structure also showed that the C6H5(CH2)8group on the imidazole ring was directed toward the previously occluded Tyr-rich hydrophobic channel, whereas the negatively charged pThr group mediated strong electrostatic interactions with the H538 and K540 residues.9 First-Phase Library Design, Synthesis, and Screening. In preliminary efforts to derive a minimal peptide from the most potent 4j peptide analogue, we found that both the His adduct and pThr residues on peptide 2 played important roles in tight binding to the Tyr-rich hydrophobic channel and the electrostatic pocket. In addition, deletion of the N-terminal ProLeu residues provided an attractive opportunity for structural elaboration with small organic moieties to target the unique broader pyrrolidine-binding pocket. The results of previous studies from our laboratory have shown that short and bulky hydrophobic functional groups at the N-terminal PLHSpT peptide are potential candidates for targeting the unique, broader pyrrolidine-binding pocket.16 Accordingly, for the firstD

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Figure 5. Crystal structures of Plk1 PBD in complex with peptidomimetic 7 or 8. (A) The crystal structure of the Plk1 PBD−peptidomimetic 7 complex (PDB ID: 4WHK) showed that the N-terminal fragment of peptidomimetic 7 was separated from the Tyr-rich hydrophobic channel. (B) The crystal structure of the Plk1 PBD−peptidomimetic 8 complex (PDB ID: 4WHL) showed that the N-terminal fragment of peptidomimetic 8 was directed toward the Tyr-rich hydrophobic channel due to the two hydrogen bonding interactions between peptidomimetic 8 and D416 from the Plk1 PBD. (C) The overlaid crystal structure of Plk1 PBD in complex with peptidomimetic 7 or 8. (D) The crystal structure of the Plk1 PBD− peptidomimetic 8 complex showing a close-up view of the two hydrogen-bonding interactions between peptidomimetic 8 and D416 from the Plk1 PBD.

reported strategy16 restricted the dual binding ability of a modified peptide using bulky hydrophobic functional groups to target specifically the unique, broader pyrrolidine-binding pocket. Similarly, our goal was to direct the N-terminus substituted benzene into the Tyr-rich hydrophobic channel through hydrogen-bonding interactions between the ethyl benzamide nitrogen atom and the backbone carbonyl group of D416. Hence, in the second phase of derivatization, we further diversified the peptidomimetic 8 skeleton (IC50 = 0.40 μM) with various substituted aryl fragments at the N-terminus and try to determine other elements critical for the binding of HSpT derivatives (Figure 6). Interestingly, we observed that the peptidomimetics 22 (IC50 = 0.04 μM) and 24 (IC50 = 0.05 μM) exhibited an approximately 10-fold increase in Plk1 PBD binding affinity in comparison to that of the p-13mer (IC50 = 0.40 μM) and, moreover, the observed IC50 values were just 2fold less active than the most potent 4j peptide analogue (IC50 = 0.02 μM). Peptidomimetic 22 possesses hydroxyl group at the para position to enhance the solubility and hydrogen bonding with PBD. Similarly, peptidomimetic 24 (IC50 = 0.05 μM) possesses an N-methylamine at the para position. This demonstrated for the first time that short, small molecule−

PBD (PDB ID: 4WHL) showed that ethylbenzamide was bound to the Tyr-rich hydrophobic channel, whereas the Nterminal fragment of peptidomimetic 7 (PDB ID: 4WHK) was separated from this channel. Moreover, the crystal structure of Plk1 PBD−peptidomimetic 8 also indicated that two hydrogenbonding interactions occurred between ligand 8 and D416 of the PBD. The first hydrogen bonding interaction occurred between a carbonyl group of ligand 8 and the nitrogen atom of D416. The hydrogen bond length was 2.8 Å. The second hydrogen bonding interaction occurred between a nitrogen of ligand 8 and the backbone carbonyl of D416. The hydrogen bond distance was 3.4 Å. Both hydrogen bonds appeared to be key factors that could be potentially exploited to improve the binding affinity of peptidomimetic 8 with Plk1 PBD (Figure 5B,D). This study demonstrates for the first time that the Tyr-rich hydrophobic channel covers the broader region to accommodate different moieties from the pThr-2 and -3 positions. Second-Phase Derivatization of Peptidomimetics. In our first library screen, we found that substituted benzene derivatives attached through 2 carbon-tethered amide bonds at the N-terminus of HSpT could enhance Plk1 PBD inhibition by occupying the long Tyr-rich hydrophobic channel. A previously E

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Figure 6. Second-phase diversification of short peptidomimetics and quantification of their inhibitory activities against Plk1 PBD. (A) Second-phase N-terminally capped peptidomimetic 8 derivatives generated using SPPS. (B) ELISA-based Plk1 PBD-binding assay result using second-phase Nterminally capped peptidomimetic 8 derivatives. Representative graphs from three independent experiments are shown (OD, optical density).

peptide hybrid analogues dramatically improved the binding affinity in close to 4j peptide. This finding suggested that additional hydrogen bondmediating substitutions are critical for enhancing Plk1 PBD binding, and this prediction was well supported by the following order of decreasing activities: 22 or 24 > 23 or 25 with a protected long alkyl chain > 21 lacking either a hydroxymethyl or an N-methyl group at the para position. Interestingly, due to the presence of an ethyl carboxamide linker, the inactive 18 (identified from the first-phase library screen) was converted into a potent analogue, peptidomimetic 20 (IC50 = 0.65 μM). On the other hand, the addition peptidomimetic 8 template on other systems showed dramatic variation in the observed binding affinity. For example, peptidomimetics 16 or 19, having less inhibitory activity than 14 or 15 in the first screen, were converted into more potent analogues 28 (IC50 = 0.16 μM) and 30 (IC50 = 0.43 μM) when compared to either 29 (IC50 = 0.33 μM) or 27 (IC50 = 0.95 μM). To verify the binding nature of peptidomimetic 22, we solved the complex structure of 22 with the PBD (PDB IB: 4WHH) and compared structural differences observed between peptidomimetics 8 and 22 (Figure 7 and Supporting Information, Table S1). According to X-ray structure, peptidomimetic 22 showed a similar binding pattern compared to peptidomimetic 8. The first hydrogen-bonding interaction occurred between the backbone carbonyl of peptidomimetic 22 and the amide of D416 as in peptidomimetic 8. The second hydrogen-bonding interaction occurred between the amidic NH linked with the phenyl group of peptidomimetic 22 and the carbonyl of D416 from the PBD. However, the length of second hydrogen bond in the case of 8 was 3.4 Å, whereas it was 3.1 Å in the case of 22. This shortened hydrogen bond

length observed with 22 increased the binding affinity with the PBD. Although we did not find specific interactions between the hydroxymethyl group of peptidomimetic 22 and the PBD, this group may increase the binding affinity by 10-fold. These results suggest that the ethyl carboxamide linker together with the hydrogen-bond donor substitution on the benzene ring are important factors for generating highly potent peptidomimetics targeting the Plk1 PBD even though the function of hydrogen-bond donor on the benzene ring remains unclear. Isoform Selectivity. One of the major obstacles hindering the disruption of protein−protein interactions is the difficulty associated with selectively targeting a specific protein. For example, the development of the most potent small-molecule Plk1 kinase inhibitor (BI 2536) into a viable drug has been hampered by its high toxicity and lack of selectivity.10 In human cells, there are five Plk subfamilies known as Plk1 to Plk5.8 Among them, Plk1, Plk2, and Plk3 show a high degree of homology. However, Plk1 is strongly associated with cancer progression, whereas Plk2 and Plk3 are responsible for the depression of cancer cells. Hence, specific inhibition of Plk1 has been considered as a promising anticancer therapy. Therefore, the peptidomimetic 8 series was designed in such way to target the Plk1 PBD specifically. We performed a specificity test for the peptidomimetic 8 series against Plk1 PBD and the closely related Plk2 and Plk3 PBDs (Figure 8). Plk4 and Plk5 PBDs were not included because of the distinct binding nature of these proteins. Our results revealed that the peptidomimetic 8 series showed specificity against Plk1 PBD compared to Plk2 and Plk3 (Figure 8). Interestingly, functional groups at the para position on the benzene ring did not influence selectivity. F

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Figure 7. Crystal structures of the Plk1 PBD in complex with peptidomimetic 22 (PDB IB: 4WHH). (A) The crystal structure of the Plk1 PBD− peptidomimetic 22 complex showed that the N-terminal fragment of peptidomimetic 22 was directed toward the Tyr-rich hydrophobic channel due to two intermolecular hydrogen-bonding interactions between the amide functional group of ethylbenzamide and the D416 backbone, whereas the C6H5(CH2)8-group on the imidazole ring was nearly superimposable with peptide 2. (B) A close-up view of the crystal structure of the Plk1 PBD− peptidomimetic 22 complex showing two hydrogen-bonding interactions between both amide functional groups of ethylbenzamide on 22 and D416 from the Plk1 PBD. (C) Simulated annealing omit |Fo − Fc| electron density map contoured at 2.0 σ. (D) The overlaid crystal structure of the Plk1 PBD in complex with peptidomimetics 7 (green), 8 (blue), or 22 (yellow).

Cell-Based Assays Using PEGylated 8 Series Peptidomimetics. Having established a notable potency in biochemical assays, we next investigated the antiproliferative effects of these analogues in HeLa cells. Because one of the major limitations of using phosphopeptides as anticancer agents is membrane permeability, we also synthesized and evaluated the antiproliferative activities of PEGylated versions of peptidomimetics 22 and 24 (Supporting Information, Figure S2). The proliferation rates of HeLa cells incubated for 24 h in the presence of peptidomimetics 22, 24, 22-Cys, 24-Cys, 22-CysPEG, and 24-Cys-PEG were determined at increasing concentrations (1−500 μM). We observed that the 22-PEG and 24-PEG inhibited cell survival in a dose-pendent manner. For instance, 22-PEG and 24-PEG (but not 22, 24, or 22-Cys, 24-Cys) showed a significant inhibition with an IC50 of 348 and 375 μM, and the treatment of cells with low concentration (98%) was assessed by RP-HPLC on an analytical Vydac C18 column (4.6 mm × 250 mm, 300 Å, 5 μm particle size). The molecular masses of purified peptides were determined using matrix-assisted laserdesorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Shimadzu, Japan).

EXPERIMENTAL SECTION

Materials and Methods. Rink amide 4-methylbenzhydrylamine (MBHA) resin and 9-fluorenylmethoxycarbonyl (Fmoc) amino acids were obtained from Calbiochem-Novabiochem (La Jolla, CA). Other reagents used for peptide synthesis included trifluoroacetic acid (TFA; Sigma), piperidine (Merck), 1-O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluoro-phosphate (HBTU; Novabiochem), 1-hydroxybenzotriazole hydrate (HOBt; Aldrich), and dimethylformamide (DMF, peptide synthesis grade; Biolab). Peptide Synthesis. All peptides were prepared by Fmoc SPPS methods using Rink amide with an initial loading of 0.61 mmol/g unless otherwise noted. Fmoc-Arg(Pbf)-OH and other Fmoc protected amino acids were purchased from Novabiochem. Resins were swollen in N,N-dimethylformamide (DMF) for 45 min prior to synthesis. For sequence extension, the Fmoc-protected amino acid (5.0 equiv) was activated by treatment with 1-O-benzotriazoleN,N,N′,N′-tetramethyluronium hexafluoro-phosphate (HBTU) (5.0 equiv) and 1-hydroxybenzotriazole (HOBt) (5.0 equiv) and N,Ndiisopropylethylamine (10.0 equiv) in DMF (2 mL) for 2 min. This I

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Linear Peptide 1. 1, MS (MALDI-TOF) m/z = 1113.18 [M + H]+; 2, MS (MALDI-TOF) m/z = 652.68 [M]; 3, MS (MALDI-TOF) m/z = 572.70 M+; 4, MS (MALDI-TOF) m/z = 1097.18 [M + H]+; 5, MS (MALDI-TOF) m/z = 636.68 M+; 6, MS (MALDI-TOF) m/z = 556.70 M+; 22-Cys, MS (MALDI-TOF) m/z = 919.47 [M + H]+; 22Cys-PEG, MS (MALDI-TOF) m/z = 2159.40 [M+2H]2+; 22-LysPEG-FITC, MS (MALDI-TOF) m/z = 1669.09 [M+2H]2+; 24-Cys, MS (MALDI-TOF) m/z = 918.54 [M + H]+; 24-Cys-PEG, MS (MALDI-TOF) m/z = 2158.13 [M+2H]2+; 24-Lys-PEG-FITC, MS (MALDI-TOF) m/z = 1667.98 [M + H]+. Linear Peptide 7. 7, MS (MALDI-TOF) m/z = 785.38 [M + H]+; 8, MS (MALDI-TOF) m/z = 786.39 [M + H]+; 9, MS (MALDITOF) m/z = 886.58 [M + H]+; 10, MS (MALDI-TOF) m/z = 796.50 [M + H]+; 11, MS (MALDI-TOF) m/z = 832.09 [M + H]+; 12, MS (MALDI-TOF) m/z = 790.41 [M + H]+; 13, MS (MALDI-TOF) m/z = 779.52 [M + H]+; 14, MS (MALDI-TOF) m/z = 801.52 [M + H]+; 15, MS (MALDI-TOF) m/z = 809.28 [M + H]+; 16, MS (MALDITOF) m/z = 781.25 [M + H]+; 17, MS (MALDI-TOF) m/z = 815.31 [M + H]+; 18, MS (MALDI-TOF) m/z = 749.62 [M + H]+; 19, MS (MALDI-TOF) m/z = 724.42 [M + H]+; 20, MS (MALDI-TOF) m/z = 792.48 [M + H]+; 21, MS (MALDI-TOF) m/z = 828.61 [M + H]+; 22, MS (MALDI-TOF) m/z = 816.44 [M + H]+; 23, MS (MALDITOF) m/z = 844.52 [M + H]+; 24, MS (MALDI-TOF) m/z = 815.43 [M + H]+; 25, MS (MALDI-TOF) m/z = 857.50 [M + H]+; 26, MS (MALDI-TOF) m/z = 864.53 [M + H]+; 27, MS (MALDI-TOF) m/z = 880.49 [M + H]+; 28, MS (MALDI-TOF) m/z = 852.44 [M + H]+; 29, MS (MALDI-TOF) m/z = 944.40 [M + Na]+; 30, MS (MALDITOF) m/z = 795.52 [M + H]+; 31, MS (MALDI-TOF) m/z = 772.45 [M + H]+. Crystallization, Data Collection, and Structure Determination. The Polo-box domain (PBD) of Plk1 was expressed and purified as previously reported.8 The PBD and peptide analogues were mixed in 1:1.5 molar ratio. Crystallization screens for the PBD−peptide analogues were carried out using the sitting drop vapor diffusion method. The crystals were soaked in cryoprotectant solution containing ethylene glycol at a final concentration of 16% (v/v) and flash cooled in the nitrogen stream at 100 K. X-ray diffraction data of the PBD and ligand complex crystals were collected using the ADSC Quantum 270 CCD detector with synchrotron radiation at Pohang Accelerator Laboratory (PAL) beamline 7A (Pohang, Korea). All data sets were integrated and scaled using HKL2000.17 All of the structures of the PBD−peptide complex were calculated by molecular replacement as the searching model 3HIH using Phaser.18 The initial model was built with COOT19 and refined with REFMAC5.20 As it was expected that the peptide analogues would be occupied similar to the 4j (PDB 3RQ7), the peptide analogue components, His adduct, Ser, and pThr were fitted manually in Fo − Fc electron density map of the 4j binding region. The ligand fitted final models were refined with REFMAC5. Parameter files for model building and refining the peptide analogues were generated using the server PRODRG2.21 Cell Viability Test. In this study, we used human cancer cell line HeLa (human epithelial carcinoma cell line) that was purchased from the American Type Culture Collection (ATCC). For identified toxicity of small molecules, 1 ×103 cells/well of HeLa cells were cultured into 96-well plates containing Dulbecco’s Modified Eagle Medium (DMEM, GIFCO BRL, Gaithersburg, MD, USA) and 10% fetal bovine serum (FBS) for 24 h, then incubated for 24 h after treatment with 1, 10, 30, 50, 80, 100, 200, and 300 μM of derivatives (22, 22-Cys, 22-PEG, 24, 24-Cys, and 24-PEG). The toxicity of small molecules was observed by the Cell Proliferation Reagent, sodium 3[1-(phenylaminocarbonyl)-d,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT, Sigma-Aldrich, USA) assay. The experiment was repeated three times in duplicate. Cellular Uptake. The uptake ability of small molecules on cancer cell lines was performed with the standard techniques with fluorescein 5(6)-isothiocyanate (FITC) conjugated derivatives (22-PEG and 24PEG). First, 2 × 104 cells/well of HeLa cells were cultured into 24-well polystyrene plates containing a plastic coverslip for 24 h. Then media were changed to FBS free DMEM media with 200 μM of derivatives and incubated for 3 h. The coverslips were washed tree times with

phosphate-buffered saline (PBS). Coverslips were mounted with mount solution (VECTOR Shield. VECTOR Corporation, USA) were imaged on a fluorescence microscope (OLYMPUS IX81, Olympus Inc., Japan). Images were processed using Image analysis program software (Metamorph, Molecular Devices Inc., USA). Plk1 Kinase Inhibition Test. For Plk1 kinase inhibition effect identification of the derivatives, we used the CycLex Polo-like kinase assay/inhibitor screening kit (MBL Inc., Japan). The cancer cells (2.5 × 105 cells/well) was cultured in 6-well plates and cultured for 24 h and incubated in added media with 200 μM of derivatives (22, 22PEG, 24, and 24-PEG) for 12 h. Then total proteins that were purified from cancer cells were resuspended using 300 μL of an appropriate extraction buffer (20 mM, Tris-HCl, pH 8.5, 150 mM NaCl, 0.2% NP40, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.2 mM, and 5 mM βmercaptoethanol). The sample (30 μg/100 μL of protein and kinase reaction buffer) was incubated at 30 °C for 30 min. Then, wells were washed to five times with wash buffer and were incubated at room temperature for 60 min with 100 μL of antiphospho-serine/threonine polyclonal antibody PPT-07 solution and incubated at room temperature for 60 min with 100 μL of HRP-conjugated antirabbit IgG and incubated at room temperature for 10 min with 100 μL of substrate reagent solution. Finally, 100 μL of stop solution was added into each well in the same order as the previously added substrate reagent. Measure absorbance of each well was read using a spectrophotometric plate reader at 450 nm/560 nm.

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic procedures of peptidomimetics 22 and 24 and data refinement statistics. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Coordinate and structure factors have been deposited with PDB under codes [2 (PL-2):4RCP], [7 (AB-102):4RCP], [8 (AB103):4RCP], and [22 (AB103-5):4RCP].

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82-43-240-5023. Fax: +82-43-240-5059. E-mail: [email protected]. Author Contributions #

M. A., Y.-H. H., and J.-E. P. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Korea Basic Science Institute’s Research Program grant T34418 (J.K.B.) and the NextGeneration BioGreen 21 Program (no. PJ009594), Rural Development Administration (NHK).

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ABBREVIATIONS USED Plk1, polo-like kinase 1; PBD, polo-box domain; PPI, protein− protein interaction inhibitors; ATP, adenosine triphosphate; PBIP1, polo-box interacting protein 1; PDB, Protein Data Bank; ELISA, enzyme-linked immunosorbent assay; SPPS, solid-phase peptide synthesis; FITC, fluorescein isothiocyantate; OD, optical density; PEG, polyethylene glycol; DAPI, 4′,6diamidino-2-phenylindole

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