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Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel

In the current work, unanticipated synthetic byproducts were obtained arising from alkylation of the δ1 nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest affinity byproduct, bearing a C6H5(CH2)8– group, a Plk1 PBD co-crystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically-stable phosphothreonyl residue (pT) bound to the Plk1 PBD with affinity equal to the non-PEGylated parent, yet it exhibited significantly less interaction with the PBDs of the two closely-related Plk2 or Plk3. Treatment of cultured cells with this PEGylated peptide resulted in Plk1 delocalization from centrosomes and kinetochores, and chromosome misalignment that effectively induced mitotic block and apoptotic cell death. This work provides new insights that may advance efforts to develop Plk1 PBD-binding inhibitors as potential Plk1-specific anticancer therapeutic agents.

Peptide cleavage and purification. Peptide resins as prepared above (200 mg) were cleaved by treatment with trifluoroacetic acid : triisopropylsilane : H 2 O (90 : 5 : 5) (5 mL, 4 h). The resin was removed by filtrations and the filtrate was concentrated under vacuum, then peptide was precipitated by the addition of diethyl ether and the precipitate washed with ether. The resulting solid was dissolved in 50% aqueous acetonitrile (5 mL) and purified by reverse phase preparative HPLC using a Phenomenex C 18 column (21 mm dia x 250 mm, cat. no: 00G-4436-P0) with a linear gradient from 0% aqueous acetonitrile (0.1% trifluoroacetic acid) to 100% acetonitrile (0.1% trifluoroacetic acid) over 30 minutes at a flow rate of 10.0 mL/minute. Lyophilization gave the products as white powders (Supplementary Tables 1, 2 and 6).

ELISA-based PBD-binding inhibition assay.
A biotinylated p--T78 peptide was first diluted with 1X coating solution (KPL Inc., Gaithersburg, MD) to the final concentration of 0.3 µM, and then 100 µl of the resulting solution was immobilized onto a 96--well streptavidin--coated plate (Nalgene Nunc, Rochester, NY). The wells were washed once with PBS plus 0.05% Tween20 (PBST), and incubated with 200 µL of PBS plus 1% BSA (blocking buffer) for 1 h to prevent non-specific binding. Mitotic 293A lysates expressing HA--EGFP--Plk1 were prepared in TBSN buffer (~ 60 µg total lysates in 100 µL buffer), mixed with the indicated amount of the competitors (p--T78 peptide and its derivative compounds), provided immediately onto the biotinylated peptide-coated ELISA wells, and then incubated with constant rocking for 1 h at 25 °C. Following the incubation, ELISA plates were washed 4 times with PBST. To detect bound HA--EGFP--Plk1, the plates were probed for 2 h with 100 µL/well of anti--HA antibody at a concentration of 0.5 µg/mL in blocking buffer and then washed 5 times. The plates were further probed for 1 h with 100 µL/well of HRP--conjugated secondary antibody (GE Healthcare, Piscataway, NJ) at a 1:1,000 dilution in blocking buffer. The plates were washed 5 times with PBST and incubated with 100 µL/well of 3,3',5,5'--tetramethylbenzidine (TMB) solution (Sigma, St. Louis, MO) as a substrate until a desired absorbance was reached. The reactions were stopped by the addition of 100 µL/well of stop solution (Cell Signaling Technology, Danvers, MA). The optical density (O.D.) was measured at 450 nm by using an ELISA plate reader (Molecular Devices, Sunnyvale, CA). Data are shown in Table 1 of the published text and Supplementary Fig. 5 and 6.

MS-MS Analysis of peptides 3j and 4j.
Tandem MS studies were undertaken to clarify the site of C 6 H 5 C 8 H 8 -adduct addition in peptides 3j and 4j. Mass spectrometry data were acquired on an Agilent 6520 Accurate-Mass Q-TOF LC/MS System, (Agilent Technologies, Inc., Santa Clara, CA) equipped with a dual electro-spray source, operated in the positive-ion mode. Separation was performed on Zorbax 300SB-C18 Poroshell column (2.1 mm x 75 mm; particle size 5 µm). The analytes were eluted with solvent system 0.1% formic acid in H 2 O/ 0.1% formic acid in acetonitrile at a flow rate of 1 ml/min with a 5 to 100% organic gradient over 4 minutes and holding organic for 1 minute. The instrument was used in either full-scan TOF mode or product ion scan (MS/MS) mode. MS source parameters were set with a capillary voltage of 4 kV, the fragmentor voltage of 175 V and skimmer 65 V. The gas temperature was 350 °C, drying gas flow 12 L/min and nebulizer pressure 55 psig. High purity nitrogen was used as a collision gas. Data were acquired at high resolution (1,700 m/z), 4 GHz. TOF-MS mass spectra were recorded across the range 100-1600 m/z. Q-TOF-MS/MS experiments were carried out in the range 50-1000 m/z with a scan rate of 1.4 spectra/s with collision energy of 35 V. Data acquisition and analysis were performed using MassHunter Workstation Software (version B.02.00). To maintain mass accuracy, an internal mass calibration solution, containing reference ions m/z 121.050873 and m/z 922.009798, was infused continuously during the LC/MS runs.
Fragmentation of peptides typically occurs sequentially along the peptide backbone at the sites of the carboxamide groups. For each residue starting from the N-terminal end of the peptide, fragments are given sequential alphabetical designations starting with "a"; for example, a1, b1, c1 for residue 1; a2, b2, c2 for residue 2, etc as shown in the Supplementary Fig. 2 (below). In similar fashion starting from the C-terminal end of the peptide, fragments for each residue are designated alphabetically starting from "z" as shown in the Figure. The most commonly observed ions are of the "a", "b" and "y" type. For peptide 3j the MS -fragments SI--10 were consistent with the C 6 H 5 C 8 H 8 -adduct being on the pThr residue, with key fragments being b 6 +H and b 5 (see the Supplementary Fig 3a and Supplementary Table 3). For peptide 4j, fragmentation was consistent with the C 6 H 5 C 8 H 16 -adduct being on the His residue, with key fragments being b4 and y4 (see Supplementary Fig. 3b and Supplementary Table 4). It should be noted that the data did not indicate the site of adduct attachment on the His residue.   140.0700 X-ray Crystallography Protein purification and crystallization. Plk1 PBD protein (residues 371--603) was purified as previously described. 2 Crystals were grown using the hanging drop vapor diffusion method. PBD protein at 12 mg/mL in 10 mM Tris pH 8, 0.5 M NaCl, 10 mM DTT, 2% DMSO and 2 mM compound 4j was mixed with an equal volume of reservoir solution consisting of 15% (w/v) PEG 3350, 0.1 M glycine pH 9 and 300 mM NaCl. Crystals appeared overnight and reached maximum size over several days.

Ac-P-L-H*-S-pT-NH
Data collection, and structure determination and refinement. Crystals were cryo--protected in 33.3% (w/v) PEG3350, 500 mM NaCl, 0.1 M glycine pH 9, 2 mM 4j, 2 % DMSO and 10 mM DTT, and data were collected at 100 K on a Mar345 image plate detector with a Rigaku RU--300 home X--ray source. The data were processed with the HKL 3 and CCP4 4 software suites. The structure was solved by molecular replacement using AMoRe 5 using chain A of structure 3FVH 2 (RCSB accession code) as a search model, and refined using PHENIX 6 with manual fitting in XtalView. 7 The final model contains 90.6%, 8.4%, 0.5% and 0.5% (Leu 490) of the protein residues in the most favorable, allowed, generously allowed, and disallowed regions of Ramachandran plot, respectively. The strained conformation of Leu 490 appears to be stabilized by ligand binding. The figure was created using Molscript 8

SI--15
Resin cleavage and HPLC purification of PEGylated peptide products. Peptides were cleaved from the resin (~ 200 mg) by treatment with trifluoroacetic acid: triisopropylsilane: H2O (4.0 mL, 0.1 mL, 0.1 mL) for 4 hours at room temperature. The resin was removed by filtration and the filtrate was concentrated under vacuum, then precipitated with diethyl ether (5.0 mL), and the precipitate was washed with diethyl ether (5 mL x 3). The resulting solid was dissolved in 50% aqueous acetonitrile (5 mL) and purified by reverse phase preparative HPLC using a Phenomenex C18 column (21 mm dia x 250 mm, cat. no: 00G--4436--P0). Lyophilization gave products as white powders (Supplementary Tables 5 and 6 and Fig. 4) Table 6. SI--23 Supplementary Figure 6. Inhibition of Plk1 PBD by various PLHSpT-derived compounds. (a-d) ELISA-based PBD inhibition assays were performed essentially as described in the Methods using HA-EGFP-Plk1-expressing mitotic 293A cell lysates. Slight differences in the inhibition curves among the graphs are due to the differences in development time. Optical densities (O.D.) for each sample were measured at 450 nm by using an ELISA plate reader. Note that for reasons unknown, 4h (n = 6) inhibited PBD reproducibly better than 4i (n = 7). Multiple panels are employed to clarify data presentation. At least more than three independent experiments were carried out for each assay. Data represent mean values +/− s.d. (bars).  with 100 nM of BI 2536, the concentration previously used to characterize its effect in cultured cells. 14 Twenty--four hours after treatment, the cells were stained with Hoechst 33342 for 10 min and analyzed as described in Supplementary Fig. 13. (b) HeLa cells were arrested with a single thymidine block and released into fresh medium. Four hours after release, cells were treated with either control DMSO or 100 nM of BI 2536. The resulting cells were then harvested at the 4, 6, 8, 10, 12, 14, 16, and 19 h time points after release to determine the percentage of mitotic cells with rounded--up morphology (See the result in Fig. 2f). The cells at the 14 h time point were photographed and representative images were provided. Note that cells treated with control DMSO reached mitosis 10 h after single thymidine release. However, cells treated with BI 2536 exhibited severely delayed cell cycle progression, but continuously accumulated mitotically round--up cells over time. (c-d) Cells released from a single thymidine block for 9 h were treated with BI 2536 for 2 h and then subjected to immunostaining analyses to examine Plk1 localization (c) and to quantify the cells with aberrant mitotic morphologies among the total mitotic population (d). In good agreement with the previous observation, 14 treatment of cells with BI 2536 induced a prometaphase arrest with drastically delocalized Plk1 signals from centrosomes (asterisks) and kinetochores, and greatly weakened spindle morphologies. Note that, as a result of delayed cell cycle progression by BI 2536 (see the results in Fig. 2f), the fraction of mitotically arrested cells in (b) and the percentage of aberrant mitotic cells in (d) were less than those from the 6--treated cells in Fig. 2c and Fig. 2e