Crystal Structure of Pim1 Kinase in Complex with a Pyrido[4,3-D]Pyrimidine Derivative Suggests a Unique Binding Mode

Human Pim1 kinase is a serine/threonine protein kinase that plays important biological roles in cell survival, apoptosis, proliferation, and differentiation. Moreover, Pim1 is up-regulated in various hematopoietic malignancies and solid tumors. Thus, Pim1 is an attractive target for cancer therapeutics, and there has been growing interest in developing small molecule inhibitors for Pim1. Here, we describe the crystal structure of Pim1 in complex with a newly developed pyrido[4,3-d]pyrimidine-derivative inhibitor (SKI-O-068). Our inhibitor exhibits a half maximum inhibitory concentration (IC50) of 123 (±14) nM and has an unusual binding mode in complex with Pim1 kinase. The interactions between SKI-O-068 and the Pim1 active site pocket residue are different from those of other scaffold inhibitor-bound structures. The binding mode analysis suggests that the SKI-O-068 inhibitor can be improved by introducing functional groups that facilitate direct interaction with Lys67, which aid in the design of an optimized inhibitor.

In contrast to other protein kinases, Pim1 kinase is constitutively active, and its activity is primarily regulated through protein expression and stability rather than posttranslational modification, although regulation by phosphorylation has been reported [4][5][6]9,10]. The most promising aspect of Pim1 is that the structural conformation of the hinge region at the ATP-binding pocket differs from that of other protein kinases, which may aid in the design of more specific and selective inhibitors [5]. Moreover, Pim1 knockout mice show a mild phenotype, which suggests that Pim1 inhibitors may have few side effects [6]. Thus, Pim1 is an attractive target for cancer therapeutics, and there has been growing interest in developing small-molecule inhibitors for Pim1 [4,11].
Many inhibitor-bound Pim1 crystal structures are available in the Protein Data Bank [5]. These inhibitors can be classified into three major classes based on their binding mode [5,12]. The first class of inhibitors interacts with the Pim1 (Glu121) hinge region, and they are ATP-mimetic inhibitors. However, the second class of inhibitors interacts with the active site lysine (Lys67) rather than the Glu121 in the hinge region, and they are non-ATP mimetic/ ATP-competitive inhibitors [5]. A third binding mode has been observed for certain inhibitors, which have characteristics of both ATP-mimetic and non-ATP mimetic binding modes [12]. A phase I evaluation was initiated for SGI-1776, which inhibits all Pim kinases; however, the clinical trials were terminated early because of cardiac toxicity [7,13]. Therefore, a new therapeutic strategy and discovery of new inhibitors is necessary for further inhibitor development.
We have focused on developing a Pim1 inhibitor and have performed an X-ray crystallographic study to gain structural insights for lead compound optimization. Here, we report a crystal structure for Pim1 in complex with a newly developed inhibitor (SKI-O-068). Our inhibitor (SKI-O-068) exhibits a 133 nM half maximum inhibitory concentration (IC 50 ) and a unique binding mode with the Pim1 kinase. Interactions between SKI-O-068 and the Pim1 active site pocket residues are different from those of previously reported inhibitor-bound Pim1 structures. We suggest that the structural information from the Pim1-inhibitor complex will be helpful in structure-based lead compound inhibitor optimization.
Biochemical kinase assays for Pim1, Syk, and Pyk2 kinase from Carna Biosciences Inc. were performed in accordance with the Perkin-Elmer TR-FRET protocol using suggested experimental materials, such as kinase and substrate. Briefly, the indicated SKI-O-068 concentrations ranged from 0.1 pM to 30 mM (final concentration in 2% DMSO) and incubation as performed with 148 mM ATP, 50 nM ULight-CREBtide peptide, and 332 pg/uL enzymes for 60 min. Following addition of a Europium-labeled anti-phospho-CREB antibody, the kinase reaction was terminated by EDTA. The Europium signal at 665 nm was detected using time-resolved fluorescence via Envision. The IC 50 was calculated using nonlinear regression and GraphPad Prism 5. Staurosporine was used as a control for the experimental conditions.

Protein Purification and Crystallization
An N-terminal truncated form (residues 292313) of the human pim1 gene was amplified using PCR and cloned into a modified pET32 vector (Novagen) using NdeI and NotI restriction sites, respectively. The residue numbering in this work followed previous structure reports [10,14]. The construct used herein included twelve extra residues (AAALEHHHHHHHH) at the carboxyl terminus of the recombinant proteins. The recombinant protein was overexpressed in E. coli Rosetta2(DE3)pLysS (Novagen) cells using Terrific-Broth culture medium. After growth to the mid-log phase at 37uC, protein expression was induced by 1 mM isopropyl 1-thio-b-D-galactopyranoside, and the cells were further incubated for 20 h at 22uC. The cells were lysed by sonication in lysis buffer (10 mM Tris-HCl at pH 7.4, 2.5 mM KCl, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM lysozyme) with 10% (v/v) glycerol. The crude lysate was centrifuged at ,36,000 g for 60 min. The supernatant was applied to a Ni 2+ -NTA column (Qiagen) for affinity purification via the C-terminal octa-histidine tag. The eluent was pooled and concentrated. The protein was further purified by gel filtration on a Superdex 75 prep-grade column (GE Healthcare), which was previously equilibrated using 20 mM Tris-HCl buffer at pH 7.5 with 200 mM NaCl and 1 mM b-mercaptoethanol. The Pim1- containing fractions were concentrated to 9.2 mg/ml for crystallization using an Amicon Ultra-15 centrifugal filter unit (Millipore).

Crystallization and Data Collection
To grow crystals of inhibitor-free and inhibitor-bound Pim1, we incubated the protein solution at 24uC for one hour after adding the inhibitor dissolved in dimethyl sulfoxide at a 5-fold molar excess. The crystals were grown using the sitting-drop vapor diffusion method at 4uC by mixing equal volumes (2 ml each) of the protein solution and reservoir solution that comprised 0.7 M sodium potassium tartrate and 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.5). The crystals were grown to approximately 0.02 mm 60.02 mm 60.4 mm within a week.
X-ray diffraction data were collected on an ADSC Quantum 210 CCD detector (Area Detector Systems Corporation, Poway, CA, USA) under cryogenic conditions at the BL-6C experimental station in Pohang Light Source, Korea. For each image, the crystal was rotated 1u, and the raw data were processed and scaled using the program suit HKL2000 (Otwinowski, Z., and W. Minor. 1997. Methods Enzymol) [15]. The crystals belonged to the hexagonal space group P6 5 . Each asymmetric crystal unit comprised a single Pim1 monomer.

Structure Determination and Refinement
The Pim1 structures were determined using molecular replacement and the program Molrep [16] by employing a Pim1 model (PDB code 1XQZ) [10] for searches. Five percent of the data were randomly used as a test set to calculate R free [17]. The models were manually constructed using the program Coot [18] and refined with using the programs Phenix [19] and Refmac [20], which included bulk solvent correction. The inhibitor (SKI-O-068) and water molecules were assigned based on mFo -DFc maps calculated using the model phases. The models demonstrated excellent stereochemistry, which was evaluated using the program MolProbity [21]. Structural deviation was calculated using Superpose [22]. Table 1 summarizes the refinement statistics.

Protein Data Bank Accession Numbers
The atomic coordinates and structure factors for human Pim1 (the inhibitor-free form and inhibitor-bound form) are in the Protein Data Bank under accession codes 4JX3 and 4JX7, respectively.

Discovery of the Pim1 Kinase Inhibitor
In the development of a Pim1 inhibitor for anticancer drug development, we discovered SKI-O-068, which was an initial hit. For this discovery, one hundred compounds from in-house library were screened against human Pim1 kinase and initial hits were selected. In a biochemical enzyme assay, SKI-O-068 inhibited Pim1 with an IC 50 value of 123 (614) nM. This compound also inhibited Syk kinase with an IC 50 of 53 (610) nM and Pyk2 kinase with an IC 50 of 55 (610) nM, which suggests that SKI-O-068 is multi-potent kinase inhibitor. Because it was a potent inhibitor for Pim1, we determined its crystal structure for lead compound optimization.

Structure Determination and Model Quality
We determined two crystal structures in this study: those of inhibitor-free Pim1 and inhibitor (SKI-O-068)-bound Pim1 (Fig. 2). The inhibitor-free Pim1 structure was refined using 2.4 Å data to R work and R free values of 0.221 and 0.249, respectively. It included 274 amino acid residues in one monomer and 68 water molecules in the asymmetric unit. The refined model for inhibitor-bound Pim1 includes 274 residues in one monomer, one consensus substrate peptide (residues KRRRH), and 104 water molecules in the asymmetric unit. It produced R work and R free values at 0.191 and 0.227 for the 20-2.40 Å data, respectively. Eleven residues (Lys29-Glu32 and Leu307-Lys313) and twelve residues from the C-terminal fusion tag are disordered in both structures. The inhibitor-free Pim1 structure is highly similar to the inhibitor-bound Pim1 structure with a 0.263 Å root mean square (r.m.s.) deviation for 223 Ca atom pairs. The refinement statistics are summarized in Table 1.

Binding Mode for Pimtide in the Substrate Binding Site
Although a synthetic peptide was not added during protein purification and crystallization, additional election density was observed at the substrate-binding site for the inhibitor-bound structure, which is not a water molecule. Various structural reports for Pim1 have demonstrated binding of a consensus substrate  c R = S | |F obs | -|F calc | |/S |F obs |, where R free is calculated for a randomly chosen 5% of reflections, which were not used for structure refinement, and R work is calculated for the remaining reflections. d Values generated using Refmac [20]. e Values generated using MolProbity [21]. doi:10.1371/journal.pone.0070358.t001 peptide (pimtide; 1 ARKRRRHPSGPPTA 14 ), and a portion of the pimtide sequence was fit to the electron density. Thus, we tentatively modeled five residues from pimtide ( 3 KRRRH 7 ) to the additional election density at the substrate-binding site (Fig. 2). In the inhibitor-bound Pim1 structure, pimtide yielded a structure identical to previously reported Pim1 structures (PDB codes 2BIL, 2BZK [23], 2C3I [24], 3CY2 [25], and 3MA3 [26]), wherein the pimtide was modeled into the substrate-binding pocket (Fig. 3A). Three residues (Arg 4 , Arg 6 , and His 7 ) of five well-defined pimtide residues interact with Pim1. Arg 4 interacts with Thr134, Asp170, and Asp234. Arg 6 contacts Asp128, Asp131, the Phe130 carbonyl moiety, and Glu171. His 7 forms interactions with Glu171 and Glu243 (Fig. 3B).

Structural Analysis of Inhibitor-Bound Pim1
Pim1 has a typical serine/threonine kinase fold comprising two domains [N-terminal domain (NTD), residues 33-120; C-terminal domain (CTD), residues 129-305], which are linked by a hinge region with a unique LERPXPX motif and the gatekeeper residue (Leu120). The ATP binding pocket is between the NTD and CTD, and it is surrounded by the hinge region, glycine-rich loop (G-loop, residues 46-54), and activation loop (A-loop, residues 191-202) [5] (Fig. 2A). For the inhibitor-bound structure, electron density was clearly observed at the ATP binding pocket and assigned as the SKI-O-068 inhibitor (Fig. 4A). The A-loop comprises the conserved DFG motif, and the A-loops for the SKI-O-068-bound and inhibitor-free Pim1 structure show an active ''DFG-in'' conformation, which is similar to other Pim1 structures. Hydrogen bonds between Lys67 and Glu89 facilitate a sustained active A-loop conformation [5]. Moreover, Lys67 is critical to Pim1 catalytic activity and in ATP-bound structures has been shown to form multiple hydrogen bonds with Asp186 (Asp residue of the 'DFG' motif), a magnesium ion, and an ATP phosphate group [10,27]. The structural conformation and hydrogen bond networks among Lys67, Glu89, and Asp186 are well conserved in our inhibitor-bound and inhibitor-free structures (Fig. 4A) [10,14,28]. Previous reports have shown that Pim1 adopts a constitutively active conformation regardless of the phosphorylation state, which suggests that its activity is regulated through expression and protein stability [4,10]. Two strategies have been suggested for kinase inhibition: inhibition of the active kinase conformation and a stabilized inactive kinase conformation [29]. Therefore, the constitutively active Pim1 conformation offers a great advantage because only one kinase structure conformation must be considered for inhibitor design. A conformational change in the G-loop, including a Phe49 flip, has been reported in certain cases [10,14,26,30]. A conformational change in the G-loop was not observed in the SKI-O-068-bound Pim1 structure.
In conclusion, we discovered a potential Pim1 inhibitor, SKI-O-068, and we produced crystal structures for both the inhibitorbound and inhibitor-free forms. In the crystal structure, the SKI-O-068 inhibitor-bound Pim1 was distinct from structurally similar scaffold inhibitors. Structural information and a binding mode analysis for SKI-O-068 will facilitate improved rational drug design. The in vitro ADME/TOX testing on SKI-O-068 as well as rational design to improve inhibitor is now in progress. Further studies on cytotoxicity and pharmacokinetics/pharmacodynamics should be completed for further development of this drug as an anticancer agent.