Human PIM1 in complex with a consensus peptide and the JNK Inhibitor V

Target ID:

PIM1A

Aliases:

PIMPIM1

Deposition Date:

Friday 25th April 2008

Families:

Protein Kinase

Related Diseases:

Chromatin and epigeneticsSignalling

Structure type:

protein-ligand

Download Coordinates:

3CY3

Authors:

Filippakopoulos, P., Bullock, A., Fedorov, O., Pike, A.C.W., von Delft, F., Arrowsmith, C.H., Edwards, A., Bountra, C., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

3CY3

tabs

Structure Details

PIM-1 is a serine/threonine kinase that was first discovered as a preferential proviral insertion site in Moloney Murine Leukemia Virus (MoMuLV) induced T-cell lymphomas. PIM-1 is over-expressed in a series of tumors but highest expression levels are found in cells of the hematopoietic and lymphoid system [1]. PIM-1 is implicated in the proliferation and differentiation of blood cells, regulates apotosis and has been linked to the development of haematopoetic malignacies and other tumors [2] [3]. Using thermal shift assays we identified AS601245 (1,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl) ethyl)amino)-4-pyrimidinyl)acetonitrile) as a potent inhibitor of PIM-1 kinase. AS601245 is a cell-permeable pyrimidinyl compound with anti-inflammatory properties. This inhibitor has been described previously to inhibit kinases of the c-Jun N-terminal kinase family (JNK, IC50 = 150, 220, and 70 nM for hJNK1, hJNK2, and hJNK3, respectively) and in vivo anti-inflammatory efficacy has been demonstrated in mice [4] [5] [6].

Materials and Methods

Entry Clone Source: TKC

Entry Clone Accession: n/a

SGC Construct ID: PIM1A-c001

GenBank GI number: gi|4505811

Entry clone accession/ sequence: The expressed protein has the sequence from gi 33304198 which differs from gi 4505811 by a single change R250G (bold in the sequence below)

Expressed protein sequence:
mhhhhhhssgvdlgtenlyfqsMLLSKIN
SLAHLRAAPCNDLHATKLAPGKEKEPLES
QYQVGPLLGSGGFGSVYSGIRVSDNLPVA
IKHVEKDRISDWGELPNGTRVPMEVVLLK
KVSSGFSGVIRLLDWFERPDSFVLILERP
EPVQDLFDFITERGALQEELARSFFWQVL
EAVRHCHNCGVLHRDIKDENILIDLNRGE
LKLIDFGSGALLKDTVYTDFDGTRVYSPP
EWIRYHRYHGRSAAVWSLGILLYDMVCGD
IPFEHDEEIIGGQVFFRQRVSSECQHLIR
WCLALRPSDRPTFEEIQNHPWMQDVLLPQ
ETAEIHLHSLSPGPS

Vector: pLIC- SGC1. Details [PDF]; Sequence [FASTA] or [GenBank]

Tags and additions: mhhhhhhssgvdlgtenlyfq*s(m). N-terminal his6 tag, TEV-protease cleavable (*).

Host: BL21(DE3)

Growth medium, induction protocol: 5 ml overnight cultures in LB, 100 µg/ml ampicillin were grown at 37°C and 0.5 ml used to inoculate 1 litre of LB medium containing 100 µg/ml ampicillin. Cultures were grown at 37°C until they reached an OD₆₀₀ of 1.2 and then cooled to 18°C over 20 minutes. Expression was then induced overnight using 0.5 mM IPTG. The cells were collected by centrifugation, transferred to 50 ml tubes, resuspended in 30 ml binding buffer, and frozen. Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol.

Extraction buffer, extraction method: The frozen cells were thawed on ice and 0.5mM TCEP and 1 mM PMSF added. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 17,000 RPM for 30 minutes. Supernatant was collected and binding buffer was added to 50 ml.

Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatmann), 10 gr of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, then washed with 20 ml binding buffer prior to loading the sample.

Buffers: Binding buffer

Procedure: Supernatant was applied at gravity flow, followed by a wash with 40 ml binding buffer. The column flow-through was collected.

Column 2: Ni-affinity. Ni-sepharose (Amersham), 5 ml of 50% slurry in 1.5 x 10 cm column, equilibrated with binding buffer.

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole, 5% Glycerol.

Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 50 ml wash buffer under gravity flow. The protein was eluted by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 250 mM); fractions were collected until essentially all protein was eluted. After elution DTT was added to a final concentration of 10 mM.

Enzymatic treatment: Dephosphorylation (a GST fusion with the lambda phosphatase) and TEV protease cleavage.
Samples containing Pim1 were pooled and treated with lambda phosphatase and TEV protease overnight at 4°C. Protein was kept in elution buffer with the addition of 10 mM DTT and 0.05 mM MnCl₂ (higher MnCl₂ concentrations caused precipitation).

Column 3: HiLoad 16/60 Superdex 200 gel filtration

Buffer: 50 mM Hepes pH 7.5, 250 mM NaCl

Procedure: Dephosphorylated Pim1 protein was concentrated to 3 ml and ran on a S200 gel filtration column collecting 1.8 ml fractions. 10 mM DTT was added to the eluted protein for overnight storage

Column 4: Ion exchange Mono Q column.

Buffers: A : 50 mM Hepes pH 7.5. B : 50 mM Hepes pH 7.5, 1000 mM NaCl.

Procedure: Pim1 was applied to MonoQ in buffer A and eluted from the column by a linear gradient.

Concentration: Pim1 fractions containing dephosphorylated protein were pooled and concentrated in Centricons (10 kDa cut off). Phosphorylation state was confirmed using LC- ESI MS-Tof.

Mass spec characterization: The purified protein was homogeneous and had an experimental mass of 35625 Da as expected from its primary structure and a single phosphorylation. Masses were determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid.

Crystallization: AS601245 (1,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl) ethyl)amino)-4-pyrimidinyl)acetonitrile) was purchased from Calbiochem (Cat. Number: 420129). A 6 ml solution with 1 mg of Pim1 buffered in 50 mM Hepes pH 7.5, 250mM NaCl, 10mM DTT was prepared containing 2 µl of a 50 mM AS601245 stock in DMSO and 20 µl of a 5 mM solution of consensus peptide (ARKRRRHPSGPPTA-amide). This sample was then concentrated to 100 µl to yield a 10 mg/ml crystal sample. Crystals were grown at 4°C in 0.5 µl sitting drops mixing 0.4 µl protein with 0.1 µl of a reservoir solution containing 9 % PEG 10K, 7.2 % Ethylene Glycol and 0.09 M HEPES pH 7.5.

Data Collection: Resolution: 2.15Å, X-ray source: rotating anode (Rigaku FR-E SuperBright), single wavelength 1.5Å.

References

1. Wang, Z., et al., Pim-1: a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis. J Vet Sci, 2001. 2(3): p. 167-79.
2. Pogacic, V., et al., Structural analysis identifies imidazo[1,2-b]pyridazines as PIM kinase inhibitors with in vitro antileukemic activity. Cancer Res, 2007. 67(14): p. 6916-24.
3. Holder, S., et al., Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase. Mol Cancer Ther, 2007. 6(1): p. 163-72.
4. Gaillard, P., et al., Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase. J Med Chem, 2005. 48(14): p. 4596-607.
5. Carboni, S., et al., Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia. J Neurochem, 2005. 92(5): p. 1054-60.
6. Ferrandi, C., et al., Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol, 2004. 142(6): p. 953-60.