Human Proviral Integration Site for MulV in Complex with an Imidazopyridazin I

Target ID:

PIM1A

Aliases:

PIMPIM1

Deposition Date:

Friday 7th October 2005

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2C3I

Authors:

P.PHILIPPAKOPOULOS,S.KNAPP,J.DEBRECZENI,A.BULLOCK,F.VON DELFT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,K.GUO,J.WEIGELT

Site:

Oxford

ICM Datapack:

ICM Datapack

2C3I

tabs

Structure Details

Recently we identified inhibitors of the i midazo[1,2-b]pyridazine and pyrazolo[1,5-a]pyrimidine type as strong inhibitors for PIM-1 (1). One of the most potent compound of these two inhibitor classes was 1-(3-{6-[(cyclopropylmethyl)amino]imidazo[1,2-b] pyridazine-3-yl}phenyl)ethanone ( imidazo[1,2-b]pyridazine 1) which had an IC 50 for PIM-1 of 61 nM and a dissociation constant measured by ITC of 25nM. Pyrazolopyrimidine-type inhibitors were first described as specific inhibitors of the Src-family of tyrosine kinases with several pyrazolo[3,4-d]pyrimidines having low nanomolar affinity for Lck and anti-cancer activity (2). One of these compounds PP1 was later crystallized in complex with the autoinhibited form of Hck, providing further guidance for drug development (3). Subsequently this scaffold yielded specific inhibitors for glycogen synthase kinase-3 (GSK-3) and members of the CDK family (4, 5). Inhibitors based on the variant pyrazolo[1,5-a]pyrimidine scaffold have also been described with specificity for CDK2 (6) and the KDR kinase domain (7). Additionally, CDK2 has been co-crystallized with both imidazo[1,2-a]pyridine and imidazo[1,2-b]pyridazine (8, 9) compounds (10).

Materials and Methods

Entry Clone Source: TKC

SGC Construct ID: PIM1A-c001

GenBank GI number: gi|4505811

Entry clone accession : The expressed protein has the sequence from gi|33304198 which has the isoform change R250G compared to gi|4505811.

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

Vector: pLIC- SGC

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

Host : BL21(DE3)

Growth medium, induction protocol: 1 ml from a 50 ml overnight culture in LB, 100µg/ml ampicillin was used to inoculate 1 litre of LB medium containing 100µg/ml ampicillin. Cultures were grown at 37°C until they reached an OD 600 of 0.25 and then cooled to 18°C. Expression was induced for 4 hours using 1 mM IPTG at an OD 600 of 0.6. The cells were collected by centrifugation, and each 2 litre equivalent transferred into 25 ml binding buffer to 50 ml tubes, and frozen at -20°C. Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol.

Extraction buffer, extraction method: The frozen cells were thawed on ice. Binding buffer (plus 1 mM PMSF) was added to a final volume of 50 ml. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 17,000 RPM for 30 minutes and the supernatant collected for purification.

Column 1 : DEAE cellulose (DE52, Whatmann), 10 g of resin in 2.5 x 20 cm column

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol

Procedure: Ion exchange - Nucleic acid removal. The resin was hydrated in 2.5M NaCl, then washed with 50 ml binding buffer prior to loading the sample. Supernatant was applied at gravity flow, followed by a wash with 50 ml binding buffer. The column flow-through was collected.

Column 2 : Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column.

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-NTA 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, 150 and 250 mM); fractions were collected until essentially all protein was eluted. After elution DTT was added to a final concentration of 10 mM.

Column 3: Ion exchange Mono Q column

Buffers: A : 50 mM Hepes pH 7.5; B : 50 mM Hepes pH 7.5, 2 M NaCl

Procedure: Ion exchange was performed after overnight enzymatic treatment at 4°C (see below). Partially dephosphorylated PIM1 was applied to a 1 ml MonoQ column in buffer A and eluted from the column by a linear gradient with buffer B. Non-phosphorylated PIM1 eluted at ~250 mM NaCl and was resolved from some residual singly phosphorylated PIM1 (at Ser261) which eluted at ~280 mM NaCl.

Concentration : Non-phosphorylated PIM1 sample was pooled and concentrated in Centricons (10 kDa cut off) to 10 mg/ml.

Enzymatic treatment : (Dephosphorylation and Histag cleavage) Prior to monoQ, samples containing Ni-NTA purified PIM1 were pooled and 20µg GST-lambda phosphatase and 20µg TEV protease added for overnight incubation at 4°C. Protein solution contained 10 mM DTT and 0.05 mM MnCl₂(higher MnCl₂ concentrations caused precipitation).

Mass spec characterization : Masses of purified proteins were confirmed 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. The purified PIM1 protein was homogeneous and had an experimental mass of 35546 Da as expected from its primary structure.

Crystallisation: Non-phosphorylated PIM1 sample was concentrated in the presence of ligands to final concentrations of 10 mg/ml PIM1, 0.7mM pimtide (ARKRRRHPSGPPTA-amide) and 1mM imidazopyridazine inhibitor. Crystals were grown in sitting drops at 4°C by mixing 100 nL of this sample with 50 nL of precipitant (40% PEG 300, 0.1M Tris pH 8.5, 0.2M Li₂SO₄). The imidazopyridazine inhibitor (1-(3-{6-[(cyclopropylmethyl)amino]imidazo [1,2-b]pyridazin-3-yl}phenyl)ethanone) was purchased from BioFocus, compound ID 229_4051_4145) and was added from a 50 mM stock in DMSO.

Data Collection/Resolution: (PDB 2C3I) 1.9Å , X-ray source: Swiss Light Source synchrotron beamline SLS -X10.

References

1. Alex N. Bullock, Judit É. Debreczeni, Oleg Y. Fedorov, Adam Nelson, Brian D. Marsden, Stefan Knapp (2005). Structural basis of inhibitor specificity of the human proto-oncogene proviral insertion site in Moloney Murine Leukemia Virus (PIM-1) kinase. J. Med. Chem., in press.

2. Hanke, J. H.; Gardner, J. P.; Dow, R. L.; Changelian, P. S.; Brissette, W. H.; Weringer, E. J.; Pollok, B. A.; Connelly, P. A. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent cell activation. J. Biol. Chem . 1996 , 271, 695-701.

3. Schindler, T.; Sicheri, F.; Pico, A.; Gazit, A.; Levitzki, A.; Kuriyan, J. Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. Mol Cell . 1999 , 3, 639-648.

4. Markwalder, J. A.; Arnone, M. R.; Benfield, P. A.; Boisclair, M.; Burton, C. R.; Chang, C. H.; Cox, S. S.; Czerniak, P. M.; Dean, C. L.; Doleniak, D.; Grafstrom, R.; Harrison, B. A.; Kaltenbach, R. F. 3 rd .; Nugiel, D. A.; Rossi, K. A.; Sherk, S. R.; Sisk, L. M.; Stouten, P.; Trainor, G. L.; Worland, P.; Seitz, S. P. Synthesis and biological evaluation of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one inhibitors of cyclin-dependent kinases. J. Med. Chem . 2004 , 18, 5894-5911.

5. Moravcova, D.; Krystof, V.; Havlicek, L.; Moravec, J.; Lenobel, R.; Strnad, M. Pyrazolo[4,3-d]pyrimidines as new generation of cyclin-dependent kinase inhibitors. Bioorg Med Chem Lett . 2003, 13, 2989-2992.

6. Williamson, D. S.; Parratt, M. J.; Bower, J. F.; Moore, J. D.; Richardson, C. M.; Dokurno, P.; Cansfield, A. D.; Francis, G. L.; Hebdon, R. J.; Howes, R.; Jackson, P. S.; Lockie, A. M.; Murray, J. B.; Nunns, C. L.; Powles, J.; Robertson, A.; Surgenor, A. E.; Torrance, C. J. Structure-guided design of pyrazolo[1,5-a]pyrimidines as inhibitors of human cyclin-dependent kinase 2. Bioorg Med Chem Lett . 2005 , 15, 863-867.

7. Fraley, M. E.; Hoffman, W. F.; Rubino, R. S.; Hungate, R. W.; Tebben, A. J.; Rutledge, R. Z.; McFall, R. C.; Huckle, W. R.; Kendall, R. L.; Coll, K. E.; Thomas, K. A. Synthesis and initial SAR studies of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines: a new class of KDR kinase inhibitors. Bioorg Med Chem Lett . 2002 , 12, 2767-2770.

8. Anderson, M.; Beattie, J. F.; Breault, G. A.; Breed, J.; Byth, K. F.; Culshaw, J. D.; Ellston, R. P.; Green, S.; Minshull, C. A.; Norman, R. A.; Pauptit, R. A.; Stanway, J.; Thomas, A. P.; Jewsbury, P. J. Imidazo[1,2-a]pyridines: a potent and selective class of cyclin-dependent kinase inhibitors identified through structure-based hybridisation. Bioorg. Med. Chem. Lett . 2003 , 13, 3021-3026.

9. Hamdouchi, C.; Zhong, B.; Mendoza, J.; Collins, E.; Jaramillo, C.; De Diego, J. E.; Robertson, D.; Spencer, C. D.; Anderson, B. D.; Watkins, S. A.; Zhang, F.; Brooks, H. B. Structure-based design of a new class of highly selective aminoimidazo[1,2-a]pyridine-based inhibitors of cyclin dependent kinases. Bioorg. Med. Chem. Lett . 2005 , 17, 1943-1947.

10. Byth, K. F.; Cooper, N.; Culshaw, J. D.; Heaton, D. W.; Oakes, S. E.; Minshull, C. A.; Norman, R. A.; Pauptit, R. A.; Tucker, J. A.; Breed, J.; Pannifer, A.; Rowsell, S.; Stanway, J. J.; Valentine, A. L.; Thomas, A. P. Imidazo[1,2-b]pyridazines: a potent and selective class of cyclin-dependent kinase inhibitors. Bioorg. Med. Chem. Letter . 2004 , 14, 2249-2252