Synthesis, biological properties and structural study of new halogenated azolo[4,5-b]pyridines as inhibitors of CK2 kinase.

The new halogenated 1H-triazolo[4,5-b]pyridines and 1H-imidazo[4,5-b]pyridines were synthesised as analogues of known CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi). Their influence on the activity of recombinant human CK2α, CK2α' and PIM1 kinases was determined. The most active inhibitors were di- and trihalogenated 1H-triazolo[4,5-b]pyridines (4a, 5a and 10a) with IC50 values 2.56, 3.82 and 3.26 µM respectively for CK2α. Furthermore, effect on viability of cancer cell lines MCF-7 (human breast adenocarcinoma) and CCRF-CEM (T lymphoblast leukemia) of all final compounds was evaluated. Finally, three crystal structures of complexes of CK2α1-335 with inhibitors 4a, 5a and 10a were obtained. In addition, new protocol was used to obtain high-resolution crystal structures of CK2α'Cys336Ser in complex with four inhibitors (4a, 5a, 5b, 10a).

Biological properties and structural study of new aminoalkyl derivatives of benzimidazole and benzotriazole, dual inhibitors of CK2 and PIM1 kinases.

The new aminoalkyl-substituted derivatives of known CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) were synthesized, and their influence on the activity of recombinant human CK2 α, CK2 holoenzyme and PIM1 kinases was evaluated. All derivatives inhibited the activity of studied kinases and the most efficient were aminopropyl-derivatives 8b and 14b. These compounds also exerted inhibition of cancer cell lines - CCRF-CEM (acute lymphoblastoid leukemia), MCF-7 (human breast cancer), and PC-3 (prostate cancer) proliferation and their EC50 is comparable with the value for clinically studied CK2 inhibitor CX-4945. Preliminary structure activity relationship analysis indicated that the spacer length affected antitumor potency, and two to three methylene units were more favorable. The complex of CK2 α1-335/8b was crystallized, both under high-salt conditions and under low-salt conditions giving crystals which diffracted X-rays to about 2.4 Šresolution, what enabled the determination of the corresponding 3D-structures.

Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights.

Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical features of the enzyme, such as its constitutive activity, acidophilic substrate specificity, dual-cosubstrate specificity and its heterotetrameric quarternary structure. Comprehensive sets of structural superimpositions reveal that both CK2alpha and CK2beta are relatively rigid molecules. In CK2beta the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP-binding region, suggesting an alternative mode of activity control.

Amino acid similarity coefficients for protein modeling and sequence alignment derived from main-chain folding angles.

A set of "similarity-parameters" was calculated that reflects the influence of the proteinogenic amino acids on the structure of the protein backbone. The parameters were derived from a detailed analysis of the amino acid specific main-chain torsion angle distributions as they are found in proteins (highly resolved protein structures from the Brookhaven Protein Data Bank). The purpose of these parameters is threefold: (1) they should help in estimating the structural effect of an amino acid substitution during the design of new mutants in protein-engineering; (2) in modeling by homology they should mark places in the protein where changes in the folding are expected; and (3) they should form a scoring matrix in protein sequence alignment superior to identity scoring. The usability of the "structure derived correlation matrix (SCM)" for these purposes is assessed and demonstrated for some examples in the paper.

Crystallization and preliminary characterization of crystals of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei.

D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimeric enzyme with a molecular mass of 74.6 kDa. It catalyzes the reduction of a wide range of 2-ketocarboxylic acids to D-2-hydroxycarboxylic acids using NADH as co-substrate. The enzyme has been crystallized by vapor diffusion using ammonium sulfate as precipitant. The crystals belong to hexagonal space group type P6(3)22 with a = b = 134.1 A, c = 124.1 A and diffract X-rays to 3.0 A resolution. Packing considerations show that there are either one or two D-HicDH monomers in the asymmetric unit.

Crystal structure of L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus at 2.2 A resolution. An example of strong asymmetry between subunits.

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus, a homotetramer with a molecular mass of 33 kDa per subunit, belongs to the protein family of the NAD(+)-dependent L-2-hydroxycarboxylate dehydrogenases. L-HicDH was crystallized with ammonium sulphate as precipitant in the presence of NAD+. The crystals belong to the trigonal space group P3(2)21, with a = 135.9 A and c = 205.9 A, and diffract X-rays to 2.2 A resolution. The crystal structure was solved by Patterson search and molecular replacement techniques and refined to an R-value of 21.4% (2.2 to 8 A). The final structure model contains one NAD+ molecule and one sulphate ion per subunit, with 309 water molecules. An unusual feature of this crystal structure is the deviation of the protein subunits from non-crystallographic symmetry, which is so strong that it can be detected globally by self-rotation calculations in reciprocal space. This asymmetry is especially pronounced in the environment of the active site; it is reflected also in the nicotinamide conformation of NAD+ and allows some conclusions to be drawn about the catalytic mechanism. In this context, an "inner active site loop" is identified as a structural element of fundamental functional importance. Furthermore, with knowledge of the crystal structure of L-HicDH the differences in substrate specificity between L-HicDH and the L-lactate dehydrogenases can be partly explained.

Crystal structure of a ternary complex of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 A resolution.

D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD+-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD+ and 2-oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range (infinity to 1.86 A). Both NAD+ and 2-oxoisocaproate were identified in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also defined by electron density. Additionally the final model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.

Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.

Crystallization, preliminary X-ray analysis of a native and selenomethionine D-hydantoinase from Thermus sp.

A D-hydantoinase from Thermus sp. was expressed in Escherichia coli, purified to homogeneity and crystallized both as native and Se-Met labelled protein. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 125.9, b = 215.8, c = 207.5 A. A three-wavelength MAD data set was collected to 2.5 A resolution and a native data set was collected to 1.7 A resolution. Crystal packing and self-rotation calculations led to the assumption of six protomers per asymmetric unit, corresponding to a V(M) value of 2.28 A(3) Da(-1) and a solvent content of 46%. As each protomer contains nine Se-Met residues, 54 selenium sites per asymmetric unit were present and could be unambigously located in the course of the MAD experiment. This selenium substructure is one of the largest selenium substructures that have been solved to date. The resulting phases obtained at a high-resolution limit of 3.0 A could be extended to 1.7 A and refined by application of density-modification techniques, especially non-crystallographic symmetry.

Crystallization and preliminary characterization of crystals of human protein kinase CK2.

The heterotetrameric recombinant holoenzyme of human protein kinase CK2 was purified to homogeneity. It degraded spontaneously to a stable and fully active state in which the catalytic subunit was about 5 kDa smaller than the wild type. The degraded enzyme was crystallized using polyethylene glycol 3350 as precipitant. The crystals belong to the hexagonal space group P6(3). They have unit-cell parameters a = b = 176.0, c = 93.6 A and diffract X-rays to at least 3.5 A resolution. The calculated crystal packing parameter is V(M) = 3.22 A(3) Da(-1), suggesting that one CK2 tetramer is contained in the asymmetric unit and that the solvent content of the unit cell is 62%.

Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme.

The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.

Characterization of CK2 holoenzyme variants with regard to crystallization.

A search for strategies was conducted in order to obtain a human protein kinase CK2 preparation which would be suitable for crystallization, despite the fact that the recombinant enzyme is abundant and can be readily purified to homogeneity. This seemingly contradiction is based on the fact that the catalytic subunit moiety of the human CK2 holoenzyme is not stable neither as a free subunit nor in the tetrameric complex. All attempts to prevent degradation failed. Hence, alternative approaches were designed in order to avoid this degradation, which was expected to hamper any crystallization efforts severely. One of the approaches chosen was the production of a chimeric holoenzyme made up from a human regulatory subunit and a catalytic subunit from Z. mays. The plant catalytic subunit, in contrast to the human counterpart is very stable and does not undergo this kind of degradation. The second strategy to tackle the problem of instability was to produce the homologous recombinant human CK2 holoenzyme and then, instead of trying to avoid degradation, attempt to accelerate degradation until all catalytic subunit material was converted to the degraded form, i.e. a 40 kDa polypeptide.

Crystallization and preliminary characterization of crystals of R-alcohol dehydrogenase from Lactobacillus brevis.

The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is a valuable catalyst for the production of chiral alcohols that can be used as synthons in asymmetric syntheses. RADH is a homotetramer with 222 symmetry and a molecular mass of 107 kDa. The recombinant enzyme has been expressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 56.5, b = 85.1, c = 115.4 A, and diffract X-rays to at least 1.8 A resolution. The calculated crystal packing parameter V(M) = 2.59 A(3) Da(-1), corresponding to a solvent content of 52.5% and suggesting that one RADH monomer is contained in the asymmetric unit. The RADH tetramer lies on a special position with its molecular dyads coinciding with the crystallographic twofold axes and with its centre of mass on the origin of the unit cell.

Crystallization and preliminary X-ray analysis of a hydantoinase from Arthrobacter aurescens DSM 3745.

L-Hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity and crystallized from polyethylene glycol solutions in a form suitable for X-ray diffraction analysis. The crystals have been grown by the sitting-drop variant of the vapour-diffusion method. X-ray diffraction studies show that the crystals belong to the monoclinic space group P2(1) with a = 111.2, b = 74.4, c = 146.5 A and beta = 106.7 degrees. Its asymmetric unit contains four monomers related by 222 symmetry. The crystals diffract to at least 2.6 A.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays.

The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters a = 142.6, b = 61.3, c = 45.6 A, beta = 103.3 degrees and diffract X-rays to at least 2.0 A resolution. The calculated crystal packing parameter is Vm = 2.47 A3 Da-1 suggesting that one CK2alpha molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.

Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A resolution.

CK2alpha is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 A resolution, of recombinant maize CK2alpha (rmCK2alpha) in the presence of ATP and Mg2+, shows the enzyme in an active conformation stabilized by interactions of the N-terminal region with the activation segment and with a cluster of basic residues known as the substrate recognition site. The close interaction between the N-terminal region and the activation segment is unique among known protein kinase structures and probably contributes to the constitutively active nature of CK2. The active centre is occupied by a partially disordered ATP molecule with the adenine base attached to a novel binding site of low specificity. This finding explains the observation that CK2, unlike other protein kinases, can use both ATP and GTP as phosphorylating agents.

GTP plus water mimic ATP in the active site of protein kinase CK2.

The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs that target CK2 or other kinases with this property.