Fluorescence polarization binding assay to develop inhibitors of inactive p38alpha mitogen-activated protein kinase.

Development of inhibitors that target inactive kinase conformations is becoming a more attractive approach to kinase inhibitor research. The major advantage of this methodology is that targeting the inactive conformation reduces competition with high intracellular adenosine triphosphate (ATP) concentrations. p38alpha Mitogen-activated protein kinase (MAPK) signaling has been identified as the principal mediator of inflammation associated with a spectrum of disorders (e.g., arthritis, Alzheimer's disease, various malignancies). To allow identification and development of p38alpha MAPK inhibitors that preferentially bind to the inactive conformation, a novel fluorescence polarization-based binding assay is presented. The assay is homogeneous, requires low amounts of the kinase and fluoroprobe, and does not rely on radioactivity. It may, therefore, offer an inexpensive alternative to current p38alpha MAPK inhibitor screening methods. The validation of the system with known p38alpha MAPK inhibitors confirmed that the binding assay, rather than the conventional enzyme activity assay, correlates with cellular efficacy. Finally, we show that pyridinyl imidazoles that potently bind to the inactive p38alpha MAPK prevent activation of p38 MAPK in living cells, suggesting that pyridinyl imidazoles other than SB203580 are able to induce the DFG-out conformation that is incompatible with activation (where DFG is a single-letter amino acid code for the aspartate-phenylalanine-glycine sequence at the start of the activation loop).

Tri- and tetrasubstituted imidazoles as p38α mitogen-activated protein kinase inhibitors.

The synthesis of 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles as potent p38α mitogen-activated protein kinase inhibitors is described. The trisubstituted imidazole series was found to be more potent than the tetrasubstituted imidazole series. Many of these compounds show low-nanomolar activities in the isolated p38α MAP kinase inhibition assay. The structure-activity relationships between these two series are different and not comparable.

N-{4-[4-(4-Fluoro-phen-yl)-1-methyl-2-[(R)-methyl-sulfin-yl]-1H-imidazol-5-yl]-2-pyridyl}acetamide dihydrate.

In the crystal structure of the title compound, C(18)H(17)FN(4)O(2)S·2H(2)O, the organic mol-ecules are linked by inter-molecular O-H⋯O, O-H⋯N and N-H⋯O hydrogen bonds with the water mol-ecules, generating a three-dimensional network. The imidazole ring system forms a dihedral angle of 24.9 (2)° with the 4-fluoro-phenyl ring. The pyridine ring is oriented approximately perpendicular [72.24 (8)°] to the imidazole ring system.

Scoliosis correction with pedicle screws in Duchenne muscular dystrophy.

This report describes the spinal fixation with pedicle-screw-alone constructs for the posterior correction of scoliosis in patients suffering from Duchenne muscular dystrophy (DMD). Twenty consecutive patients were prospectively followed up for an average of 5.2 years (min 2 years). All patients were instrumented from T3/T4 to the pelvis. Pelvic fixation was done with iliac screws similar to Galveston technique. The combination of L5 pedicle screws and iliac screws provided a stable caudal foundation. An average of 16 pedicle screws was used per patient. The mean total blood loss was 3.7 l, stay at the intensive care unit was 77 h and hospital stay was 19 days. Rigid stabilisation allowed immediate mobilisation of the patient in the wheel chair. Cobb angle improved 77% from 44 degrees to 10 degrees, pelvic tilt improved 65% from 14 degrees to 3 degrees. Lumbar lordosis improved significantly from 20 degrees to 49 degrees, thoracic kyphosis remained unchanged. No problems related to iliac fixation, no pseudarthrosis or implant failures were observed. The average percentage of predicted forced vital capacity (%FVC) of the patients was 55% (22-94%) preoperatively and decreased to 44% at the last follow-up. There were no pulmonary complications. One patient with a known cardiomyopathy died intraoperatively due to a sudden cardiac arrest. The rigid primary stability with pedicle screws allowed early mobilisation of the patients, which helped to avoid pulmonary complications.

Synthesis, biological testing, and binding mode prediction of 6,9-diarylpurin-8-ones as p38 MAP kinase inhibitors.

Based on the purine scaffold of ATP, derivatives of 6,9-diarylpurine-8-one were prepared and tested for their ability to inhibit p38 MAP kinase, a key enzyme in the cellular regulation of proinflammatory cytokines. The inhibitor design combines the purine system of the authentic cosubstrate ATP with various phenyl moieties to explore the selectivity for the two hydrophobic regions of the kinase's ATP-binding cleft. The present study indicates a new binding mode of our scaffold to p38 MAP kinase, which comprises the desired structural features of ATP and the N-phenyl-N-purin-6-yl ureas previously published by Wan et al. Combinations of Autodock and FlexX docking with different scoring functions were used to assess the postulated binding mode. The predictive power of different docking-scoring combinations was determined. The presented results may form a solid basis for further optimization cycles since our theoretical findings are consistent with our experimental binding data and supported by the literature.

Synthesis and biological testing of purine derivatives as potential ATP-competitive kinase inhibitors.

On the basis of ATP adenine, a series of adenine and purine derivatives was prepared and tested for their ability to inhibit a spectrum of disease-related kinases. There has been scant research investigating the potential of cosubstrate derived kinase inhibitors for other kinases than CDKs. Our inhibitor design combined the purine system from the original cosubstrate ATP and phenyl moieties in order to explore possible interactions with the different regions of the ATP binding site in several disease-related protein kinases. There have been a number of hits for the assayed substances, which led us to conclude that the spectrum of compounds may prove to be a valuable tool kit for the evaluation of bonding and selectivity patterns for a wide variety of kinases.

Synthesis and biological testing of N-aminoimidazole-based p38alpha MAP kinase inhibitors.

The p38 mitogen-activated protein (MAP) kinase alpha plays a central role in the regulation of cellular responses such as differentiation, proliferation, apoptosis, and inflammation. Inhibition of p38 results in decreased synthesis of pro-inflammatory cytokines. To date, diverse p38alpha inhibitors are in phase II clinical trials for numerous cytokine-dependent diseases. 2-Sulfanylimidazole derivatives offer advantages over the prototype inhibitor SB 203580, including fewer cytochrome P450 interactions and better kinetic properties. The aim of this study was to develop novel 1,2,4,5-tetrasubstituted pyridinylimidazoles with acyl residues at the imidazole N1 position that can interact with the kinase's hydrophobic region II (HR II) or sugar pocket (SP) to improve both selectivity and activity. The substitution pattern was optimized by variation of the acyl moiety at the N1 position of the N-aminoimidazole core. Acylation of the amino function was used for optimization and led to potent p38alpha MAPK inhibitors.

3,4-Diaryl-isoxazoles and -imidazoles as potent dual inhibitors of p38alpha mitogen activated protein kinase and casein kinase 1delta.

In this study, we report on the discovery of isoxazole 1 as a potent dual inhibitor of p38alpha (IC(50) = 0.45 microM) and CK1delta (IC(50) = 0.23 microM). Because only a few effective small molecule inhibitors of CK1 have been described so far, we aimed to develop this structural class toward specific agents. Molecular modeling studies comparing p38alpha/CK1delta suggested an optimization strategy leading to design, synthesis, biological characterization, and SAR of highly potent compounds including 9 (IC(50) p38alpha = 0.006 microM; IC(50) CK1delta = 1.6 microM), 13 (IC(50) p38alpha = 2.52 microM; IC(50) CK1delta = 0.033 microM), 17 (IC(50) p38alpha = 0.019 microM; IC(50) CK1delta = 0.004 microM; IC(50) CK1epsilon = 0.073 microM), and 18 (CKP138) (IC(50) p38alpha = 0.041 microM; IC(50) CK1delta = 0.005 microM; IC(50) CK1epsilon = 0.447 microM) possessing differentiated specificity. Selected compounds were profiled over 76 kinases and evaluation of their cellular efficacy showed 18 (CKP138) to be a highly potent and dual-specific inhibitor of CK1delta and p38alpha.

2-Acylaminopyridin-4-ylimidazoles as p38 MAP kinase inhibitors: Design, synthesis, and biological and metabolic evaluations.

Targeting cytokines has become an important focus in the treatment of many inflammatory disorders. p38 MAP kinase (MAPK) is the key enzyme in regulating the biosynthesis and release of pro-inflammatory cytokines such as IL-1beta and TNFalpha. Inhibition of p38 MAPK results in decreased expression of these cytokines. Tri- and tetrasubstituted pyridinylimidazoles are potent inhibitors of p38 MAPK. Substitution on the pyridinyl moiety allows the design of inhibitors that show increased selectivity and activity by targeting the enzyme's hydrophobic region II. The objective of this study was to synthesize novel 1,2,4,5-tetrasubstituted imidazole derivates and to characterize them not only for their ability to inhibit p38 MAPK and modulate cytokine release in human whole blood, but also to evaluate their metabolic stability. Biological data and metabolic studies demonstrate that the introduction of a 2-acylamino function at C2 of the pyridine results in highly efficient and metabolically stable inhibitors relative to C2-alkylamino derivatives. A series of novel candidates was investigated for metabolic stability in human liver microsomes and in human whole blood. Additionally, metabolic S-oxidation was investigated, and possible metabolites were synthesized.

Design, synthesis and characterization of N9/N7-substituted 6-aminopurines as VEGF-R and EGF-R inhibitors.

In this study we report on the design, synthesis and biological characterization of novel N(9) or N(7) arylethanone-substituted 6-aminopurines and 6-methoxypurines, respectively, as EGF-R and VEGF-R inhibitors. The compounds were initially profiled in a panel of 24 cancer-relevant protein kinases. Dependent on the regio-substitution of the purine core we found inhibition activity for EGF-R and VEGF-R with IC(50) values in the microM range. The two novel N(9)/N(7) 2-(6-amino-purine)-1-(1H-indole-3-yl)ethanone derivatives were characterized in an enhanced panel of 78 kinases showing the N(9) derivative to also inhibit MNK1 and IRR while the N(7) isomer was found to be specific for VEGF-R2.

Design, synthesis, and biological evaluation of novel Tri- and tetrasubstituted imidazoles as highly potent and specific ATP-mimetic inhibitors of p38 MAP kinase: focus on optimized interactions with the enzyme's surface-exposed front region.

The synthesis, biological testing, and SAR of novel 2,4,5- and 1,2,4,5-substituted 2-thioimidazoles are described. Amino, oxy, or thioxy substituents at the 2-position of the pyridinyl moiety were evaluated for their contributions to inhibitor potency and selectivity against p38 mitogen activated protein kinase (p38 MAPK) as well as for the ability to minimize cytochrome P450 (CYP450) inhibition. Incorporation of polar substituted (cyclo)aliphatic amino substituents (e.g., tetrahydropyranylamino), which positively interacted with the surface-exposed front region (hydrophobic region II) of the enzyme led to the identification of extremely potent p38 MAPK inhibitors with p38 IC 50 values in the low nanomolar range. Approximately 90 pyridinylimidazole-based compounds with a range of potencies against p38alpha MAP kinase were further investigated for their ability to inhibit the release of tumor necrosis factor-alpha (TNFalpha) and/or interleukin-1beta (IL-1beta) from human whole blood. Some of the most promising drug candidates underwent selectivity profiling against a panel of 17 different kinases besides p38alpha and/or were tested for their interaction potential toward a number of metabolically relevant CYP450 isozymes.