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1.
J Med Chem ; 59(16): 7544-60, 2016 08 25.
Article in English | MEDLINE | ID: mdl-27502541

ABSTRACT

Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.


Subject(s)
Drug Discovery , Imidazoles/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Quinolines/pharmacology , Animals , Crystallography, X-Ray , Dogs , Dose-Response Relationship, Drug , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , MAP Kinase Kinase Kinases/metabolism , Male , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins/metabolism , Quinolines/chemical synthesis , Quinolines/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/antagonists & inhibitors
2.
J Biol Chem ; 290(24): 15210-8, 2015 Jun 12.
Article in English | MEDLINE | ID: mdl-25918157

ABSTRACT

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.


Subject(s)
MAP Kinase Kinase Kinases/chemistry , Protein Folding , Proto-Oncogene Proteins/chemistry , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry
3.
PLoS One ; 9(2): e89615, 2014.
Article in English | MEDLINE | ID: mdl-24586913

ABSTRACT

Chronic low-grade inflammation in adipose tissue often accompanies obesity, leading to insulin resistance and increasing the risk for metabolic diseases. MAP3K8 (TPL2/COT) is an important signal transductor and activator of pro-inflammatory pathways that has been linked to obesity-induced adipose tissue inflammation. We used human adipose tissue biopsies to study the relationship of MAP3K8 expression with markers of obesity and expression of pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8). Moreover, we evaluated obesity-induced adipose tissue inflammation and insulin resistance in mice lacking MAP3K8 and WT mice on a high-fat diet (HFD) for 16 weeks. Individuals with a BMI >30 displayed a higher mRNA expression of MAP3K8 in adipose tissue compared to individuals with a normal BMI. Additionally, high mRNA expression levels of IL-1ß, IL-6 and IL-8, but not TNF -α, in human adipose tissue were associated with higher expression of MAP3K8. Moreover, high plasma SAA and CRP did not associate with increased MAP3K8 expression in adipose tissue. Similarly, no association was found for MAP3K8 expression with plasma insulin or glucose levels. Mice lacking MAP3K8 had similar bodyweight gain as WT mice, yet displayed lower mRNA expression levels of IL-1ß, IL-6 and CXCL1 in adipose tissue in response to the HFD as compared to WT animals. However, MAP3K8 deficient mice were not protected against HFD-induced adipose tissue macrophage infiltration or the development of insulin resistance. Together, the data in both human and mouse show that MAP3K8 is involved in local adipose tissue inflammation, specifically for IL-1ß and its responsive cytokines IL-6 and IL-8, but does not seem to have systemic effects on insulin resistance.


Subject(s)
Adipose Tissue/immunology , Adipose Tissue/pathology , Blood Proteins/analysis , Inflammation/etiology , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Kinase Kinases/physiology , Obesity/complications , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Adipose Tissue/metabolism , Animals , Blotting, Western , Body Mass Index , Cells, Cultured , Diet, High-Fat , Glucose Tolerance Test , Humans , Immunoenzyme Techniques , Inflammation/metabolism , Inflammation/pathology , Insulin/metabolism , Insulin Resistance , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , MAP Kinase Kinase Kinases/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/metabolism , Obesity/pathology , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
4.
Magn Reson Med ; 67(2): 499-509, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21656559

ABSTRACT

Magnetic resonance imaging (MRI) has been used to follow the course of bleomycin-induced lung injury in mice and to investigate two knockout mouse lines with the aim of providing potential therapeutic targets. Bleomycin (0.25 mg/kg) was administered intranasally six times, once a day. MRI was carried out on spontaneously breathing animals up to day 70 after bleomycin. Neither cardiac nor respiratory gating was applied during image acquisition. A long lasting response following bleomycin has been detected by MRI in the lungs of male C57BL/6 mice. Histology showed that, from day 14-70 after bleomycin, fibrosis was the predominant component of the injury. Female C57BL/6 mice displayed a smaller response than males. Bleomycin-induced injury was significantly more pronounced in C57BL/6 than in Balb/C mice. MRI and histology demonstrated a protection against bleomycin insult in female heterozygous and male homozygous cancer Osaka thyroid kinase knockout animals. In contrast, no protection was seen in cadherin-11 knockout animals. In summary, MRI can quantify, in spontaneously breathing mice, bleomycin-induced lung injury. With the ability for repetitive measurements in the same animal, the technique is attractive for in vivo target analysis and compound profiling in this murine model.


Subject(s)
Antibiotics, Antineoplastic/toxicity , Bleomycin/toxicity , Image Enhancement , Image Processing, Computer-Assisted , Lung/drug effects , Magnetic Resonance Imaging , Pulmonary Fibrosis/chemically induced , Administration, Intranasal , Alleles , Animals , Cadherins/genetics , Dose-Response Relationship, Drug , Female , Genetic Carrier Screening , Genetic Predisposition to Disease , Lung/pathology , MAP Kinase Kinase Kinases/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Proto-Oncogene Proteins/genetics , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/pathology , Sex Factors
5.
Arthritis Rheum ; 60(12): 3651-62, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19950280

ABSTRACT

OBJECTIVE: Caspase 1, a known cysteine protease, is a critical component of the inflammasome. Both caspase 1 and neutrophil serine proteases such as proteinase 3 (PR3) can process pro-interleukin-1beta (proIL-1beta), a crucial cytokine linked to the pathogenesis of rheumatoid arthritis. This study was undertaken to establish the relative importance of caspase 1 and serine proteases in mouse models of acute and chronic inflammatory arthritis. METHODS: Acute and chronic arthritis were induced in caspase 1-/- mice, and the lack of caspase 1 was investigated for its effects on joint swelling, cartilage metabolism, and histopathologic features. In addition, caspase 1 activity was inhibited in mice lacking active cysteine proteases, and the effects of dual blockade of caspase 1 and serine proteases on arthritis severity and histopathologic features were evaluated. RESULTS: Surprisingly, caspase 1-/- mice, in a model of acute (neutrophil-dominated) arthritis, developed joint swelling to an extent similar to that in wild-type control mice. Joint fluid concentrations of bioactive IL-1beta were comparable in caspase 1-/- mice and controls. In contrast, induction of chronic arthritis (characterized by minimal numbers of neutrophils) in caspase 1-/- mice led to reduced joint inflammation and less cartilage damage, implying a caspase 1-dependent role in this process. In mice lacking neutrophil serine PR3, inhibition of caspase 1 activity resulted in decreased bioactive IL-1beta concentrations in the synovial tissue and less suppression of chondrocyte anabolic function. In addition, dual blockade of both PR3 and caspase 1 led to protection against cartilage and bone destruction. CONCLUSION: Caspase 1 deficiency does not affect neutrophil-dominated joint inflammation, whereas in chronic arthritis, the lack of caspase 1 results in reduced joint inflammation and cartilage destruction. These findings suggest that inhibitors of caspase 1 are not able to interfere with the whole spectrum of IL-1beta production, and therefore such inhibitors may be of therapeutic value only in inflammatory conditions in which limited numbers of neutrophils are present.


Subject(s)
Arthritis, Rheumatoid/enzymology , Caspase 1/deficiency , Interleukin-1/metabolism , Myeloblastin/metabolism , Protein Precursors/metabolism , Animals , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Caspase 1/genetics , Caspase 1/metabolism , Disease Models, Animal , Enzyme Activation , Joints/metabolism , Joints/pathology , Leukocyte Elastase/metabolism , Macrophages/enzymology , Macrophages/immunology , Mast Cells/enzymology , Mast Cells/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/enzymology , Neutrophils/immunology , Serine Endopeptidases , Synovial Fluid/metabolism , Synovial Membrane/chemistry , Synovial Membrane/metabolism
6.
Top Curr Chem ; 273: 1-14, 2008.
Article in English | MEDLINE | ID: mdl-23605457

ABSTRACT

Key processes in molecular biology are regulated by interactions between biomolecules. Protein-proteinand protein-ligand interactions, e.g., in signal transduction pathways, rely on the subtle interactionsbetween atoms at the binding interface of the involved molecules. Because biomolecules often havemany interacting partners, these interactions are not necessarily strong. The study of molecularrecognition gives insight into the complex network of signaling in life and is the basis of structure-baseddrug design.In the situation where the interaction is weak, one of the traditional methods that can be appliedto obtain structural information (internuclear distances) of the bound ligand is the so-called transferredNOE (trNOE) method. Recently, it became possible to use transferred cross-correlated relaxation (trCCR)to directly measure dihedral angles. The combined use of these two techniques significantly improvesthe precision of the structure determination of ligands weakly bound to macromolecules.The application of these techniques will be discussed in detail for a peptide derived fromIKKß bound to the protein NEMO that plays an important rolein the NFκB pathway.

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