Microwave-assisted synthesis of a MK2 inhibitor by Suzuki-Miyaura coupling for study in Werner syndrome cells

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells

Chromatin structure and DNA methylation

The DNA of eukaryotes has been shown to contain the minor base 5-methylcytosine. This arises by the enzymic modification of cytosine already incorporated into DNA. The distribution of methylation in chromatin is not random but there are distinct methylated and unmethylated domains, one such unmethylated domain consists of transcriptionally active DNA. The work undertaken during the tenure of this award has been to examine the effects of chromatin structure and chromatin constituents upon DNA methylation, with the aim of finding a specific inhibitor of methylation which may be responsible for the undermethylation of transcribing regions of chromatin. When the distribution of methyl groups in chromatin is examined, it is found that nucleosomal core DNA is enriched in these groups when compared to linker DNA. This DNA is also enriched in the bases cytosine and guanine. When nuclei from log phase cells are methylated in vitro with the endogenous methylase, the methyl groups are added predominantly to core DNA. As DNA synthesis is not continuing in vitro, and most of the methylation has already occurred in vivo, the methylation in these nuclei is "delayed methylation" Evidence is presented that this methylation is occurring in the nuclear matrix, and the methylation pattern observed simply reflects the pattern in vivo. But when mouse ascites methylase is added to these nuclei, the methyl groups go on to linker DNA, showing that this DNA is more susceptible to methylation than core DNA. These two observations suggest that the methylation pattern found in vivo is not a function of chromatin structure, and it is thought that nucleosomes bind to DNA enriched in cytosine and guanine, and that the enrichment of methylcytosine is a consequence of this. This idea is reinforced by several lines of evidence; i) Histones are very effective inhibitors of DNA methylation in vitro when added to a methylase assay. ii) Chromatin is a poor acceptor of methyl groups compared to DNA. Core particles, which have a higher histone to DNA ratio, are not as good acceptors as chromatin. iii) When native mouse DNA and histones are reconstituted into chromatin, the nucleosomes are again found on methyl rich and cytosine and guanine rich DNA. When transcriptionally competent chromatin is examined, the DNA is found to be deficient in 5-niethylcytosine compared to total chromatin. When nuclei from log phase cells are incubated in vitro the methylation occurs predominantly in the fraction of chromatin containing transcribing regions. As this fraction is also thought to contain DNA associated with the nuclear matrix it is not certain which DNA is being methylated. As the methylation in isolated nuclei is not affected by extraction with 0.2 M NaCl, which removes all of the soluble DNA methylase, this methylation is a product of a bound DNA methylase which is thought to be in the nuclear matrix. Adding soluble DNA methylase to nuclei in vitro results in an elevated methylation of DNA in this fraction, and again the methyl groups are being added to transcribing (matrix) chromatin. When the effects of nuclear non-histone proteins on DNA methylation in vitro are examined no inhibition is observed by either high mobility group proteins or nuclear non-histone proteins in general. These results suggest that there are no specific proteins in transcribing regions which inhibit DNA methylation in vivo

Microwave-assisted synthesis of a MK2 inhibitor by Suzuki-Miyaura coupling for study in Werner syndrome cells

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells

The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells

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Evaluating the role of p38 MAPK in the accelerated cell senescence of Werner syndrome fibroblasts

Progeroid syndromes show features of accelerated ageing and are used as models for human ageing, of which Werner syndrome (WS) is one of the most widely studied. WS fibroblasts show accelerated senescence that may result from p38 MAP kinase activation since it is prevented by the p38 inhibitor SB203580. Thus, small molecule inhibition of p38-signalling may be a therapeutic strategy for WS. To develop this approach issues such as the in vivo toxicity and kinase selectivity of existing p38 inhibitors need to be addressed, so as to strengthen the evidence that p38 itself plays a critical role in mediating the effect of SB203580, and to find an inhibitor suitable for in vivo use. In this work we used a panel of different p38 inhibitors selected for: (1) having been used successfully in vivo in either animal models or human clinical trials; (2) different modes of binding to p38; and (3) different off-target kinase specificity profiles, in order to critically address the role of p38 in the premature senescence seen in WS cells. Our findings confirmed the involvement of p38 in accelerated cell senescence and identified p38 inhibitors suitable for in vivo use in WS, with BIRB 796 the most effective

Assessing the role of stress signalling via p38 MAP kinase in the premature senescence of Ataxia Telangiectasia and Werner syndrome fibroblasts

The premature ageing Ataxia Telangiectasia (AT) and Werner syndromes (WS) are associated with accelerated cellular ageing. Young WS fibroblasts have an aged appearance and activated p38 MAP kinase, and treatment with the p38 inhibitor SB230580 extends their lifespan to within the normal range. SB203580 also extends the replicative lifespan of normal adult dermal fibroblasts, however, the effect is much reduced when compared to WS cells, suggesting that WS fibroblasts undergo a form of stress-induced premature senescence (SIPS). A small lifespan extension is seen in AT cells, which is not significant compared to normal fibroblasts, and the majority of young AT cells do not have an aged appearance and lack p38 activation, suggesting that the premature ageing does not result from SIPS. The lack of p38 activation is supported by the clinical manifestation, since AT is not associated with inflammatory disease, whereas WS individuals are predisposed to atherosclerosis, type II diabetes and osteoporosis, conditions known to be associated with p38 activation