BIX 02189

BIX 02189 is a selective inhibitor of MEK5 with IC50 value of 1.5 nM ¹.

BIX 02189 belongs to the indolinone kinase inhibitor series. It selectively inhibited the catalytic activity of MEK5 but not other closely related kinases such as MEK1, MEK2, ERK2 and JNK2. BIX 02189 also inhibited ERK5 with IC50 value of 59 nM. In HeLa cells, treatment of BIX 02189 inhibited the phosphorylation of ERK5 but not ERK1/2. Besides that, BIX 02189 prevented the transcription of the downstream substrate MEF2C in HeLa and HEK293 cells. Moreover, in a three-dimensional lymphangiogenic sprouting assay, BIX 02189 resulted in an inhibition of lymphangiogenic sprouting with a minimal effective concentration of 1 μM ^1,2.

References:
1. Tatake R J, O’Neill M M, Kennedy C A, et al. Identification of pharmacological inhibitors of the MEK5/ERK5 pathway. Biochemical and biophysical research communications, 2008, 377(1): 120-125.
2. Schulz M M P, Reisen F, Zgraggen S, et al. Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis. Proceedings of the National Academy of Sciences, 2012, 109(40): E2665-E2674.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.[Pubmed:28114389]

PLoS One. 2017 Jan 23;12(1):e0169092.

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells' chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 muM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

Beneficial effects of diazepin-quinazolin-amine derivative (BIX-01294) on preimplantation development and molecular characteristics of cloned mouse embryos.[Pubmed:27477633]

Reprod Fertil Dev. 2017 Jun;29(6):1260-1269.

Somatic cell nuclear transfer is frequently associated with abnormal epigenetic modifications that may lead to the developmental failure of cloned embryos. BIX-01294 (a diazepine-quinazoline-amine derivative) is a specific inhibitor of the histone methyltransferase G9a. The aim of the present study was to investigate the effects of BIX-01294 on development, dimethylation of histone H3 at lysine 9 (H3K9), DNA methylation and the expression of imprinted genes in cloned mouse preimplantation embryos. There were no significant differences in blastocyst rates of cloned embryos treated with or without 0.1muM BIX-01294. Relative to clone embryos treated without 0.1muM BIX-01294, exposure of embryos to BIX-01294 decreased histone H3K9 dimethylation and DNA methylation in cloned embryos to levels that were similar to those of in vivo-fertilised embryos at the 2-cell and blastocyst stages. Cloned embryos had lower expression of octamer-binding transcription factor 4 (Oct4) and small nuclear ribonucleoprotein N (Snrpn), but higher expression of imprinted maternally expressed transcript (non-protein coding) (H19) and growth factor receptor-bound protein 10 (Grb10) compared with in vivo-fertilised counterparts. The addition of 0.1muM BIX-01294 to the activation and culture medium resulted in lower H19 expression and higher cyclin dependent kinase inhibitor 1C (Cdkn1c) and delta-like 1 homolog (Dlk1) expression, but had no effect on the expression of Oct4, Snrpn and Grb10. The loss of methylation at the Grb10 cytosine-phosphorous-guanine (CpG) islands in cloned embryos was partially corrected by BIX-01294. These results indicate that BIX-01294 treatment of cloned embryos has beneficial effects in terms of correcting abnormal epigenetic modifications, but not on preimplantation development.

Effect of BIX-01294 on H3K9me2 levels and the imprinted gene Snrpn in mouse embryonic fibroblast cells.[Pubmed:26285804]

Biosci Rep. 2015 Aug 18;35(5). pii: BSR20150064.

Histone H3 lysine 9 dimethylation (H3K9me2) hypermethylation is thought to be a major influential factor in cellular reprogramming, such as somatic cell nuclear transfer (SCNT) and induction of pluripotent stem cells (iPSCs). The diazepin-quinazolin-amine derivative (BIX-01294) specifically inhibits the activity of histone methyltransferase EHMT2 (euchromatic histone-lysine N-methyltransferase 2) and reduces H3K9me2 levels in cells. The imprinted gene small nuclear ribonucleoprotein N (Snrpn) is of particular interest because of its important biological functions. The objective of the present study was to investigate the effect of BIX-01294 on H3K9me2 levels and changes in Snrpn DNA methylation and histone H3K9me2 in mouse embryonic fibroblasts (MEFs). Results showed that 1.3 muM BIX-01294 markedly reduced global levels of H3K9me2 with almost no cellular toxicity. There was a significant decrease in H3K9me2 in promoter regions of the Snrpn gene after BIX-01294 treatment. A significant increase in methylation of the Snrpn differentially methylated region 1 (DMR1) and slightly decreased transcript levels of Snrpn were found in BIX-01294-treated MEFs. These results suggest that BIX-01294 may reduce global levels of H3K9me2 and affect epigenetic modifications of Snrpn in MEFs.

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.[Pubmed:26604326]

Reproduction. 2016 Jan;151(1):39-49.

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

BIX02189 is a selective inhibitor of MEK5 with IC50 of 1.5 nM, also inhibits ERK5 catalytic activity with IC50 of 59 nM in cell-free assays, and does not inhibit closely related kinases MEK1, MEK2, ERK2, and JNK2.

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