Stereoselective Synthesis of Allele-Specific BET Inhibitors

Developing stereoselective synthetic routes that are efficient and cost-effective is important to allow easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) bromodomain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was confirmed by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitor and a viable route that will facilitate wider access to this compound class.

Stereoselective Synthesis of Allele-Specific BET Inhibitors

Developing stereoselective synthetic routes that are efficient and cost-effective is important to allow easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) bromodomain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was confirmed by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitor and a viable route that will facilitate wider access to this compound class.

Association of MAOA-uVNTR gene polymorphism with hostility in an open population of males aged 45-64 in Russia / Siberia (international program: WHO «MONICA-psychosocial», «HAPIEE»)

Objective: to study the association of hostility with high and low-active variants of the MAOA gene in an open population of men 45-64 years old. Using the methods proposed by the WHO International Program “MONICA-psychosocial” and “HAPIEE”, a representative sample of men aged 45–64 years old (n = 781 men, average age was 56.48 ± 0.2 years) in 2003-2005. All respondents independently completed a questionnaire on hostility. From the surveyed sample, using the random number method, 156 men were selected who were genotyped for MAOA-uVNTR polymorphism. It was approved that the level of hostility in the population of men was 60.3%. It was revealed that among persons with low-active alleles of the MAOA-L gene (allele 2 and 3) a high level of hostility was more common — 50.9%. The results of building a logistic regression model showed that the presence of low-active alleles (2; 3) of the MAOA gene increases the likelihood of hostility OR = 2,103 (95% CI 1,137-3,889, p = 0.018). The results obtained allow us to conclude that the low-active allele of the MAOA-L gene is associated with hostility.

DNA methylation is a key mechanism for maintaining monoallelic expression on autosomes

In diploid cells, maternal and paternal copies of genes usually have similar transcriptional activity. Mammalian allele-specific epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. The discovery of mitotically stable monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. However, despite its prevalence, the mechanistic basis of MAE remains unknown. To uncover the mechanism of MAE maintenance, we devised a small-molecule screen for reactivation of silenced alleles across multiple loci using targeted RNA sequencing. Contrary to previous reports, we identified DNA methylation as a key mechanism of MAE mitotic maintenance. In contrast with the binary choice of the active allele in XCI, stringent transcriptome-wide analysis revealed MAE as a regulatory mode with tunable control of allele-specific expression, dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to changes in DNA methylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance, reveal tunability of this mode of gene regulation, and provide the essential platform for probing the biological role of MAE in development and disease.

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.

A constitutive active allele of the transcription factor Msn2 mimicking low PKA activity dictates metabolic remodeling in yeast

In yeast, protein kinase A (PKA) adjusts transcriptional profiles, metabolic rates, and cell growth in accord with carbon source availability. PKA affects gene expression mostly via the transcription factors Msn2 and Msn4, two key regulators of the environmental stress response. Here we analyze the role of the PKA-Msn2 signaling module using an Msn2 allele that harbors serine-to-alanine substitutions at six functionally important PKA motifs (Msn2A6) . Expression of Msn2A6 mimics low PKA activity, entails a transcription profile similar to that of respiring cells, and prevents formation of colonies on glucose-containing medium. Furthermore, Msn2A6 leads to high oxygen consumption and hence high respiratory activity. Substantially increased intracellular concentrations of several carbon metabolites, such as trehalose, point to a metabolic adjustment similar to diauxic shift. This partial metabolic switch is the likely cause for the slow-growth phenotype in the presence of glucose. Consistently, Msn2A6 expression does not interfere with growth on ethanol and tolerated is to a limited degree in deletion mutant strains with a gene expression signature corresponding to nonfermentative growth. We propose that the lethality observed in mutants with hampered PKA activity resides in metabolic reprogramming that is initiated by Msn2 hyperactivity.

Overactivation of hedgehog signaling in the developing Müllerian duct interferes with duct regression in males and causes subfertility

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2cre). Previous studies showed that development of the oviduct and uterus are abnormal in femaleAmhr2cre/+SmoM2mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes.Amhr2mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.

Genomic Imprinting

BACKGROUND: Genomic imprinting is the inheritance out of Mendelian borders. Many of inherited diseases and human development violates Mendelian law of inheritance, this way of inheriting is studied by epigenetics.AIM: The aim of this review is to analyze current opinions and options regarding to this way of inheriting.RESULTS: Epigenetics shows that gene expression undergoes changes more complex than modifications in the DNA sequence; it includes the environmental influence on the gametes before conception. Humans inherit two alleles from mother and father, both are functional for the majority of the genes, but sometimes one is turned off or “stamped” and doesn’t show in offspring, that gene is imprinted. Imprinting means that that gene is silenced, and gene from other parent is expressed. The mechanisms for imprinting are still incompletely defined, but they involve epigenetic modifications that are erased and then reset during the creation of eggs and sperm. Genomic imprinting is a process of silencing genes through DNA methylation. The repressed allele is methylated, while the active allele is unmethylated. The most well-known conditions include Prader-Willi syndrome, and Angelman syndrome. Both of these syndromes can be caused by imprinting or other errors involving genes on the long arm of chromosome 15.CONCLUSIONS: Genomic imprinting and other epigenetic mechanisms such as environment is shown that plays role in offspring neurodevelopment and autism spectrum disorder.