Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation.

Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.

Analysis of Methylation and mRNA Expression of Lin28B Gene Promoter Region in the Hypothalamus of Dolang Sheep During Pubertal Initiation.

Lin28B plays an important role in puberty initiation in sheep. This study aimed to discuss the correlation between different growth periods and the methylation status of cytosine-guanine dinucleotide (CpG) islands in the promoter region of the Lin28B gene in the Dolang sheep's hypothalamus. In this study, the sequence of the Lin28B gene promoter region in Dolang sheep was obtained by cloning and sequencing, and methyl groups of the CpG island of the Lin28B gene promoter in the hypothalamus were detected by bisulfite sequencing PCR during the three periods of prepuberty, adolescence, and postpuberty in Dolang sheep. Lin28B expression in the Dolang sheep's hypothalamus was detected by fluorescence quantitative PCR at three stages: prepuberty, puberty, and postpuberty. In this experiment, the 2993-bp Lin28B promoter region was obtained, and it was predicted that there was a CpG island containing 15 transcription factor binding sites and 12 CpG sites, which may play a role in gene expression regulation. Overall, methylation levels increased from prepuberty to postpuberty, while Lin28B expression levels decreased, indicating that Lin28B expression was negatively correlated with promoter methylation levels. Variance analysis showed significant differences in the methylation status of CpG5, CpG7, and CpG9 between pre- and postpuberty (p < 0.05). Our data show that Lin28B expression is increased by demethylation of promoter CpG islands, with CpG5, CpG7, and CpG9 implicated as critical regulatory sites.

Population structure, genetic diversity and prolificacy in pishan red sheep under an extreme desert environment.

Extreme environmental conditions are a major challenge for livestock production. Changes in climate conditions, especially those that lead to extreme weather, can reduce livestock production. The screening of genes and molecular markers is of great significance to explore the genetic mechanism of sheep prolificacy traits in Taklimakan Desert environment. We selected healthy adult Pishan Red Sheep (PRS) and Qira Black Sheep (QR) which live in Taklimakan Desert environment, collected blood from jugular vein, extracted DNA, and prepared Illumina Ovine SNP50 chip. For PRS, linkage disequilibrium (LD) was calculated using the ovine SNP50 Beadchip and the effective population size (Ne) was estimated using SMC++. The genetic characteristics of PRS were analyzed by integrated haplotype score (iHS) and fixation index (F ST ). The result showed that r 2 of PRS was 0.233 ± 0.280 in the range of 0-10 Kb and decreased with increasing distances. SMC++ tested that the Ne of PRS remained at 236.99 in recent generations. 184 genes were screened out under iHS 1% threshold, and 1148 genes were screened out with F ST under the 5% threshold, and 29 genes were obtained from the intersection of the two gene sets. In this study, the genetic characteristics of PRS and QR were compared by ovine genome chip, and the related excellent genes were searched, providing reference for the protection of sheep germplasm resources and molecular breeding in a desert environment.

Exploring the growth trait molecular markers in two sheep breeds based on Genome-wide association analysis.

Growth traits are quantitative traits controlled by multiple micro-effect genes. we identified molecular markers related to sheep growth traits, which formed the basis of molecular breeding. In this study, we randomly selected 100 Qira Black sheep and 84 German Merino sheep for the blood collection the jugular vein to genotype by using the Illumina Ovine SNP 50K Bead Chip. quality control criteria for statistical analysis were: rejection detection rate < 90% and minimum allele frequency (MAF) < 5%. Then, we performed Genome-wide association studies (GWAS) on sheep body weight, body height, body length, and chest circumference using mixed linear models. After getting 55 SNPs with significant correlation, they were annotated by reference genome of Ovis aries genome (Oar_v4.0) and We obtained a total of 84 candidate genes associated with production traits (BMPR1B, HSD17B3, TMEM63C, etc.). We selected BMPR1B for population validation and found a correlation between the FecB locus and body weight traits. Therefore, this study not only supplements the existing knowledge of molecular markers of sheep growth traits, but also has important theoretical significance and reference value for the mining of functional genes of sheep growth traits.

Landscape genomics reveals adaptive divergence of indigenous sheep in different ecological environments of Xinjiang, China.

Sheep are important livestock animals that have evolved under various ecological pressures. Xinjiang is a region with diverse and harsh environments that have shaped many local sheep breeds with unique characteristics and environmental adaptability. However, these breeds are losing ecological flexibility due to the promotion of intensive farming practices. Here we sequenced 14 local sheep breeds from Xinjiang and analyzed their genetic structure and gene flow with other sheep breeds from neighboring regions. The Tibetan Plateau was the geographic origin of Xinjiang native sheep evolution. We performed genome-environment association analysis and identified Bio9: Mean Temperature of Driest Quarter and Bio15: Precipitation Seasonality as the key environmental factors affecting Xinjiang local sheep and the key genes involved in their survival and adaptation. We classified Xinjiang native sheep breeds into six groups based on their differential genes by pairwise selective sweep analysis and Community Network Analysis. We analyzed transcriptome expression data of 832 sheep tissues and detected tissue-specific enrichment of six group-specific genes in different biological systems. Our results revealed the genetic basis of year-round estrus, drought tolerance, hypoxia resistance, and cold tolerance traits of Xinjiang sheep breeds. Moreover, we proposed conservation strategies for Xinjiang local sheep breeds and provided theoretical guidance for breeding new sheep breeds under global extreme environments.

Expression and functional analysis of GnRH at the onset of puberty in sheep.

Gonadotropin-releasing hormone (GnRH) is a key factor at the onset of puberty. This decapeptide has been found in mammalian ovaries, but its regulatory mechanism in the ovary of sheep at the onset of puberty is not clear. This study investigated the coding sequence (CDS) of the GnRH gene in the ovary of Duolang sheep and the expression of GnRH mRNA in different tissues at the onset of puberty, and analyzed the effect of GnRH on ovarian granulosa cells (GCs) of Duolang sheep. The results showed that the GnRH CDS of sheep was cloned, the full length of the GnRH CDS in sheep ovary was 279 bp, and the nucleotide sequence was completely homologous to that in the hypothalamus. The expression of GnRH mRNA was highest in the hypothalamus and ovary. The expression of related hormones and receptors in GCs of Duolang sheep treated with different concentrations of GnRH for 24 h was affected. GnRH significantly inhibited LH synthesis and LHR expression in GCs. Low concentration (100 ng mL - 1 ) had the most obvious therapeutic effect on follicle-stimulating hormone (FSH) and FSHR. Higher concentration (250 ng mL - 1 ) significantly promoted estradiol and ER ß mRNA. These findings provide strong evidence that ovarian GnRH is an important regulatory factor at the onset of puberty in sheep.

Transcriptome Sequencing-Based Mining of Genes Associated With Pubertal Initiation in Dolang Sheep.

Improving the fertility of sheep is an important goal in sheep breeding as it greatly increases the productivity. Dolang sheep is a typical representative breed of lamb in Xinjiang and is the main local sheep breed and meat source in the region. To explore the genes associated with the initiation of puberty in Dolang sheep, the hypothalamic tissues of Dolang sheep prepubertal, pubertal, and postpubertal periods were collected for RNA-seq analysis on the Illumina platform, generating 64.08 Gb clean reads. A total of 575, 166, and 648 differentially expressed genes (DEGs) were detected in prepuberty_vs._puberty, postpuberty_vs._prepuberty, and postpuberty_vs._puberty analyses, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, the related genes involved in the initiation of puberty in Dolang sheep were mined. Ten genes that have direct or indirect functions in the initiation of puberty in Dolang sheep were screened using the GO and KEGG results. Additionally, quantitative real-time PCR was used to verify the reliability of the RNA-Seq data. This study provided a new approach for revealing the mechanism of puberty initiation in sheep and provided a theoretical basis and candidate genes for the breeding of early-pubertal sheep by molecular techniques, and at the same time, it is also beneficial for the protection, development, and utilization of the fine genetic resources of Xinjiang local sheep.

Identification of Signatures of Selection for Litter Size and Pubertal Initiation in Two Sheep Populations.

Fecundity is an important economic trait in sheep that directly affects their economic and productive efficiency. Our study aimed to identify SNP loci associated with sheep puberty or litter size which could be used in future breeding programs to improve fertility. Genomic DNA was obtained from Hetian and Cele Black sheep breeds and used for reduced-representation genome sequencing to identify SNP loci associated with pubertal initiation and litter size. Selective signatures analysis was performed based on the fixation index and nucleotide diversity, followed by pathway analysis of the genes contained in the selected regions. The selected SNP loci in the genes associated with pubertal initiation and litter size were validated using both sheep breeds. In total, 384,718 high quality SNPs were obtained and 376 genes were selected. Functional annotation of genes and enrichment analysis identified 12 genes associated with pubertal initiation and 11 genes associated with litter size. SNP locus validation showed that two SNP on PAK1 and four on ADCY1 may be associated with pubertal initiation, and one SNP on GNAQ gene (NC_040253.1: g.62677376G > A) was associated with litter size in Cele Black sheep. Our results provide new theoretical support for sheep breeding.

Analysis on the desert adaptability of indigenous sheep in the southern edge of Taklimakan Desert.

The southern margin of the Taklimakan Desert is characterized by low rainfall, heavy sandstorms, sparse vegetation and harsh ecological environment. The indigenous sheep in this area are rich in resources, with the advantages of perennial estrus and good resistance to stress in most sheep. Exploring the molecular markers of livestock adaptability in this environment will provide the molecular basis for breeding research to cope with extreme future changes in the desert environment. In this study, we analyzed the population genetic structure and linkage imbalance of five sheep breeds with three different agricultural geographic characteristics using four complementary genomic selection signals: fixation index (FST), cross-population extended haplotype homozygosity (xp-EHH), Rsb (extended haplotype homozygosity between-populations) and iHS (integrated haplotype homozygosity score). We used Illumina Ovine SNP 50K Genotyping BeadChip Array, and gene annotation and enrichment analysis were performed on selected regions of the obtained genome. The ovary of Qira Black sheep (Follicular phase, Luteal phase, 30th day of pregnancy, 45th day of pregnancy) was collected, and the differentially expressed genes were screened by transcriptomic sequencing. Genome-wide selective sweep results and transcriptome data were combined for association analysis to obtain candidate genes associated with perennial estrus and stable reproduction. In order to verify the significance of the results, 15 resulting genes were randomly selected for fluorescence quantitative analysis. The results showed that Dolang sheep and Qira Black sheep evolved from Kazak sheep. Linkage disequilibrium analysis showed that the decay rate of sheep breeds in the Taklimakan Desert was higher than that in Yili grassland. The signals of FST, xp-EHH, Rsb and iHS detected 526, 332, 308 and 408 genes, respectively, under the threshold of 1% and 17 overlapping genes under the threshold of 5%. A total of 29 genes were detected in association analysis of whole-genome and transcriptome data. This study reveals the genetic mechanism of perennial estrus and environmental adaptability of indigenous sheep breeds in the Taklimakan Desert. It provides a theoretical basis for the conservation and exploitation of genetic resources of indigenous sheep breeds in extreme desert environment. This provides a new perspective for the quick adaptation of sheep and other mammals to extreme environments and future climate changes.

3,5-diarylazoles as novel and selective inhibitors of protein kinase D.

The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD/HDAC5 signaling.

Compound set enrichment: a novel approach to analysis of primary HTS data.

The main goal of high-throughput screening (HTS) is to identify active chemical series rather than just individual active compounds. In light of this goal, a new method (called compound set enrichment) to identify active chemical series from primary screening data is proposed. The method employs the scaffold tree compound classification in conjunction with the Kolmogorov-Smirnov statistic to assess the overall activity of a compound scaffold. The application of this method to seven PubChem data sets (containing between 9389 and 263679 molecules) is presented, and the ability of this method to identify compound classes with only weakly active compounds (potentially latent hits) is demonstrated. The analysis presented here shows how methods based on an activity cutoff can distort activity information, leading to the incorrect activity assignment of compound series. These results suggest that this method might have utility in the rational selection of active classes of compounds (and not just individual active compounds) for followup and validation.

Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED.

Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.

Matrix-Based Activity Pattern Classification as a Novel Method for the Characterization of Enzyme Inhibitors Derived from High-Throughput Screening.

One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S × 4I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.

Discovery and Characterization of Allosteric WNK Kinase Inhibitors.

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability.

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

Probing the primary screening efficiency by multiple replicate testing: a quantitative analysis of hit confirmation and false screening results of a biochemical assay.

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.

Evaluation of division-arrested cells for cell-based high-throughput screening and profiling.

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.

Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor alpha.

The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331