Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain–containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal–regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.     

Production of 3′,3′-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme,Bd0367, regulates exit from prey by gliding motility

Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3′,3′-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.     

庞泉沟自然保护区寒温性针叶林演替优势种格局动态分析

以华北落叶松和青杆为主的寒温性针叶林是庞泉沟自然保护区的重要林型,也是主要保护对象之一。通过空间代时间构建庞泉沟自然保护区寒温性针叶林演替的时间序列,序列1是华北落叶松单优群落稳定发展的过程,序列2是从华北落叶松单优群落演替至华北落叶松-青杆共优群落再到青杆单优群落的过程,采用点格局分析法和与Monte-Carlo拟合检验对演替过程中华北落叶松和青杆的分布格局及其相互关系进行了研究。结果表明:(1)序列1在华北落叶松单优群落稳定发展过程中,华北落叶松的分布格局由集群分布趋向于随机分布,甚至在0—2.5 m上表现为均匀分布,驱动力是种内竞争引起的自疏现象。序列2从华北落叶松单优群落演替至青杆单优群落过程中,华北落叶松的分布格局同样是由集群分布趋向于随机分布,驱动力主要来自于由于青杄侵入扩散而形成的种间竞争;青杄集群的尺度逐步增大,驱动力主要是种群拓殖和种内竞争。(2)二者种间关系,在华北落叶松单优群落阶段无明显相关,共优阶段由于竞争在0—6.5 m尺度上呈显著正相关,在青杄单优群落,青杄竞争获胜,在0—2.5 m尺度上无明显相关,二者种间关系的变化主要来自于群落剩余资源驱动下的种内种间竞争。     

Wet-chemical postcolumn reaction and fluorescence detection analysis of the reference interval of endogenous serum vitamin K1(20)

The reference interval for serum vitamin K1(20) levels was assayed in healthy fasting adults by a method based on high-performance liquid chromatography (HPLC). The isolation procedure involves a solvent extraction of the plasma lipids followed by two chromatographic steps, consisting of a purification of the extract on a semipreparative adsorption column and a final quantitation on a reverse-phase column. Vitamin K1(20) and vitamin K1(25), the internal standard, are monitored by fluorescence detection after postcolumn reduction with a methanolic solution of tetramethylammonium octahydridotriborate. This reaction is performed in an open tubular reaction coil at elevated temperature. The median plasma concentration in 50 healthy fasting adults was 247 pg/ml. The levels showed a skewed distribution with a range of 62 to 980 pg/ml [log x +/- 2 SD (log x)]. The method is linear over the entire physiological range and has a within-run precision of 3.6% (n = 5, mean = 311 pg/ml). The minimum detectable amount in serum is 50 pg/ml. Other extraction procedures resulted in lower recoveries or in interferences in the final measurement. The vitamin K1(20) levels as reported by other research groups are also discussed.     

Changes in snowmelt date and summer precipitation affect the flowering phenology of Erythronium grandiflorum (glacier lily; Liliaceae)

? Premise of the study: Climate change has affected species worldwide, including alterations in phenology, migration patterns, distribution, and survival. Because Erythronium grandiflorum is an early-season bloomer, alterations in its phenology may have serious implications for many North American Rocky Mountain communities, including changes in resource availability for pollinators and herbivores. ? Methods: We investigated whether changes in the snowmelt date, summer temperature, and summer precipitation have altered the timing and abundance of flowering in E. grandiflorum by collecting long-term data on floral abundance from 1975-2008 in a series of 2 × 2 m plots at the Rocky Mountain Biological Laboratory (RMBL) in Gothic, Colorado in the United States. ? Key results: Snowmelt date and mean summer temperature were negatively correlated. Over the 30-yr study, the snowmelt date advanced by 4.14 d/decade, and mean summer temperature increased by 0.38°C/decade. Summer precipitation was variable, showing no change. The first, peak, and last flowering dates of E. grandiflorum advanced an average of 3.2 d/decade. Furthermore, earlier snowmelt and greater summer precipitation in the previous year led to earlier flowering in E. grandiflorum. There was no change in flowering abundance in this species, indicating it may be controlled by a complex set of abiotic and biotic variables. ? Conclusions: Our study indicates that snowmelt is arriving earlier at the RMBL, which has caused earlier flowering in E. grandiflorum. Because alterations in phenology can disrupt important ecological interactions, information on potential phenological shifts in species that interact with E. grandiflorum is essential in determining the net effect of climate-driven alterations in phenology.     

Use of Gas Chromatography for Determining Catabolic Products of Arginine by Bacteria

A rapid and sensitive procedure for determining catabolic products of arginine metabolism by bacteria was developed. The method consists of inoculating a solution of L-arginine with a heavy cell suspension of the test organism. After a 2-hr incubation period, dissimilation products (citrulline, ornithine, agmatine, putrescine) are converted to volatile derivatives and analyzed by gas-liquid chromatography. Compared with conventional microbiological tests, the new procedure is rapid and can be used for sensitive quantitative measurements of specific metabolites from arginine.     

The three-dimensional structures of mutants of porphobilinogen deaminase: toward an understanding of the structural basis of acute intermittent porphyria.

Mutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3-dimensional structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.