Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow

PI3Kδ mediates key immune cell signaling pathways and is a target of interest for multiple indications in immunology and oncology. Here we report a structure-based scaffold-hopping strategy for the design of chemically diverse PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can be combined to generate new PI3Kδ inhibitors with high potency and isoform selectivity. In particular, an oxindole-based scaffold was found to impart exquisite selectivity when combined with several hinge binding motifs.     

Osmoregulation of leaf motor cells

"Osmotic Motors"--the best-documented explanation for plant leaf movements--frequently reside in specialized motor leaf organs, pulvini. The movements result from dissimilar volume and turgor changes in two oppositely positioned parts of the pulvinus. This Osmotic Motor is powered by a plasma membrane proton ATPase, which drives KCl fluxes and, consequently, water, across the pulvinus into swelling cells and out of shrinking cells. Light signals and signals from the endogenous biological clock converge on the channels through which these fluxes occur. These channels and their regulatory pathways in the pulvinus are the topic of this review.     

Low-dimensional compounds containing cyano groups. XIX. Crystal structure, spectroscopic, thermal and magnetic properties of {[Cu(tn)2]3[Pt(CN)4]2}[Pt(CN)4] (tn = 1,3-diaminopropane) complex

Violet prismatic crystals of {[Cu(tn)₂]₃[Pt(CN)₄]₂}[Pt(CN)₄] (tn = 1,3-diaminopropane) were crystallized from the water-methanol solution containing CuCl₂·2H₂O, tn and K₂[Pt(CN)₄]·3H₂O. Prepared complex was characterized using elemental analysis, infrared and UV-Vis spectroscopy, magnetic measurement and thermal analysis. X-ray analysis revealed an ionic character of the complex containing mononuclear square planar [Pt(CN)₄]²⁻ complex anions and penta-nuclear [Cu(tn)₂-Pt(CN)₄-Cu(tn)₂-Pt(CN)₄-Cu(tn)₂]²⁺ complex cations. The inner Cu(II) atom of the complex cation is hexa-coordinated, whereas two crystallographically equivalent peripheral Cu(II) atoms are penta-coordinated in the shape of a deformed square pyramid. Four v(CN) absorption bands observed in the IR spectrum are in agreement with the higher number of crystallographically different cyano groups and a broad highly asymmetric band observed in the reflectance UV-Vis spectrum is consistent with the presence of both hexa- and penta-coordinated Cu(II) atoms in the structure. The temperature dependence of the inverse susceptibility suggests the presence of a weak antiferromagnetic exchange coupling between Cu(II) ions. The complex is stable up to 210 °C when its two-stage thermal decomposition starts.     

Human p38δ MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38α, β, γ and δ. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38α and β, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38γ and/or δ was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38δ attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38δ with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38δ isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38α and/or β isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.     

猪胰蛋白酶自溶产物中δ-、γ-和σ-胰蛋白酶的分离与结晶

将1％猪胰蛋白酶溶于0.05M,pH9.0硼酸缓冲液中,在25℃自溶6小时后,上大豆胰蛋白酶抑制剂亲和层析柱STI-Sepharose4B,用pH5.0—3.0的磷酸钾缓冲液梯度洗脱,可以有效地分开各种自溶活性产物。从得到的三个活性峰S_1、S_2和S_3中,用DNS-Cl Edman法和有色Edman法测定酶分子肽键断裂后N末端的部分氨基酸顺序,从而鉴定出三种不同形式的自溶活性产物:δ~-、γ~-和σ~-胰蛋白酶。S_3主要含有完整的单链β-胰蛋白酶;S_2主要含有Arg_(105)—Val_(156)断裂的双链δ-胰蛋白酶;S_1含有Arg_(105)—Val_(106),Lys_(145)—Arg_(146)和Arg_(105)—Val_(106),Lys_(131)—Ser_(132)断裂的三链γ-和σ-胰蛋白酶。活性产物的几个N端氨基酸顺序与已知猪胰蛋白酶氨基酸顺序完全相符。 与β-胰蛋白酶相比,δ~-、γ~-和σ~-胰蛋白酶的等电点稍有降低,约为10.5左右。 对分离出的自溶活性产物的结晶条件进行了摸索,用悬滴法已经得到δ~-、γ~-和σ~-胰蛋白酶与苯甲脒复合物的结晶。这是关于分离出胰蛋白酶自溶活性产物结晶体的首次报道。     

Chloroplast culture IX chlorophyll(ide) a biosynthesis invitro at rates higher than inMvivo

Chlorophyll $\text{a}$ is the plant pigment which in nature catalyzes the conversion of solar energy into chemical energy. By pretreating etiolated cucumber cotyledons with kinetin and gibberellic acid in the dark, it was observed that the plastids which were isolated from such tissues, and incubated in a cofactor-fortified medium, under a repetitive light-dark regime, were capable of synthesizing chlorophyll(ide) $\text{a}$ from exogenous δ-aminolevulinic acid at a rate about twice as high as the highest rates observable in greening tissues $\text{in}\text{vivo}$.     

Determination of the sites of synthesis of chlorophyll synthesizing enzymes in cell cultures of Nicotiana tabacum

The activity of a sequence of enzymes involved in chlorophyll biosynthesis (δ-aminolevulinic acid synthetase (ALAS), δ-aminolevulinic acid dehydratase, porphobilinogenase and chlorophyllase) was followed during greening of tobacco cell cultures under the influence of chloramphenicol (CAP). The photosynthetic enzymes ribulose diphosphate carboxylase (RuDPCO) and NADP linked glyceraldehyde dehydrogenase (NADP-GDH) were used as markers for penetration and action of the inhibitor. RuCPCO was inhibited at concentrations of CAP which still allowed good chlorophyll accumulation. The enzymes of chlorophyll biosynthesis, the activity of which increased during illumination and CAP treatment, behaved like NADP-GDH which is known to be synthesized in the cytoplasm. The results suggest that synthesis of enzymes of chlorophyll biosynthesis takes place in the cytoplasm. Decreasing light induced increment of ALAS activity caused by CAP may possibly be taken as an indication that things are more complicated with this enzyme.     

Effect of pyridoxal phosphate and cysteine on δ-aminolaevulinate synthetase and dehydratase in soya

The specific activity of ALA-S extracts prepared from lyophilized soya callus growing in light or dark showed a striking increase when tissue cultures were grown in the presence of pyridoxal phosphate (PyP) in order to complex aminothiols. By contrast ALA-D specific activity in the light grown callus was reduced on incorporation of PyP into the growth medium. Cysteine (Cy) added to the culture medium did not influence the specific activities of either enzyme.     

Ultrafiltration of raw sewage using an immobilized enzyme membrane

Ultrafiltration of raw sewage was performed using multiple enzymes immobilized on non-cellulosic, ultrafiltration membranes. An increase of 12% in the permeate flux rate at quasi-steady state was observed due to the action of the immobilized enzymes. Enzymes were immobilized by physical sorption to minimize the loss of ultrafiltration capability of the membrane, due to the immobilization process. A mathematical model based on diffusive transport and enzymatic action is presented. A standard Marquardt algorithm and a fourth-order Runge-Kutta integration routine were used for estimation of the non-linear parameters in the model. A comparison of data presented here with the data reported earlier on the ultrafiltration of NFDM (non-fat dry milk), showed that the enzyme-membrane has a longer half-life in the case of NFDM than for raw sewage. Furthermore, the first-order enzyme decay rate is much faster in the multiple enzyme system used in raw sewage filtration than in the single enzyme system used in the ultrafiltration of NFDM.     

Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue

The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis can be bioassayed in vitro using cultured insect tissue. Larval cells of the spruce budworm, Choristoneura fumiferana, are damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity is accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue is inversely proportional to the amount of toxin added. Tissue cells from the spruce budworm exhibited maximum susceptibility to activated δ-endotoxin after 120 hr incubation. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 μg or less of toxin protein per 2 × 10⁵ insect tissue cells. Activated δ-endotoxin was entomocidal to insects as well, as detemined by mortality studies with second-instar larvae of the European corn borer. Electron microscopic observations of insect tissue treated with activated δ-endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.