人类蛋白激酶组的多靶点分子对接系统

蛋白激酶在信号转导、基因转录和蛋白翻译等生物过程起关键性作用,因而与大量人类疾病密切相关。所以,蛋白激酶的抑制剂筛选是抗肿瘤药物开发的热点,正在向基于全激酶组的高通量多靶点筛选模式发展。为了降低大规模实验筛选的成本,提高成功率,本文构建人类蛋白激酶组的多靶点分子对接系统,对抑制剂-激酶组的相互作用进行预测。我们首先利用同源模建方法,对人类激酶组约500个激酶变异体的催化域进行结构建模;接着以催化域结构模型为受体,用已知激酶抑制剂进行分子对接,对抑制剂与各激酶变异体的结合亲和力进行了定量计算。结果显示,本文所建立的多靶点分子对接系统可以准确预测抑制剂与激酶变异体的相互作用,结合自由能的计算值与实验值有很强的相关性。所以,该分子对接系统可用于多靶点激酶抑制剂的计算筛选,为激酶抑制剂开发与抗肿瘤药物设计提供理论依据。     

Method development and measurements of endogenous serine/threonine Akt phosphorylation using capillary electrophoresis for systems biology

Signal transduction studies have indicated that Akt is essential for transducing the signals originating from extracellular stimuli. An exploration of the Akt signal transduction mechanism depends on the ability to assay its activation states by determining the ability of Akt to phosphorylate various substrates. This paper describes a CE-based kinase assay for Akt using a UV detection method. The RPRAATF peptide was used as the specific substrate to determine the Akt activity. Under the CE separation conditions used, the phosphorylated and nonphosphorylated forms of the RPRAATF peptide were rapidly resolved in the Akt reaction mixture within 20 min. Using this method for measuring the Akt activity, the incubation time for the Akt reactions as well as the kinetic parameters (KM) were examined. Furthermore, the developed method was applied to a PC12 cell system to assess the dynamics of the Akt activity by examining the effectiveness of the RPRAATF peptide substrate under various cytokine-stimulated environments. These results highlight the feasibility of the CE method, which is a simple and reliable technique for determining and characterizing various enzyme reactions particularly kinase enzymes.     

Resorcylic acid lactones as new lead structures for kinase inhibition

The naturally occurring resorcylic acid lactones LL-Z1640-2 (1), hypothemycin (2), L-783277 (3), radicicol A (4) and, to a somewhat lesser extent, aigialomycin D (5) have recently emerged as new lead structures for kinase inhibition. Total syntheses have now been reported for all of these natural products, most of which are based on macrocyclization through ester bond formation. However, RCM-based approaches have also been described and a variety of strategies have been pursued to obtain the requisite seco acids or dienes, respectively, as precursors for macrocyclization.     

A 2D NMR method to study peptide phosphorylation

We demonstrate a 2D NMR method which distinguishes between phosphorylated and non-phosphorylated amino acids. The method is capable of monitoring the amino acid and site-specific enzymatic phosphorylation and dephosphorylation of peptides. The method was developed using O-phosphorylated amino acids and its potential is shown with a peptide fragment of the myelin basic protein (MBP).     

Targeting the Dark Lipid Kinase PIP4K2C with a Potent and Selective Binder and Degrader

Phosphatidylinositol 5-phosphate 4-kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy-dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non-lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX-4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX-4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX-4102 and TMX-4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.     

Design and chemical synthesis of [1,2,4]triazol[1,5-c]pyrimidin-5-yl amines, a novel class of VEGFR-2 kinase inhibitors

The Letter describes a facile approach to 7,8-dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-yl amines, a novel class of potent inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). The synthetic sequence is centered around preparation of the key 3(5)-cyanomethyl-1,2,4-triazole intermediates and their Knoevenagel condensation with aromatic aldehydes. A subsequent three-step conversion of Knoevenagel adducts involving a reduction of vinyl nitriles followed by the reaction of the resulting amines with aryl isothiocyanates and cyclization of the respective thioureas yielded targeted heterocycles as a 1:1 mixture of tautomers. A representative molecule featured sound activity against VEGFR-2 in both enzymatic and cellular assays.     

Computational systematic selectivity of the Fasalog inhibitors between ROCK-I and ROCK-II kinase isoforms in Alzheimer’s disease

Human Rho-associated coiled-coil forming kinase (ROCK) is a class of essential neurokinases that consists of two structurally conserved isoforms ROCK-I and ROCK-II; they have been revealed to play distinct roles in the pathogenesis of Alzheimer’s disease (AD) and other neurological disorders. Selective targeting of the two kinase isoforms with small-molecule inhibitors is a great challenge due to the surprisingly high homology in kinase domain (92 %) and the full identity in kinase active site (100 %). Here, we describe a computational protocol to systematically profile the selectivity of Fasudil and its 25 analogs (termed as Fasalogs) between the two kinase isoforms. It is suggested that the substitution of Fasudil’s 1,4-diazepane moiety with rigid ring such as Ripasudil and Dimehtylfasudil would render the resulting inhibitors of ROCK-II over ROCK-I (II-o-I) selectivity, while the substitution with long, flexible group such as H-89 and BDBM92607 tends to have I-o-II selectivity. Structural analysis reveals that the inhibitor affinity is not only determined by the identical active site, but also contributed from the non-identical first and second shells of the site as well as other non-conserved kinase regions, which can indirectly influence the active site and inhibitor binding through allosteric effect. A further kinase assay basically confirms the computational findings, which also exhibits a good consistence with theoretical selectivity over 10 tested samples (R_p = 0.89). In particular, the Fasalog compounds Dimehtylfasudil and H-89 are identified as II-o-I and I-o-II selective inhibitors. They can be considered as promising lead molecular entities to develop new specific ROCK isoform-selective Fasalog inhibitors.     

Synthesis of aminopyrimidylindoles structurally related to meridianins

The synthesis of new meridianin derivatives substituted at the C-5′ position of the 2-aminopyrimidine ring by various aryl groups and substituted or not by a methyl group on the indole nitrogen is described. The 2-aminopyrimidine ring was obtained via a Bredereck synthesis. Aryl groups were introduced by Suzuki cross-coupling after bromination of the 2-aminopyrimidine ring at the C-5′ position.     

Synthesis and biological evaluation of novel 2,4,5-triaryl-1 H-pyrazol-3（2H）-ones as inhibitors of ALK5

A series of 2,4,5-triaryl substituted 1H-pyrazol-3（2H）-ones,as ALK5 inhibitors,were desigened,synthesized and evaluated in vitro.Most compounds exhibited noticeable ALK5 inhibition activities at 1μmol/L and displayed no significant cytotoxicities at 30μmol/L.