ASD v3.0: unraveling allosteric regulation with structural mechanisms and biological networks.

Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries.

ASBench: benchmarking sets for allosteric discovery.

Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a 'Core set' with 235 unique allosteric sites and a 'Core-Diversity set' with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand-protein interactions to guide allosteric drug design.

ASD v2.0: updated content and novel features focusing on allosteric regulation.

Allostery is the most direct and efficient way for regulation of biological macromolecule function and is induced by the binding of a ligand at an allosteric site topographically distinct from the orthosteric site. AlloSteric Database (ASD, http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information on allostery. Owing to the inherent high receptor selectivity and lower target-based toxicity, allosteric regulation is expected to assume a more prominent role in drug discovery and bioengineering, leading to the rapid growth of allosteric findings. In this updated version, ASD v2.0 has expanded to 1286 allosteric proteins, 565 allosteric diseases and 22 008 allosteric modulators. A total of 907 allosteric site-modulator structural complexes and >200 structural pairs of orthosteric/allosteric sites in the allosteric proteins were constructed for researchers to develop allosteric site and pathway tools in response to community demands. Up-to-date allosteric pathways were manually curated in the updated version. In addition, both the front-end and the back-end of ASD have been redesigned and enhanced to allow more efficient access. Taken together, these updates are useful for facilitating the investigation of allosteric mechanisms, allosteric target identification and allosteric drug discovery.

Discovery of novel nonpeptide allosteric inhibitors interrupting the interaction of CDK2/cyclin A3 by virtual screening and bioassays.

Serine/threonine-specific cyclin-dependent kinases (CDKs) are key regulatory elements in eukaryotic cell cycle progression, and the dysregulation of CDKs has been implicated in cancers. Therefore, CDKs have been identified as anti-cancer targets for the development of small-molecule drugs. In this Letter, virtual screening and biological evaluation were performed to identify novel lead structures that allosterically disrupt the interaction between CDK2 and cyclin A3, which are directed toward a noncatalytic binding pocket of CDK2. Ultimately, B2 was identified as exhibiting superior CDK2/cyclin A3 inhibition activity. In addition, our results indicated that B2 exhibited antiproliferative activities against a broad spectrum of human cancer cell lines. Significantly, B2 certainly interrupted the interaction between CDK2 and cyclin A3 and exhibited a concentration-dependent trend. In summary, our results suggest that B2 is the first effective allosteric chemical small-molecule CDK2 inhibitor to be discovered, and further lead optimization may result in a series of novel anti-CDK2 agents.

Prediction of human clearance based on animal data and molecular properties.

Human clearance is often predicted prior to clinical study from in vivo preclinical data by virtue of interspecies allometric scaling methods. The aims of this study were to determine the important molecular descriptors for the extrapolation of animal data to human clearance and further to build a model to predict human clearance by combination of animal data and the selected molecular descriptors. These important molecular descriptors selected by genetic algorithm (GA) were from five classes: quantum mechanical, shadow indices, E-state keys, molecular properties, and molecular property counts. Although the data set contained many outliers determined by the conventional Mahmood method, the variation of most outliers was reduced significantly by our final support vector machine (SVM) model. The values of cross-validated correlation coefficient and root-mean-squared error (RMSE) for leave-one-out cross-validation (LOOCV) of the final SVM model were 0.783 and 0.305, respectively. Meanwhile, the reliability and consistency of the final model were also validated by an external test set. In conclusion, the SVM model based on the molecular descriptors selected by GA and animal data achieved better prediction performance than the Mahmood method. This approach can be applied as an improved interspecies allometric scaling method in drug research and development.

The mechanism of allosteric inhibition of protein tyrosine phosphatase 1B.

As the prototypical member of the PTP family, protein tyrosine phosphatase 1B (PTP1B) is an attractive target for therapeutic interventions in type 2 diabetes. The extremely conserved catalytic site of PTP1B renders the design of selective PTP1B inhibitors intractable. Although discovered allosteric inhibitors containing a benzofuran sulfonamide scaffold offer fascinating opportunities to overcome selectivity issues, the allosteric inhibitory mechanism of PTP1B has remained elusive. Here, molecular dynamics (MD) simulations, coupled with a dynamic weighted community analysis, were performed to unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site in PTP1B. This result revealed that the allosteric inhibitor compound-3 induces a conformational rearrangement in helix α7, disrupting the triangular interaction among helix α7, helix α3, and loop11. Helix α7 then produces a force, pulling helix α3 outward, and promotes Ser190 to interact with Tyr176. As a result, the deviation of Tyr176 abrogates the hydrophobic interactions with Trp179 and leads to the downward movement of the WPD loop, which forms an H-bond between Asp181 and Glu115. The formation of this H-bond constrains the WPD loop to its open conformation and thus inactivates PTP1B. The discovery of this allosteric mechanism provides an overall view of the regulation of PTP1B, which is an important insight for the design of potent allosteric PTP1B inhibitors.

Geographical origin discriminant of Dendrobium officinale based on stable isotope ratios / 中国中药杂志

The isotopic ratios of strontium isotope (Sr) and light elements (C/H/O/N) in Dendrobium officinale from different producing areas (Shaoguan, Guangdong; Yulin, Guangxi; Shibing, Guizhou; Wenshan, Yunnan and Zhejiang province) were determined with thermal ionization mass spectrometry (TI-MS) and isotope ratio mass spectrometry (IRMS)．The differences of the stable isotope ratios in D. officinale were obtained by the variance analysis and the correlation analysis, and pattern recognition techniques with principal component analysis (PCA) was used to classify the geographical origins of D. officinale from different producing areas．The isotopic ratios of strontium isotope can be used to identified D. officinale in Zhejiang province, and the isotopic ratios of light elements showed the difference followed with the different producing areas. For δD and δ¹⁸ O in samples, maybe influenced by the environment effect. A positive correlation was found between δD and δ ¹⁸O.The principal component analysis was used to discern the samples of D. officinale from different producing areas based on detection technology of stable isotope ratios.These results revealed that it was possible and feasible to classify the geographical origin of D. officinale by the method of determination of isotopes,and provided a new method to identificate origin information of Chinese medicinal materials.

Changes of chemical compositions and sulfur dioxide residues of Gastrodiae Rhizoma with different storage times / 中国中药杂志

To study the effect of different storage time on the chemical compositions and sulfur dioxide residues of sulfur-fumigated Gastrodiae Rhizoma (GR), and provide scientific basis for solving the quality and safety issues of sulfur-fumigated traditional Chinese medicinal materials. GR, sulfur-fumigated GR and its medicinal slices were stored under the same conditions, and then 8 active ingredients and sulfur dioxide residues were measured respectively. The results showed that the content of gastrodins in sulfur-fumigated GR and its medicinal slices was significantly lower than that in the non-fumigated GR. Moreover, the content of sulfur dioxide residue in sulfur-fumigated GR was significantly higher than that in its medicinal slices. That is to say, sulfur fumigation degree had significantly higher effect on GR quality as compared with its medicinal slices. During the whole storage time (8 months), the content of the eight chemical components in GR was not changed greatly in general. However, after the storage for 4 months, the content of 8 components and sulfur dioxide residues in all of GR samples were significantly changed. In particular, the content of sulfur dioxide residue in GR medicinal materials decreased up to 50% or more.

Quality changes in Gastrodia Rhizoma of different origins and forms before and after sulfur fumigation / 中国中药杂志

As Gastrodiae Rhizoma (GR) is one of the herbs more seriously affected by sulfur fumigation, so its quality has been always of a great concern. In this paper, GR samples collected from eight main producing areas and in three forms were fumigated with sulfur and quantitatively and qualitatively analyzed based on UPLC-Q-TOF-MS/MS. The results showed that the contents of gastrodin, parishin, parishin B and parishin C were decreased, while the content of parishin E was increased after sulfur fumigation treatment. Besides, a new sulfur marker named p-hydroxybenzyl hydrogen sulfite was produced in sulfur-fumigated GR samples. As compared with producing origins, forms had a greater impact on the quality of GR, especially in Hongtianma and Wutianma. Besides, the contents of gastrodins and parishins in Hongtianma from Jilin were lowest as compared with those in other producing areas. This might be correlated with planting patterns and environmental factors. In conclusion, sulfur fumigation has a more obvious impact on the quality of GR than origins and forms, which is attributed to the generation of new sulfur fumigated markers.

Quantitative analysis and stability of -hydroxybenzyl hydrogen sulfite in sulfur-fumigated Gastrodiae Rhizoma / 中国中药杂志

Studies on the characteristic chemical markers of sulfur fumigation have become an effective auxiliary way for quality control of traditional Chinese medicine. This study established a quantitative analysis method for the determination of -hydroxybenzyl hydrogen sulfite (-HS) in Gastrodiae Rhizoma (GR) based on UPLC-MS/MS. Then, 37 batches of GR were screened, and the results showed that 27 batches of them were sulfur-fumigated. Especially, the GR samples in Yunnan producing areas were sulfur-fumigated more seriously. Based on the stability of -HS after different storage time and heat treatment methods, it was found that the content of -HS was stable and reliable in the storage period of 8 months and under normal heat treatment (50, 60, 70, 80 °C) conditions. In conclusion, this study shows a high sensitivity, good selectivity and good stability of -HS, which can provide reference for the quality control and sulfur fumigation screening of GR.

Sulfur dioxide limit standard and residues in Chinese medicinal materials / 中国中药杂志

The traditional sulfur fumigation processing method has been widely used in the initial processing and storage of traditional Chinese medicinal materials due to its economy, efficiency, convenience, high operability and effect on mold and insect prevention. However, excessive sulfur fumigation of traditional Chinese medicinal materials would lead to the changes in chemical compositions, and even endanger human health. This study showed that traditional Chinese medicinal materials were sulfur fumigated directly after being harvested for quick drying, or fumigated after being weted in the storage process for preventing mold and insects. We found that the sulfur dioxide limits for traditional Chinese medicinal materials were stricter than those for foods. Based on the existing limit standards, we obtained the data of sulfur dioxide residues for 35 types of traditional Chinese medicinal materials in a total of 862 batches. According to the limit standard in the Chinese Pharmacopoeia (150， 400 mg·kg⁻¹), the average over-standard rate of sulfur dioxide was as high as 52.43%, but it was reduced to 29.47% if calculated based on the limit for vegetable additive standard (500 mg·kg⁻¹). Sulfur fumigation issue shall be considered correctly: sulfur dioxide is a type of low toxic substance and less dangerous than aflatoxin and other highly toxic substances, and a small amount of residue would not increase the toxicity of traditional Chinese medicinal materials. However, sulfur fumigation might change the content of chemical substances and affect the quality of traditional Chinese medicinal materials. Furthermore, the exposure hazards of toxic substances are comprehensively correlated with exposure cycle, exposure frequency, and application method. In conclusion, it is suggested to strengthen the studies on the limit standard of traditional Chinese medicinal materials, formulate practical and feasible limit standard for sulfur dioxide residues in traditional Chinese medicinal materials that are consistent with the medication characteristics of traditional Chinese medicinal materials and can guarantee people's demand for safe medication.

Secondary metabolic responses to treatment of 2-aminoindan-2-phosphonic acid in cell lines from Arnebia euchroma / 中国中药杂志

The accumulation of rosmarinic acid, acetylshikonin, deoxyshikonin, β, β'-dimethylacrylshikonin and isovalerylshikonin was investigated in cell suspension cultures of Arnebia euchroma (Royle) Johnst under the influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), and of the effector methyl jasmonate (MeJA). The results showed that methyl jasmonate promoted the accumulation of rosmarinic acid and shikonin derivatives. Conversely, 2-aminoindan-2-phosphonic acid suppressed the formation of rosmarinic acid, which indicated that AIP, indeed, was able to inhibit the phenylpropanoid pathway in A. euchroma. Meanwhile, the content of total shinkonins and other four kinds of shikonin derivatives, though varied in degrees, was also inhibited. And the inhibition was dose-dependent and time-dependent. Acetylshikonin responsed most rapidly to the treatment of AIP, the content reduced after 24 h of treatment and decreased to only half of those untreated control 48 h after teratment. β, β'-Dimethylacrylshikonin, difffer from acetylshikonin, responded much slowly to the treatment, inhibition could only be observed 96 h later. These suggest that phenylpropanoid pathway plays an important role in the shikoninsbiosynthesis, and this study provides a reference for the further research in metabolic regulation of producing shikonins by cell culture technology and biosynthesis pathways of shikonin derivatives. Still, shikonins biosynthesis pathways is complicated, the exact dose- and time-effect relationship of AIP and interaction between AIP and other effectors like MeJA need further research.

Transcriptome-based gene mining and bioinformatics analysis of p-hydroxybenzoate geranyltransferase genes in Arnebia euchroma / 中国中药杂志

The p-hydroxybenzoate geranyltransferases(PGT) play an important role in the biosynthesis pathways of shikonin derivatives. Six PGTs were obtained from transcriptome datebase of Arnebia euchroma by using bioinformatics methods and the proteins＇physiochemical properties they encoded were predicted. The result of protein domain prediction showed all of the six protein sequences contained the conserved domain of Ubia prenyltransferase family and possessed the motif NDxxDxxxD for prenyldiphosphate binding and a GX(K/Y)STAL sequence for putative aromatic ring binding. The phylogenetic tree showed that PGT and p-hydroxybenzoate polyprenyltransferase(PPT) belonged to two different clades. The results of gene expression analyses showed that the expression levels in the red shikonin-proficient line and the overground part of A. euchroma that could produce shikonin derivatives was much higher than the white shikonin-deficient line and the underground part, which suggested a positive correlation between the expression levels of PGT genes and shikonin production. This study aims to lay a foudation for further understanding of the function and enzymatic properties of PGT and provide a basis for the biosynthesis pathways and metabolic regulation of shikonin derivatives.

Mitogen-activated protein kinase genes of Artemisia annua and their expression analysis under Cadmium stress / 中国中药杂志

In order to study Artemisia annua under cadmium stress, whether there are corresponding MAPK genes involved in transduction of the cadmium signal. 17 AaMAPK genes, named AaMAPK1-AaMAPK17 repectively, were finally obtained by using Trinity method for de novo assembly of transcripts from SRA database and BLAST search against AtMAPK genes and determing conserved domain using a series of bioinformatics tools. There exist 16 MAPK genes contained T[D/E]Y conserved domains among the obtained genes. The expressions of these genes were analyzed by Real-time PCR under cadmium stress. The results showed that the expressions level of AaMAPK3 and AaMAPK10 were down-regulated and MAPK7, MAPK9 and MAPK12 were up-regulated. These indicated that there exist corresponding MAPK genes involved in transduction of the cadmium signal.