Preclinical three-dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies.     

A novel identification approach for discovery of 5-HydroxyTriptamine 2A antagonists: combination of 2D/3D similarity screening,molecular docking and molecular dynamics

5-HydroxyTriptamine 2A antagonists are potential targets for treatment of various cerebrovascular and cardiovascular disorders. In this study, we have developed and performed a unique screening pipeline for filtering ZINC database compounds on the basis of similarities to known antagonists to determine novel small molecule antagonists of 5-HydroxyTriptamine 2A. The screening pipeline is based on 2D similarity, 3D dissimilarity and a combination of 2D/3D similarity. The shortlisted compounds were docked to a 5-HydroxyTriptamine 2A homology-based model, and complexes with low binding energies (287 complexes) were selected for molecular dynamics (MD) simulations in a lipid bilayer. The MD simulations of the shortlisted compounds in complex with 5-HydroxyTriptamine 2A confirmed the stability of the complexes and revealed novel interaction insights. The receptor residues S239, N343, S242, S159, Y370 and D155 predominantly participate in hydrogen bonding. π–π stacking is observed in F339, F340, F234, W151 and W336, whereas hydrophobic interactions are observed amongst V156, F339, F234, V362, V366, F340, V235, I152 and W151. The known and potential antagonists shortlisted by us have similar overlapping molecular interaction patterns. The 287 potential 5-HydroxyTriptamine 2A antagonists may be experimentally verified.     

中国红螯蛛属两新种及两种雄蛛新发现(蜘蛛目:管巢蛛科)

本文记述红螯蛛属２新种,即宁明红螯蛛Ｃｈｅｉｒａｃａｎｔｈｉｕｍｎｉｎｇｍｉｎｇｅｎｓｉｓ,ｓｐ．ｎｏｖ,和思茅红螯蛛Ｃｈｅｉｒａｃａｎｔｈｉｕｍｓｉｍａｏｅｎｓｉｓ,ｓｐ．ｎｏｖ及２种雄性的补充描述,即粗美红螯蛛ＣｈｅｉｒａｃａｎｔｈｉｕｍｅｘｑｕｅｓｔｉｕｍＺｈａｎｇｅｔＺｈｕ,１９９３和纤红螯蛛ＣｈｅｉｒａｃａｎｔｈｉｕｍｆｉｂｒｏｓｕｍＺｈａｎｇｅｔａｌ,１９９４模式标本保存于湖南省生物研     

Is allostery an intrinsic property of all dynamic proteins?

Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.     

Effective use of preparative chiral HPLC in a preclinical drug synthesis

Access to a key 3-aryl-delta-lactone intermediate in enantiopure form using preparative chiral chromatography allowed expedited preparation of an important drug discovery target. A preclinical drug discovery strategy that combines rapid route discovery with effective use of preparative chiral chromatography can result in significant savings of both time and labor.     

HPLC-based bioactivity profiling of plant extracts: a kinetic assay for the identification of monoamine oxidase-A inhibitors using human recombinant monoamine oxidase-A

An assay for the HPLC-based search for monoamine oxidase-A (MAO-A) inhibitors in plant extracts was established. It combines human recombinant MAO-A, expressed as GST-fusion protein in yeast, with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline. Substrate selectivity and kinetic parameters of the GST-fusion protein were comparable to the wild-type enzyme. The applicability of the assay to HPLC-based activity profiling was tested with plant extracts spiked with small amounts of known MAO inhibitors.     

A microplate assay specific for the enzyme aggrecanase

We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.     

Thresholding rules for recovering a sparse signal from microarray experiments

We consider array experiments that compare expression levels of a high number of genes in two cell lines with few repetitions and with no subject effect. We develop a statistical model that illustrates under which assumptions thresholding is optimal in the analysis of such microarray data. The results of our model explain the success of the empirical rule of two-fold change. We illustrate a thresholding procedure that is adaptive to the noise level of the experiment, the amount of genes analyzed, and the amount of genes that truly change expression level. This procedure, in a world of perfect knowledge on noise distribution, would allow reconstruction of a sparse signal, minimizing the false discovery rate. Given the amount of information actually available, the thresholding rule described provides a reasonable estimator for the change in expression of any gene in two compared cell lines.     

Statistical inference for simultaneous clustering of gene expression data

Current methods for analysis of gene expression data are mostly based on clustering and classification of either genes or samples. We offer support for the idea that more complex patterns can be identified in the data if genes and samples are considered simultaneously. We formalize the approach and propose a statistical framework for two-way clustering. A simultaneous clustering parameter is defined as a function theta=Phi(P) of the true data generating distribution P, and an estimate is obtained by applying this function to the empirical distribution P(n). We illustrate that a wide range of clustering procedures, including generalized hierarchical methods, can be defined as parameters which are compositions of individual mappings for clustering patients and genes. This framework allows one to assess classical properties of clustering methods, such as consistency, and to formally study statistical inference regarding the clustering parameter. We present results of simulations designed to assess the asymptotic validity of different bootstrap methods for estimating the distribution of Phi(P(n)). The method is illustrated on a publicly available data set.