A novel high-throughput screening format to identify inhibitors of secreted acid sphingomyelinase

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.     

Virtual screening to identify novel potential inhibitors for Glutamine synthetase of Mycobacterium tuberculosis

Abstract

Glutamine synthetase (GS) of Mycobacterium tuberculosis (Mtb) is an essential enzyme which is involved in nitrogen metabolism and cell wall synthesis. It is involved in the inhibition of phagosome-lysosome fusion by preventing acidification. Targeting GS can be helpful to control the infection of Mtb. In order to identify potential inhibitors, we screened chemical libraries (56,400 compounds of ChEMBL anti-mycobacterial, 1596 FDA approved drugs, 419 Natural product and 916 phytochemical) against this target using the virtual screening approach. Screening by molecular docking identified ten top-ranked compounds as GSMtb inhibitors and they were compared with known inhibitors (as control). Since GS enzyme (GSHs) is also present in human. We have compared the protein sequence of GS from Mtb and human using the P-BLAST in NCBI. We found ～27% identity in between these two sequences, so we also compared the binding affinity of inhibitor between Mtb and human. Finally, we identified top two compounds namely CHEMBL387509, CHEMBL226198 from ChEMBL anti-mycobacterial dataset, and Eriocitrin and Malvidin from phytochemical dataset which showed lees binding affinity towards GSHs whereas Pamidronate, and Phentermine from FDA approved drugs and (-)-Quinic Acid, Hexopyranuronic acid, Quebrachit, and Castanospermine from natural product showed protein-ligand interaction with Mtb protein while no interaction with GSHs. The top two docked complexes were subjected to molecular dynamic simulation to understand the stability of the molecule. Further, we calculated the binding free energy of the docked complex and analyzed hydrogen bond, salt bridge, pie stacking, and hydrophobic interaction in the docking region. These ligands exhibited very good binding affinity GSMtb enzymes. Therefore, these ligands are novel and drug-likeness compounds, and they may be potential inhibitors of M tuberculosis.

Communicated by Ramaswamy H. Sarma     

Phage display as a novel screening method to identify extracellular proteins

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.     

Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.     

Nonaggregating refolding of ribonuclease A using reverse micellar dialysis

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.     

A PiggyBac-based recessive screening method to identify pluripotency regulators

Phenotype driven genetic screens allow unbiased exploration of the genome to discover new biological regulators. Bloom syndrome gene (Blm) deficient embryonic stem (ES) cells provide an opportunity for recessive screening due to frequent loss of heterozygosity. We describe a strategy for isolating regulators of mammalian pluripotency based on conversion to homozygosity of PiggyBac gene trap insertions combined with stringent selection for differentiation resistance. From a screen of 2000 mutants we obtained a disruptive integration in the Tcf3 gene. Homozygous Tcf3 mutants showed impaired differentiation and enhanced self-renewal. This phenotype was reverted in a dosage sensitive manner by excision of one or both copies of the gene trap. These results provide new evidence confirming that Tcf3 is a potent negative regulator of pluripotency and validate a forward screening methodology to identify modulators of pluripotent stem cell biology.     

A high-throughput screen to identify novel calcineurin inhibitors

Calcineurin is a eukaryotic protein phosphatase important for many signalling and developmental processes in cells. Inhibitors of this enzyme are used clinically and there is interest in identifying novel inhibitors for therapeutic applications. This report describes a high-throughput assay that can be used to screen natural or chemical libraries of compounds to identify new calcineurin inhibitors. The microtitre plate assay is based on a yeast reporter strain and was validated with known inhibitors and tested in a pilot screen of bacterial extracts.     

Structure-based virtual screening approach to identify novel classes of Cdc25B phosphatase inhibitors

Discovery of Cdc25B phosphatase inhibitors has been actively pursued with the aim to develop anticancer agents. We have been able to identify eight novel Cdc25B inhibitors by means of a computer-aided drug design protocol involving the virtual screening with docking simulations under consideration of the effects of ligand solvation in the binding free energy function. Structural features relevant to the interactions of the newly identified inhibitors with the active-site residues of Cdc25B are also discussed in detail.     

A novel protein refolding method using a zeolite

We have succeeded in developing a simple and effective protein refolding method using the inorganic catalyst, beta-zeolite. The method involves the adsorption of proteins solubilized with 6M guanidine hydrochloride from inclusion body (IB) preparations onto the zeolite. The denaturant is then removed, and the proteins in the IBs are released from the zeolite with polyoxyethylene detergent and salt. All of the IBs tested (11 different species) were successfully refolded under these conditions. The refolded proteins are biochemically active, and NMR analysis of one of the proteins (replication protein A 8) supports the conclusion that correct refolding does occur. Based on these results, we discuss the refolding mechanism.     

In vitro cytotoxicity screening to identify novel anti-osteosarcoma therapeutics targeting pyruvate dehydrogenase kinase 2

Pyruvate dehydrogenase kinases (PDKs) act as negative modulator of mitochondrial pyruvate dehydrogenase complex (PDC) and play a crucial role in the regulation of oxidative glycolysis, which recently have been considered as a potential drug target for varying types of cancer and diabetes. Herein, we describe the discovery and biological validation of novel anti-osteosarcoma therapeutics targeting PDK2. We identified 14 anti-osteosarcoma compounds from an in-house small molecule library, which were then evaluated in a PDK2 kinase inhibition assay. We found that compounds with 2-((4-oxo-6-((4-phenylpiperazin-1-yl)methyl)-4H-pyran-3-yl)oxy)acetamide moiety showed promising inhibitory potencies to PDK2. Especial for 12, which bound to PDK2 with a K_d value of 2.3 µM, and inhibited PDK2 activity with an EC₅₀ value of 1.1 µM. In addition, 12 selectively inhibited PDK2, the selectivity indexes are 10.6, 22.0, and 60.9 for PDK2 as compared to PDK1, 2 and 4, respectively. The MTT assay suggested that 12 reduced MG-63 cancer cell proliferation with an IC₅₀ value of 4.7 µM. All these observations indicated that 12 was a novel anti-osteosarcoma therapeutic, which deserved for further investigation.     

A novel screening strategy to identify biocontrol fungi using protease production or chitinase activity against Meloidogyne root-knot nematodes

We isolated 137 fungal strains from females and eggs of Meloidogyne spp. collected from plant roots and infested soil in Jiangsu, China, and examined their in vitro protease production and chitinase activities. We then selected 20 strains, each with a protease production higher than 2 (protease production was measured as the ratio of diameters of the zone of clearing to that of the colony) and a chitinase activity higher than 0.2 U/min mL. For these strains, we examined their egg parasitic rates, mortality rates of stage 2 juvenile (J2), egg hatching rates and evaluated their biocontrol efficacies under greenhouse conditions. The biocontrol efficacies of these 20 potential biocontrol fungal strains ranged from 29 to 58%. The actual biocontrol efficacy against Meloidogyne root-knot was highly correlated with the in vitro protease production (r=0.82), chitinase activity (r=0.80), and mortality of J2 (r=0.76), but poorly correlated with the egg parasitic rate (r=0.51) and the egg hatch rate (r=0.52). Based on these results, we suggest that in vitro protease production or chitinase activities of the fungal strains could be an applicable indicator for assessing biocontrol efficacy against nematodes. This notion was also supported by our field experiments on tomatoes with Paecilomyces lilacinus strains XT18, XCS46 and Pochonia chlamydosporia strain XT124, which reached the biocontrol efficacies of 60.7, 51.5 and 48.1% in 2007; and 67.8, 57.3 and 53.2% in 2008, respectively.     

A disease-relevant high-content screening assay to identify anti-inflammatory compounds for use in cystic fibrosis

Chronic lung inflammation caused by bacterial pathogenesis through activation of nuclear factor kappa B (NFκB)-responsive proinflammatory genes is a major hurdle in the management of lung disease in cystic fibrosis (CF) patients. The authors generated a disease-relevant cell-based high-content screen to identify novel anti-inflammatory compounds for treating lung inflammation in CF. The human bronchial epithelial cell line KKLEB, harboring the most common form of mutation that causes CF, was modified to express an NFκB-responsive green fluorescent protein (GFP) reporter. After creation, the cell line was tested for its ability to respond to disease-relevant inflammatory stimuli elicited by treatment of cells with filtrates of Pseudomonas aeruginosa isolated from the airways of a CF patient. P. aeruginosa filtrates potently activated NFκB-responsive GFP reporter expression in cells. Subsequently, the assay was optimized for high-throughput screening (HTS) through generation of a Z factor (~0.5) and by testing its tolerance to the commonly used solvents ethanol and DMSO. A pilot library of clinically approved compounds was screened for assay validation. Several compounds with known NFκB inhibitory activity were identified, including several steroidal compounds that have been clinically tested in CF. Thus, the assay can be used in a broader HTS campaign to find anti-inflammatory agents for use in CF.     

Structure-based virtual screening against SARS-3CL(pro) to identify novel non-peptidic hits

Severe acute respiratory syndrome is a highly infectious upper respiratory tract disease caused by SARS-CoV, a previously unidentified human coronavirus. SARS-3CL(pro) is a viral cysteine protease critical to the pathogen's life cycle and hence a therapeutic target of importance. The recently elucidated crystal structures of this enzyme provide an opportunity for the discovery of inhibitors through rational drug design. In the current study, Gold docking program was utilized to conduct extensive docking studies against the target crystal structure to develop a robust and predictive docking protocol. The validated docking protocol was used to conduct a structure-based virtual screening of the Asinex Platinum collection. Biological evaluation of a screened selection of compounds was carried out to identify novel inhibitors of the viral protease.     

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.     

A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways

Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.     

A novel method to identify topological domains using Hi-C data

Over the last decade the 3C-based (Chromosome Conformation Capture, 3C) approaches have been developed to describe the frequency of chromatin interaction. The invention of Hi-C allows us to obtain genome-wide chromatin interaction map. However, it is challenging to develop efficient and robust analytical tools to interpret the Hi-C data. Here we present a new method called Clustering based Hi-C Domain Finder (CHDF), which is based on the difference of interaction intensity inside/outside domains, to identify Hi-C domains. We also compared CHDF with existing methods including Direction Index (DI) and HiCseg. CHDF can define more chromatin domains validated by higher resolution local chromatin structure data (Chromosome Conformation Capture Carbon Copy (5C) data). Using Hi-C data of lower sequencing depth, chromatin structure identified by CHDF is closer to that discovered by data of higher sequencing depth. Furthermore, the implement of CHDF is faster than the other two. Using CHDF, we are potentially able to discover more hints and clues about chromatin structural elements at domain level.     

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.