Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.     

Functional nucleic acids in high throughput screening and drug discovery

In vitro selection can be used to generate functional nucleic acids such as aptamers and ribozymes that can recognize a variety of molecules with high affinity and specificity. Most often these recognition events are associated with structural alterations that can be converted into detectable signals. Several signaling aptamers and ribozymes constructed by both design and selection have been successfully utilized as sensitive detection reagents. Here we summarize the development of different types of signaling nucleic acids, and approaches that have been implemented in the screening format.     

A whole-cell assay for the high throughput screening of calmodulin antagonists

Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.     

基于报告基因技术的MDR1转录抑制剂高通量筛选模型的建立和应用

MDR1基因是引起肿瘤多药耐药的主要基因,其编码的P-gp蛋白可持续将药物由胞内排出胞外以降低胞内药物浓度导致多药耐药,MDR1基因的转录抑制剂可抑制MDR1基因在癌细胞中的表达,从而逆转肿瘤多药耐药.通过克隆MDR1基因的启动子,将其插入pGL3-basic质粒构建MDR1-luc＋报告基因载体,再将重组载体转染入HepG2肝癌细胞并筛选单克隆细胞株,构建了MDR1启动子的高通量筛选模型,Z′因子为0.75;通过对中药样品库的筛选,得到两种中药提取物高良姜水提物、红豆蔻醇提物有明显耐药逆转效果,EC50值分别为高良姜水提物16.37mgL-1和红豆蔻醇提物14.96mgL-1,RT-PCR验证上述两种阳性样品具有明显的抑制MDR1基因表达的作用.以上结果为MDR1基因的转录抑制剂高通量筛选奠定了基础.     

Immunoassays: biological tools for high throughput screening and characterisation of combinatorial libraries

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.     

Design of a compound screening collection for use in high throughput screening

In this paper we introduce a quantitative model that relates chemical structural similarity to biological activity, and in particular to the activity of lead series of compounds in high-throughput assays. From this model we derive the optimal screening collection make up for a given fixed size of screening collection, and identify the conditions under which a diverse collection of compounds or a collection focusing on particular regions of chemical space are appropriate strategies. We derive from the model a diversity function that may be used to assess compounds for acquisition or libraries for combinatorial synthesis by their ability to complement an existing screening collection. The diversity function is linked directly through the model to the goal of more frequent discovery of lead series from high-throughput screening. We show how the model may also be used to derive relationships between collection size and probabilities of lead discovery in high-throughput screening, and to guide the judicious application of structural filters.     

Applications of high throughput microsomal stability assay in drug discovery

High throughput in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with high throughput assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between high throughput in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds in order to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. In this study the relationship between in vitro single time point high throughput microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies was examined using 306 real world drug discovery compounds. The results showed that in vitro Phase I microsomal stability t(1/2) is significantly correlated to in vivo clearance with a p-value<0.001. For compounds with low in vitro rat microsomal stability (t(1/2)<15 min), 87% showed high clearance in vivo (CL>25 mL/min/kg). This demonstrates that high throughput microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability (t(1/2)>15 min), no significant differentiation was observed between high and low clearance compounds. This is likely owing to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance. This finding supports the strategy used by medicinal chemists and drug discovery teams of applying the in vitro data to triage compounds for in vivo PK and efficacy studies and guide structural modification to improve metabolic stability. When in vitro and in vivo data are both available for a compound, potential in vivo clearance pathways can be diagnosed to guide further discovery studies.     

Integration of virtual and high throughput screening in lead discovery settings

In the last decade mass screening strategies became the main source of leads in drug discovery settings. Although high throughput (HTS) and virtual screening (VS) realize the same concept the different nature of these lead discovery strategies (experimental vs theoretical) results that they are typically applied separately. The majority of drug leads are still identified by hit-to-lead optimization of screening hits. Structural information on the target as well as on bound ligands, however, make structure-based and ligand-based virtual screening available for the identification of alternative chemical starting points. Although, the two techniques have rarely been used together on the same target, here we review the existing prominent studies on their true integration. Various approaches have been shown to apply the combination of HTS and VS and to better use them in lead generation. Although several attempts on their integration have only been considered at a conceptual level, there are numerous applications underlining its relevance that early-stage pharmaceutical drug research could benefit from a combined approach.     

Developing the molecular modelling of diffusion in zeolites as a high throughput catalyst screening technique

Molecular modelling techniques have been used to screen zeolite catalysts for their suitability for organic synthesis. For example, we have used these techniques to study the alkylation of aromatic molecules which are important in the fine-chemical and drug industries. A survey of all such efforts is reviewed in this article. The application of molecular modelling techniques in a systematic manner is an efficient first step in the design of zeolite catalysts. As a qualitative screening tool, molecular graphics is used to visualize how well the reactant and product molecules fit inside the pores of the zeolites. Using a hybrid of several molecular modelling methods, which combines molecular dynamics (MD) and Monte Carlo methods with energy minimization, it is possible to determine the minimum energy locations of the molecules inside the zeolites cages. The minimum energy configurations determined by this hybrid method are taken as a starting point for diffusion of the molecules through the zeolite channels. When a molecule is allowed to diffuse through zeolite channel, the molecule attains some maxima and minima in its diffusion energy profile. From the differences between a maximum and a minimum energy configuration, the diffusion energy barrier for the molecule can be calculated in the zeolites. By comparing the diffusion energy barriers for various isomers of a molecule in different zeolites, it is possible to find out the most suitable zeolite for achieving the required shape-selectivity. In addition, factors influencing the diffusivity of the molecules and consequently the shape selectivity are derived. The list of factors and their relative importance are analysed to derive valuable guidelines to design shape-selective zeolite catalysts for a given reaction. Thus, the ultimate aim of these studies is to develop a high throughput computational screening process for the selection of shape-selective zeolite catalysts for various reactions. The dynamic behaviour of molecules inside the pores of zeolites can be studied using MD methods. Since MD is computationally time consuming, it is more efficient to screen the possible zeolite catalysts by energy minimization methods and then perform MD in specific zeolites. More accurate values of diffusivity of the molecules can be calculated using MD methods, and these values can be correlated with the shape-selectivity observed experimentally and /or derived from diffusion energy barrier calculations.     

Discovery of a new family of chromium ethylene polymerisation catalysts using high throughput screening methodology

Following a discovery that salicylaldimines bearing bulky ortho-phenoxy substituents and small imine substituents give very active chromium catalysts for ethylene polymerisation, High Throughput Screening (HTS) methodology has been employed to facilitate a further discovery of exceptionally active catalysts based on tridentate salicylaldimine ligands with bulky triptycenyl groups.     

Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting molecules

Targeting of drugs and genes to specific cell types is an emerging paradigm in the treatment of many medical conditions. However, targeting structures such as peptides are susceptible to rapid inactivation in vivo. To address this problem, novel targeting molecules can now be rapidly synthesized using a combinatorial approach. Methods to screen the large libraries created in this process are often lacking or compatible only with solution-based screening. This report describes a high-throughput cell-based method utilizing flow cytometry, capable of rapidly screening large libraries of molecules simultaneously for biological functionality and stability. In this method, each library molecule is attached to a microsphere exhibiting a unique set of optical properties, or "fingerprint", conferring modularity and multiplex capability. We investigated the multiplex capability of our flow cytometric method to determine its capacity for high-throughput screening. Current instrumentation in our laboratory allows the screening of at least 75 unique compounds in a single well, a number comparable to available solution-based assays. In state-of-the-art configuration, however, this methodology can support the screening of up to 1875 compounds per well, achieving high-throughput potential in a single multiwell plate. We also investigated the binding capability of targeted microspheres to adherent target cells. These microspheres exhibited a 12-fold increase in binding over control, untargeted microspheres. Competitive inhibition experiments with soluble ligand confirmed the specificity of microsphere binding. Overall, the methodology proposed here is capable of quickly and effectively screening large libraries of targeting molecules using instrumentation readily available to the greater research community.     

Development of a novel 96-microwell assay with high throughput for determination of olmesartan medoxomil in its tablets

A novel 96-microwell-based spectrophotometric assay has been developed and validated for determination of olmesartan medoxomil (OLM) in tablets. The formation of a colored charge-transfer (CT) complex between OLM as a n-electron donor and 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquinone (p-chloranilic acid, pCA) as a π-electron acceptor was investigated, for the first time, and employed as a basis in the development of the proposed assay. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 490 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 1-200 μg ml^-1. The limits of detection and quantitation were 0.3 and 1 μg ml^-1, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from hydrochlorothiazide and amlodipine that are co-formulated with OLM in some formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has great practical value in the routine analysis of OLM in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for OLM, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.     

Design of new plasmepsin inhibitors: a virtual high throughput screening approach on the EGEE grid

Though different species of the genus Plasmodium may be responsible for malaria, the variant caused by P. falciparum is often very dangerous and even fatal if untreated. Hemoglobin degradation is one of the key metabolic processes for the survival of the Plasmodium parasite in its host. Plasmepsins, a family of aspartic proteases encoded by the Plasmodium genome, play a prominent role in host hemoglobin cleavage. In this paper we demonstrate the use of virtual screening, in particular molecular docking, employed at a very large scale to identify novel inhibitors for plasmepsins II and IV. A large grid infrastructure, the EGEE grid, was used to address the problem of large computation resources required for docking hundreds of thousands of chemical compounds on different plasmepsin targets of P. falciparum. A large compound library of about 1 million chemical compounds was docked on 5 different targets of plasmepsins using two different docking software, namely FlexX and AutoDock. Several strategies were employed to analyze the results of this virtual screening approach including docking scores, ideal binding modes, and interactions to key residues of the protein. Three different classes of structures with thiourea, diphenylurea, and guanidino scaffolds were identified to be promising hits. While the identification of diphenylurea compounds is in accordance with the literature and thus provides a sort of "positive control", the identification of novel compounds with a guanidino scaffold proves that high throughput docking can be effectively used to identify novel potential inhibitors of P. falciparum plasmepsins. Thus, with the work presented here, we do not only demonstrate the relevance of computational grids in drug discovery but also identify several promising small molecules which have the potential to serve as candidate inhibitors for P. falciparum plasmepsins. With the use of the EGEE grid infrastructure for the virtual screening campaign against the malaria causing parasite P. falciparum we have demonstrated that resource sharing on an eScience infrastructure such as EGEE provides a new model for doing collaborative research to fight diseases of the poor.     

Essential attributes identified in the design of a Laboratory Information Management System for a high throughput siRNA screening laboratory

In recent years high throughput screening operations have become a critical application in functional and translational research. Although a seemingly unmanageable amount of data is generated by these high-throughput, large-scale techniques, through careful planning, an effective Laboratory Information Management System (LIMS) can be developed and implemented in order to streamline all phases of a workflow. Just as important as data mining and analysis procedures at the end of complex processes is the tracking of individual steps of applications that generate such data. Ultimately, the use of a customized LIMS will enable users to extract meaningful results from large datasets while trusting the robustness of their assays. To illustrate the design of a custom LIMS, this practical example is provided to highlight the important aspects of the design of a LIMS to effectively modulate all aspects of an siRNA screening service. This system incorporates inventory management, control of workflow, data handling and interaction with investigators, statisticians and administrators. All these modules are regulated in a synchronous manner within the LIMS.     

Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview

Diagnosis of inherited diseases or cancer predispositions frequently involves determination of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is significantly rising every year. As a result, an increasing number of patient samples with a rising complexity of genetic diseases require molecular diagnostics. In order to apply genetic analyses to large groups of patients or population screening, automation of a sensitive and precise method is highly desirable. Capillary electrophoresis (CE) facilitates the development of methods which can rapidly process large number of patient samples in an automated fashion. In contrast, conventional techniques including Southern blotting, sequencing or standard gel electrophoresis are time consuming, cost ineffective and require substantial amounts of each specimen. Robustness, ease of operation, good reproducibility and low cost are the main advantages of CE. Currently, most protocols adapted to automated CE represent (i) analyses of DNA fragment length or DNA restriction patterns (RFLP), (ii) analyses of single-strand conformation polymorphism (SSCP) and (iii) microsatellite analyses. Recently, automated detection of variations in the FRAXA (CGG)n region (fragile X syndrome), LDL receptor gene, p53 gene, MTHFR (methylenetetrahydrofolate reductase) gene, HFE gene and others has been established on CE systems. These applications clearly demonstrate the suitability of CE for high throughput screening in medical applications.     

In vitro high throughput screening of compounds for favorable metabolic properties in drug discovery

Drug metabolism can have profound effects on the pharmacological and toxicological profile of therapeutic agents. In the pharmaceutical industry, many in vitro techniques are in place or under development to screen and optimize compounds for favorable metabolic properties in the drug discovery phase. These in vitro technologies are meant to address important issues such as: (1) is the compound a potent inhibitor of drug metabolising enzymes (DMEs)? (2) does the compound induce the expression of DMEs? (3) how labile is the compound to metabolic degradation? (4) which specific enzyme(s) is responsible for the compound's biotransformation? and (5) to which metabolites is the compound metabolized? Answers to these questions provide a basis for judging whether a compound is likely to have acceptable pharmacokinetic properties in vivo. To address these issues on the increasing number of compounds inundating the drug discovery programs, high throughput assays are essential. A combination of biochemical advances in the understanding of the function and regulation of DMEs (in particular, cytochromes P450, CYPs) and automated analytical technologies are revolutionizing drug metabolism research. Automated LC-MS based metabolic stability, fluorescence, radiometric and LC-MS based CYP inhibition assays are now in routine use. Automatible models for studying CYP induction based on enzyme activity, quantitative RT-PCR and reporter gene systems are being developed. We will review the utility and limitations of these HTS approaches and highlight on-going developments and emerging technologies to answer metabolism questions at the different stages of the drug discovery process.     

Design and use of electrochemical sensors in enantioselective high throughput screening of drugs. A minireview

The importance of reliable detection systems for enantiomeric assays increases with the necessity of high throughput screening analysis of raw materials for the pharmaceutical industry. The utilization of electrochemical sensors in enantioselective analysis is an accurate and precise alternative to chromatographic techniques. The reliability of the response characteristics as well as of the analytical information obtained by using electrochemical sensors is strictly correlated with the design of the sensors. The designs evaluated for sensors have been based on PVC, imprinting polymers and carbon paste matrices. Among these, carbon paste sensors have been the most reliable and have been utilized for the construction of potentiometric, enantioselective membrane electrodes as well as for amperometric biosensors, and immunosensors. There are two ways to use the electrochemical sensors in enantioselective screening analysis: selective binding and catalyst selectivity. A molecule with a special chemical architecture is required for selective binding: a lock for a key. The high reliability of analytical information obtained using these sensors has made possible the automation of potentiometric and amperometric techniques by integration of enantioselective sensors as detectors in flow injection analysis and sequential injection analysis techniques.