Bio Screening Industry News

European chemists have discovered that both mirror-image forms of a particular compound can bind at the same time in the same site of an enzyme, a phenomenon that has never been seen before. The finding has significance for drug discovery screening and studies of how small molecules interact with proteins.

Rolf Breinbauer from Graz University of Technology, Austria, and Wulf Blankenfeldt from the Max Planck Institute of Molecular Physiology in Dortmund, Germany, were studying a metabolic enzyme from a species of the bacterium Burkholderia cepacia, using racemic mixtures of chiral probe molecules to find ones that bound in the enzyme’s active site. In most cases only one form of a chiral (or ‘handed’) molecule would bind at once, but they found that in one instance both enantiomeric forms occupied the binding site at the same time.

‘If you read the textbooks about enantiomers,’ says Breinbauer, ‘there’s a simplified notion that one enantiomer is good and the other is either bad or just idle.’ He explains that for most proteins (apart from certain enzymes that have evolved to cope with wide ranges of substrate molecules) either only one enantiomer will bind, or both can bind individually - with the assumption that one form will be significantly more active than the other. ‘Our findings show that the world is more complicated,’ he adds.

While each individual enantiomer can bind to the enzyme seperately, Breinbauer notes that the arrangement of the molecules within the binding site is quite different when both bind together. This could lead to cooperative effects, producing either an enhanced or diminished response relative to the individual enantiomers.

He adds that this could have relevance in drug discovery screening, where mixtures of both enantiomers of chiral compounds are routinely screened together to find initial hits. ‘People need to consider more options when interpreting binding data from racemic mixtures.’

Dafydd Owen of Pfizer Research Chemistry in  Sandwich, UK, agrees that the finding is an important reminder that chemists need to be open-minded about interpreting screening data. It also highlights the inherent trade-offs made when screening mixtures - particularly in high-throughput screens when mixtures of several compounds are tested at once.

Owen sees most interest in the discovery in the area of fragment-based drug discovery, where small ‘fragment’ molecules found to bind to a drug target are linked together to make potential drug molecules. ‘As a medicinal chemist,’ he adds, ‘my immediate thought was to join the two structures together to incorporate the best of each and make a hybrid.’ He points out, however, that from a fragment point of view it is almost irrelevant to the enzyme that the two molecules happen to be mirror images of each other, ‘despite their apparent similarity, nature views enantiomers as very different molecules’.

Phillip Broadwith

Source: rsc.org

High-content screening (HCS) and the technology to do it faster, on more compounds in a shorter period of time, and to generate quantitative, multiparametric data took center stage at CHI’s “High Content East” meeting held in Boston last month. Presenters described how they are implementing enhanced screening systems, image-analysis methods, and data-management strategies to achieve daily HCS runs on tens of thousands of wells and screening campaigns totaling 200,000 to 3 million wells.

High throughput HCS—albeit not yet reaching the numbers common for conventional high-throughput screening (HTS) and with lingering limitations and challenges related to live-cell imaging over time—is making its mark and being used to probe the biological basis of disease and to detect even subtle phenotypic changes in response to experimental compounds.

Determining whether a cell looks like a cancer cell, for example, typically requires being able to detect subtle morphological changes, such as small alterations in size or structure, changes in the connections a cell makes with neighboring cells, or variations in the texture of staining. These have, historically, been mainly qualitative parameters detected by studying and comparing images of cells.

In her talk at the conference, Anne Carpenter, Ph.D., director of the imaging platform at the Broad Institute of Harvard University and MIT, presented her group’s work using HCS and image analysis to quantify difficult phenotypes and differentiate disease states such as leukemia.

Not only do HCS systems and image-analysis software automate the screening process, enabling theanalysis of many more cells in less time and increasing the chances of detecting even small numbers of altered cells, they can also utilize algorithms that evaluate defined combinations of parameters in a quantifiable manner and apply techniques to distinguish between clumping or closely juxtaposed cells. Relying on computer-based image analysis also standardizes the process, eliminating factors such as variability in human expertise and experience, consistency, and fatigue.

Dr. Carpenter’s group uses machine-learning methods to train image-analysis software to identify subtle phenotypic changes. Biologists work with the software in an iterative fashion in a process called supervised machine learning. They teach and correct the computers on a series of test images, refining the system’s knowledge base in a process that typically takes less than a day. The group developed the algorithms used by the biologists and has made them available as open-source software.

A recent paper published in PNAS by T. R. Jones, et al., documents the use of a trained image-analysis system to discriminate 15 different cellular phenotypes. Other projects involve teaching the software to discriminate leukemic from normal cells, to identify liver cells that are growing normally in culture—to aid in the development of physiologic models of liver function for use in drug testing—and training computers to detect subtle changes that signal the initiation of cell division for studying cell-cycle regulation in cancer.

Neil Carragher, Ph.D., senior scientist in the advanced science and technology laboratory at AstraZeneca, described how the company is applying high-content and live-cell imaging techniques and integrating the results with data derived from in vivo imaging and proteomic studies to improve clinical predictability.

Dr. Carragher’s group combines the results of high-content in vitro and in vivo assays to generate mechanistic information about phenotypic responses on candidate therapeutic compounds. The goal is to create a multiparametric fingerprint of a phenotype from images generated by HCS and to use this knowledge to enhance predictions of efficacy and toxicity early in drug discovery and reduce attrition later in development.

The phenotypic signatures are based on measurements of approximately 150 different parameters per cell for each assay. Data from multiple assays is collated for every test compound and compared with data obtained using well-characterized reference compounds to generate mechanistic hypotheses.

Only recently has open-source and commercial software become available “that allows you to quantitate more complex phenotypes, subtle changes, and heterogeneous responses from images,” Dr. Carragher said.

His group is employing two main approaches—each with different advantages and limitations. The first strategy relies on Definiens’ Cognition Network Technology™ software that allows users to develop algorithms that capture, computationally, what researchers can see visually. “It is very much context-based” and identifies objects based on how they are related to others in the image, rather than as individual pixels, explained Dr. Carragher. The in-house algorithm-development process depends on iterative programming steps. The other approach involves machine-learning tools using software such as the CellProfiler developed at the Broad Institute.

Redirecting Approved Drugs

While studying an HIV protein that plays an essential role in AIDS progression, researchers at the University of Pittsburgh School of Medicine have discovered compounds that show promise as novel treatments for the disease.

HIV drug discovery efforts have met with little success in finding compounds that interact with an important HIV virulence factor, called Nef, because it lacks biochemical activity that can be directly measured, explained Thomas E. Smithgall, Ph.D., William S. McEllroy Professor and Chair, Department of Microbiology and Molecular Genetics, and senior author of the paper, which was published last week in the early, online version of ACS Chemical Biology.

To get around that problem, Dr. Smithgall’s team developed an assay to measure Nef function indirectly by coupling it to another protein, called Hck, which Nef activates in HIV-infected cells. Because Hck activity can be easily measured, the investigators were able to use it as a reporter for Nef activity in an automated high-throughput screening process. In collaboration with the University of Pittsburgh Drug Discovery Institute, they screened a library of 10,000 chemical compounds against the coupled proteins to see which ones influenced Nef-induced activation of Hck.

After further testing, they confirmed that three compounds inhibited the activity of the Nef-Hck complex and, more importantly, all of them also interfered with HIV replication. One compound was so effective that it suppressed HIV replication to undetectable levels in cell culture experiments.

“So we now have a way to rapidly and efficiently screen for inhibitors of Nef signaling through Hck,” Dr. Smithgall said. “But the surprise was that some of those inhibitors also showed strong antiviral activity in cell culture models.”

There is evidence that people infected with HIV variants that have mutations in the Nef gene take substantially longer to develop disease symptoms or AIDS, he said. In animal models, disrupting the production of Nef from the virus or its interaction with Hck also delays or prevents disease symptoms. The next challenge for the researchers will be to determine whether these compounds also interfere with progression of AIDS-like disease in animal models by blocking Nef function.

“Most current therapies for HIV infection use drugs that interfere with the function of viral enzymes such as reverse transcriptase or with the interaction of the virus and the host cell,” Dr. Smithgall said. “Targeting Nef represents an entirely new approach that could be useful to deal with issues such as drug-resistant HIV strains, and may slow the progression to AIDS.”

He added that Nef is just one of several so-called “accessory proteins” encoded by HIV which are important virulence factors in AIDS. Inhibitory compounds against some of the others might be revealed using a similar coupled protein approach for high throughput screening.

Source: labspaces.net

This story originally ran on Oct. 6.

By Tony Fong

Researchers from the University of Cincinnati and MDS Analytical Technologies have used mass spectrometry to develop a high-throughput screening method for drug discovery they say can be more precise and cost-effective than existing techniques.

The technique is based on a MALDI triple-quadrupole platform and exploits the selective multiple-reaction monitoring transition features of the platform. By doing so, the new method is able to lower the cost of high-throughput screening for drug compounds to pennies per well from as much as $1 per well currently, Ken Greis, associate professor of cancer and cell biology and director of proteomics and mass spectrometry at the University of Cincinnati College of Medicine, told ProteoMonitor recently.

A study detailing the method was published Sep. 15 in the online edition of Rapid Communications in Mass Spectrometry.

In the paper, Greis and his co-authors said that drug discovery typically begins with a validated target enzyme “with the initial goal of finding appropriate molecular scaffolds with inhibitory activity via high-throughput screening.” The scaffolds are then subsequently used for lead compound optimization, and “ideally for the development of a safe and effective therapeutic compound.”

The most common methods of high-throughput screening have been fluorescence- and chemiluminescence-based approaches. Such approaches, Greis said, have been “very successful” because the same reagents can be used for many different enzymes.

But that same characteristic also creates a risk for interference.

“When one’s evaluating a compound repository for inhibitors, you often have a series of compounds that will fluoresce themselves,” Greis said. “If they fluoresce, they’re going to give you a false signal. Alternatively … there are compounds that inhibit the fluorescent properties, or what’s called quenching fluorescence, [that] also give false read-outs.”

Another problem is in the way the assays get generalized so that the reagents work for a wide range of enzymes. Such assays are called coupled assays: “You have a product being formed from your enzyme reaction but that’s not what actually triggers the fluorescence,” Greis said. “That product gets converted to another enzyme to another product through another enzyme to another product that then can be fluoresced.”

This series of enzyme step, or coupled assays, ultimately results in a read-out. “The problem is any compound that interferes with any of those steps along the way also gives you false read-outs,” which tend to be false positives, he added.

But by using mass spectrometry to measure enzyme activity, Greis and his colleagues are able to get a direct read-out, “so a mass spectrometer effectively can give you a quantitation and a mass of a compound.”

By taking a ratio of the substrate being converted to a product — the essence of an enzyme assay, Greis said — and measuring that directly on a mass spec, there is no interference either from quenching or auto-fluorescence.

“And what we’ve found thus far is we’ve not seen any false positive read-outs. If we get a compound that shows that it’s active, even in single-point assays, it’s been demonstrated that it’s a dose-dependent inhibitor.”

And because the method uses native peptides or small-molecule substrates, the method can be done for “at most, pennies per sample well,” Greis said. By comparison, fluorescent and chemiluminescence reagents cost between 50 cents to $1 per well.

“So if you run a million compounds, you can run up a half-million dollars of reagents costs right away, whereas the label-free read-out is going to cost you maybe a couple thousand dollars for the reagents,” he said. “That’s a mass spec advantage.”

A prior study by researchers in China had demonstrated the utility of a MALDI-Fourier transform mass spectrometer for high-throughput screening of small-molecule substrate/product conversion.

Nowadays, more and more novel enzymes can be easily found in the whole enzyme pool with the rapid development of genetic operation. However, experimental work for substrate screening of a new enzyme is laborious, time consuming and costly.

On the other hand, many computational methods have been widely used in lead screening of drug design. Seeing that the ligand-target protein system in drug design and the substrate-enzyme system in enzyme applications share the similar molecular recognition mechanism, we aim to fulfill the goal of substrate screening by in silico means in the present study.

Results: A computer-aided substrate screening (CASS) system which was based on the enzyme structure was designed and employed successfully to help screen substrates of Candida antarctica lipase B (CALB).

In this system, restricted molecular docking which was derived from the mechanism of the enzyme was applied to predict the energetically favorable poses of substrate-enzyme complexes. Thereafter, substrate conformation, distance between the oxygen atom of the alcohol part of the ester (in some compounds, this oxygen atom was replaced by nitrogen atom of the amine part of acid amine or sulfur atom of the thioester) and the hydrogen atom of imidazole of His224, distance between the carbon atom of the carbonyl group of the compound and the oxygen atom of hydroxyl group of Ser105 were used sequentially as the criteria to screen the binding poses.

223 out of 233 compounds were identified correctly for the enzyme by this screening system. Such high accuracy guaranteed the feasibility and reliability of the CASS system.

Conclusions: The idea of computer-aided substrate screening is a creative combination of computational skills and enzymology.

Although the case studied in this paper is tentative, high accuracy of the CASS system sheds light on the field of computer-aided substrate screening.

Author: Tao XuLujia ZhangXuedong WangDongzhi WeiTianbi Li
Credits/Source: BMC Bioinformatics 2009, 10:257

Source: 7thspace.com

San Francisco, CA - GTCbio Announces its 4th Annual Assay Development and Screening Conference taking place June 8-9, 2009. As compounds derived from high throughput screening increasingly find their way into clinical trials, drug screening has become widely accepted as a critical step in the drug discovery process. After more than a decade of rapid growth, tremendous progress has been made in assay technology, laboratory automation, and informatics. These technological developments have not only facilitated a drastic increase in throughput and efficiency in drug screening, but have also provided novel solutions in other areas of drug discovery and development. As screening has also become prominent in biological research, screening facilities have become increasingly popular in academic institutions.

As the pharmaceutical industry continues to face the challenges of developing more new chemical entities and reducing the cost of R&D, the demand for novel technologies and creative approaches for improving the efficiency of screening has intensified. Cell-based assays used in compound screening and high-content screening technologies have gained popularity in the industry. Years of intensive research have finally resulted in label-free technologies in the drug screening market place. These technologies provide new ways of interrogating cellular and molecular binding events and enable orthogonal screening approaches to drug targets.

The goal of the 4th annual Assay and Screening Technologies Conference is to provide a forum for academics and professionals in the drug discovery industry to stay abreast of exciting new developments in assay technologies while exchanging ideas and developing more efficient approaches to the drug discovery and development process.

For more information, visit http://gtcbio.com/conferenceDetails.aspx?id=123

Munich (D) and Leiden (NL), November 24, 2008 / b3c newswire /  – CRELUX and ZoBio announced today that they have successfully executed their first fragment based screening projects from a jointly established platform.

One of the first targets, which also will be made accessible to customers, was Pim1, a kinase that has been implicated in the progression of several haematological malignancies. In addition to the “off the shelf” data on Pim1, tailor made fragment based screening projects are available for customers upon request.

The joint technology platform combines ZoBio’s proprietary Target Immobilized NMR Screening (TINS) technology with CRELUX’s high performance kinase crystallography platform. In the first campaign Pim1 was screened by TINS using ZoBio’s fragment library, hits were assessed in an in vitro kinase assay and the top 50 hits have been soaked into protein crystals. 37 out of these 50 fragments showed defined binding modes. Together with this high hit rate the structural diversity within this group generated multiple points for optimization and clearly proved the power of this technology combination.

“We are delighted to have found a perfect partner for entering into high performance fragment based screening. The collaboration with ZoBio adds another crucial drug discovery technology to our service portfolio”, commented Dr. Michael Schäffer, CEO of CRELUX.

“This project demonstrates the power of the combination of TINS with top notch crystallography. I am absolutely convinced that together we can provide our customers with critical starting point to jump start their challenging or failed targets.” noted Dr. Gregg Siegal, CSO of ZoBio.

CRELUX has used its state-of-the-art structural biology platform to solve more than 270 crystal and co-crystal structures for pharma and biotech companies. This platform encompasses all steps – from target cloning and expression all the way to high-throughput protein crystallization and in-house x-ray crystallography.

ZoBio provides fragment discovery and characterization services to the pharmaceutical and biotech industries using its proprietary Target Immobilized NMR Screening (TINS) platform. TINS, with its unparalleled sensitivity and reliability, has been used to discovery highly diverse, efficient ligands for a variety of targets including kinases, protein-protein interactions, viral targets and membrane proteins.

Cell-based assays provide one of the most valuable tools in drug discovery.  They are routinely used in target validation, HTS campaigns, structure activity relationship analyses and ADMET studies. Many factors need to be considered in designing, developing and running relevant cell based assays to progress discovery programs.  Significant advances continue to be made in assay design and cell supply processes that incorporate biologically relevant cell types and novel detection technologies. This symposium is designed to capture successful techniques and practices that enable high quality cell-based assays using commonly employed cell types such as CHO, HEK and U-2 OS, as well as the expanding application of additional cell types. The symposium will be of great interest to cell culture scientists, assay developers, screeners, medicinal chemists, ADMET and therapeutic teams.

Session Overviews:

Session 1: Cells as Reagents - Fact or Fiction?
This session will cover various aspects of the most important material used in cell-based assays: the cells. The talks will address the topics such as: how to characterize cell lines to ensure their identity? What are the impacts to the cells after transfection? What are the issues during scale up process? This session will also highlight advances in cell culture automation and material tracking system for cell-based assays.

Session 2: Assay Development - Present Realities
Topics covered include the application of BacMam virus based gene delivery in assay development, the development of high-content cellular assays, dielectric spectroscopy technology and photoprotein aqueorin based GPCR assay platforms and considerations in screening for antibody based therapeutics.

Session 3: Cell-Based Screening - The How, Why & Where
This session will include topics on the implementation of cell-based assays and screens across the discovery process focusing on the challenges, solutions and issues to consider. Presentations include experiences with high throughput screening for lead identification, profiling with cellular panels, ADME applications, and the use of high content screening in both drug discovery and to probe complex cellular systems.

Session 4: Cell-Based Assays - Emerging Trends
The topics in this session will address emerging trends in supply of cells that attempts to bridge the gap between traditional target based in vitro assays and in vivo measurements. The presentations will highlight potential applications of cells to build model systems that can offer the combined benefits of traditional in vitro and ex vivo approaches. These emerging technologies and methodologies hold the promise of addressing a major gap in using target based approaches to discover new biological tools and drugs and will challenge the supply of cell reagents.

http://www.sbsonline.com/

The FINANCIAL — Sanofi-aventis announced on September 26 the launch of the dScreen Consortium, a research initiative conducted with RainDance Technologies, Inc., Lexington, Massachusetts, and Louis Pasteur University, Strasbourg, France, to develop the next generation of High-Throughput Screening (HTS) for drug discovery applications.

The consortium was founded with the assistance of the Alsace BioValleyTM Cluster, France, which helped secure financing and support for the program.
The dScreen Consortium assembles:
- the renowned drug screening expertise of sanofi-aventis
- the unique expertise in droplet-based microreactors of the Chemical Biology Laboratory at the Institute for Science and Supramolecular Engineering (ISIS) of Louis Pasteur University
- and RainDance Technologies’ unique capabilities to apply droplet-based microfluidic technologies to human health and disease research.
“We are delighted to enter this partnership with two highly innovative research groups in this rapidly advancing field,” said Martin Galvan Ph.D., Scientific Director at the sanofi-aventis research site in Strasbourg. “The expected gains in terms of productivity and knowledge should significantly accelerate our drug discovery programs”.
Based in the Alsace BioValley in Strasbourg, the consortium will utilize the pico-liter volumes and ultra-high speed capabilities of RainDance’s technology and systems to achieve breakthrough performance in high-throughput drug screening methodologies.
“This exciting project represents the first research collaboration undertaken by our new RainDance Technologies France SARL subsidiary,” said Chris McNary, President and Chief Executive Officer of RainDance Technologies. “The speed, simplicity, and minute volume of our droplets eliminate the need for unnecessarily complex automation solutions in high-throughput screening. Our technology will process 10 million droplets per hour on a single benchtop instrument, dramatically accelerating the drug discovery process while conserving precious screening compounds,” added McNary.

“This project is an excellent opportunity to develop the compartmentalisation of reactions in emulsion droplets for an entirely new field of application: HTS for drug discovery” said Andrew Griffiths, head of the Chemical Biology Laboratory at ISIS. ”University Louis Pasteur is proud to be part of this consortium which will open new scientific routes while generating top scientific lectures to our students” said Jean-Marc Jeltsch, Vice-President, Louis Pasteur University.
“The dScreen Consortium is a great illustration of the “Pôle de Compétitivité” policy in France: the development of breakthrough innovations in drug screening through collaborative R&D programs results in strengthening local actors such as the sanofi-aventis research site and in the creation of a subsidiary of an US company in Alsace. Furthermore, the establishment of a drug screening services platform based on the results of the program will reinforce the capabilities of our cluster” underlined Pascal Neuville, President of Alsace BioValley.

Services to accelerate programs from biological target to first-in-man use Idealp-Pharma is launching fully integrated drug discovery and preclinical development services combining medicinal chemistry, cheminformatics,
screening, early ADMET and preclinical development capabilities to speed up
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“Idealp-Pharma manages its customers’ hit discovery and validation, hit-to-lead
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More information about integrated drug discovery services can be found at www.idealp-pharma.com
About Idealp-Pharma

Idealp-Pharma’s aim is to expand partner’s drug pipeline by accelerating drug
discovery process from the biological target to first-in-man use. Idealp-Pharma
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Idealp-Pharma’s purpose-built lab covers a total of 2000 square meters. Idealp-Pharma now employs 60 staff. More information about Idealp-Pharma can be found at www.idealp-pharma.com