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Feature Articles : Aug 1, 2007 (Vol. 27, No. 14)

Cell-Based Research Gains Momentum

Assays Advance Knowledge of Drug and Protein-protein Interactions in Humans
  • Elizabeth Lipp

Cell-based assays have become the rage once again, and there are a number of avenues for researchers to utilize to get the work they need done. At Cambridge Healthtech’s “World Pharmaceutical Congress,” the focus was on new and improved technologies for cell-based assays for high-throughput screening (HTS). The Cell-Based Assays for HTS track highlighted new screening tools and recent developments for cell-based assays, label-free screening, and novel approaches and technologies for cell-based assays and drug screening.

Genomic approaches such as microarray expression analyses and ChIP-CHIP transcription factor binding assays have yielded many new discoveries and insights into genetic pathways. These technologies provide valuable observational data but do not identify the functional connections within networks or explain the mechanism of gene regulation. SwitchGear Genomics (www.switchgeargenomics.com), a company spun out of Stanford University, provides new tools to characterize the functional connections in biological networks

A More Complete Picture

“When it comes to understanding the function of the human genome and specifically how gene regulatory networks are controlled, SwitchGear’s tools can be applied in a powerful way,” noted Nathan D. Trinklein, cofounder and CEO of SwitchGear. “We not only distribute novel experimental research tools, but we also provide services using our platform.”

Trinklein’s presentation described data and analysis of the hypoxia pathway in living cells upon different compound treatments using the SwitchGear platform. “Hypoxia is a great pathway to study when you’re thinking about creating drug programs for cancer, cardiovascular disease, stroke, or anemia,” said Trinklein. “In transcriptionally regulated pathways like hypoxia, companies can look to target a master transcription factor.

In this scenario it is important to characterize how the pathway is activated in tumor growth compared to a stroke, for example. What companies want to see is a detailed picture of how different compounds affect a pathway, and in this same way, researchers can also determine ideal dosages and quantify the subtle differences between compound analogs.”

SwitchGear has produced libraries of thousands of human promoters, UTRs, and other regulatory elements encompassing diverse disease-related pathways. SwitchGear will soon release a tool to characterize nuclear hormone receptor pathways. Its regulatory element libraries are built using luciferase reporter technology and are made available as ready-to-use tools for cell-based studies. Trinklein notes that their customers cover the spectrum of labs from academia to big pharma.

“The tools we offer enable researchers to utilize high-throughput screening to describe a more complete picture of what’s happening inside a cell,” said Trinklein. “This is a cutting edge approach that should be useful in targeting compounds, discovering off-target effects, and getting more effective therapies to market sooner.”

RNAi Screen for Huntington's

BioFocus DPI (www.biofocusdpi.com) in conjunction with the High Q Foundation developed a high-content screen to identify genes that modulate the survival of cells over-expressing mutant forms of the Huntington’s protein.

“This assay was used to screen the human neuroblastoma cell line SH-SY5Y with a collection of adenoviral shRNAs and full-length human cDNAs totaling over 33,000 data-points,” said Richard Janssen, senior director, BioFocus. “We developed analysis algorithms using InCell Developer suite factoring in the number of cells per field, integrated polyQ-GFP levels, ratio of dead (PI-positive cells over total cell number), and the ratio of cells carrying inclusions, based on GFP density.”

Not only did they observe a successful cell survival screen using adenoviral shRNA on a high-content platform, but they also found that the high-content data can be used for pathway analysis, “and you can extend this to target and compound discovery in other disease mechanisms as well,” said Janssen.

“A logical extension of these target and drug discovery programs for patient foundations has been the development of similar studies in cystic fibrosis and ALS. It will be important to have good cell assays, preferably based on human primary cells, to accommodate this kind of research. And, as we’re already seeing, stem cells are also going to become important in drug development.”

One of the key points in Janssen’s stem cell argument is their versatility. “You can work with a large range of human primary cells, but the problem is that not all cell types are available in sufficient amounts. They don’t live long enough.” Human stem cells will provide a solution to this problem. “The human primary cells, either derived from donors or from stem cells, are the most relevant with regard to research and discovery.”

More than half of all primary drug screening assays now use cell-based assay formats. Consequently, the increased cell use poses a number of issues, namely batch variation, cell production scheduling, and capacity management.

Bulk Production of Cells

“There has been a great deal of interest recently in the use of cryopreserved cells in ready-to-go format as a means for addressing these issues,” said Stephen M. Game, technology manager, GE Healthcare (www.gehealthcare.com). “This format offers the potential for the manufacture of single batches of cells sufficient for complete screening campaigns and effectively decouples cell production from screening.”

Game described the use of microcarriers to culture cells in bulk for HTS. This 3-D culture system, used in combination with appropriately configured bioreactors, provides a robust and reproducible format for single batch bulk cell production. “Bulk cell production, is one part of a range of services we offer,” said Game. “It’s one less job for the researcher to do.”

Cryopreserved cells cultured by this method have been validated in a number of HTS-relevant functional assays and meet required performance criteria for primary HTS assays. “We’ve had a lot of interest in the concept of outsourcing cryopreserved cell production, which makes sense, given that many labs would have to add considerable infrastructure to meet the increasing demand for cells,” said Game.

The approach also has great potential to facilitate the bulk production of transiently transfected or transduced cells opening the door to their wider use in HTS. “Basically, we aim to produce cryopreserved cells that can be used just like any other assay reagent, ready for use straight from the freezer,” Game said.

Automation

Tim Ward, director of strategic marketing for The Automation Partnership (www.automationpartnership.com), described new developments in automated cell culture and how they can be used to support a wide range of different applications that require consistent, high-quality cells for functional assays. “The use of more compact automation platforms together with high-density culture vessels has provided increased flexibility and improved productivity in supporting both bulk cell supply and maintenance of a wide range of cell lines for high-throughput assays,” said Ward.

“The whole dilemma of cell production is that these cells are produced in an unnatural way,” Ward noted. “We want to have a lot of cells, and they need to be happy and healthy. Creating cells unnaturally is not a perfect process, as outputs can vary widely.” This is why automation is perfectly suited to cell production. “It is much easier to create a reproducible reagent when you are using consistent processes. It’s difficult to do that manually.”

Ward noted that these technology advances are demonstrating similar benefits where small quantities of cells are required over extended time periods for profiling and follow-up studies. “The way we identify and find solutions is collaborative. When we work with companies, we ask them where the breaks in their processes are, where their pain points are, and we work together to find the best solution. Our goal is to make a valuable, viable business.”

Protein-protein Interactions

Keith R. Olson, vp, DiscoveRx (www.discoverx.com) presented a novel assay technology for monitoring protein activity in whole cells that is based on enzyme fragment complementation (EFC) using b-galactosidase. “Unlike most split enzyme approaches, our assay utilizes a small 4-kD peptide for complementation that can be implemented like any epitope tag in a cell-based assay,” said Olson. “The benefit in this case is that there are no antibodies or imaging methods required, allowing the user to monitor complex cell signaling pathways with a simple, homogeneous assay with excellent screening characteristics provided by the added enzyme-based signal amplification inherent to this approach.”

A new peptide tag variant called ProLink™ shows weak affinity for inactive b-gal, allowing it to be used for analysis of protein-protein interaction events, said Olsen. Previous versions of this tag bound to the complementing portion of b-Gal tightly, and were in effect irreversible, making them poor candidates for studying protein interactions.

“Protein-protein interactions represents a fairly untapped area of research,” noted Olson. “Developing this approach has helped us bridge a gap. We’ve taken barriers away that can accelerate research and hopefully bring forward new kinds of targets and assays based on our improved screening performance.”

Using this new approach, a productive binding interaction between two target proteins is required to deliver the b-gal components together and accomplish complementation. Olson’s team applied this initially to a targeted set of interactions, namely the binding between b-arrestin and activated GPCRs.

“It was a purely commercial decision. After all, how do you choose which of the millions of protein-protein interactions to target?” Olson posited. “Given the importance of GPCRs for screening and the amount of previous research that had gone into validation of arrestin for GPCR assays, this was clearly an area where we could get a high return on investment for our research dollars.”

Looking forward, Olson added, “The question is still fairly common as to whether or not you can develop a drug capable of disrupting a protein-protein interaction. It’s been done successfully, so we are hopeful that our technology will be a key driver in advancing the field. The next step is staging whole screening campaigns around this concept.”

Embryonic Stem Cells for HTS

Invitrogen (www.invitrogen.com) is currently developing a platform for rapidly engineering human and mouse embryonic stem cells for use in screening. “This platform consists of a toolbox of genetic elements that can be rapidly assembled and integrated into the stem cell genome at specific loci,” said Jon Chesnut, director, stem cells and regenerative medicine.

“Cell screening platforms can be created with lineage- and tissue-specific promoters driving reporter genes as well as with gene perturbation and overexpression constructs.”

These platforms could then be used for screening compounds in stem cells or in cell populations differentiated from the stem cells. The tangible, near-term goal is to use these platforms in drug screening to provide more relevant tissues and compounds.

The aim of Chesnut’s group is to provide relevant platforms for driving screening that would allow companies to do their screening in human, normal adult tissue rather than primary or cadaveric cells. “We want to be able to either fail drugs for safety reasons sooner or get those compounds that can be applied to market sooner,” said Chesnut.

“This is the future of the business; testing on transformed cells simply doesn’t work the same way. Moving toward working with stem cell-derived adult-human cells should reduce the necessity of using whole-animal models, lowering the development cost of a drug as well as the time to market.”

Ryan McGuinness from MDS Analytical Technologies, formerly MDS Sciex (www.mdssciex.com), presided over a roundtable discussion about the strong points of competing label-free assays.

“Label-free cell-based platforms, with their abilities to monitor receptor activation in a native setting, further the push toward biorelevant and disease relevant screening in a manner that is easy for the user to set up, straightforward to perform, and provides results in rich, real-time manner. The early adopters, mostly big pharma, have seen the advantages of these technologies in bringing more biorelevance into the drug discovery pipeline earlier.” said McGuinness.

“Label-free technologies are still in their early stages, and the feedback we have gotten from our early adopters has been crucial. The collaborative effort is key.”