September 15, 2006 (Vol. 26, No. 16)

Better Characterization Is the Key to Finding a Successful Target

Biomolecular screening broadly encompasses biological and biochemical screening approaches in the quest to find new drugs. It typically involves the use of cells, chemistry, genomics, and proteomics in one or many aspects of the screening assay. Biomolecular screening can be applied at various screening stages and with multiple assay formats.

“The current challenge for biomolecular screening, whether biochemical or cell-based, is to translate the wealth of genomic data and the hundreds of potential disease target proteins into novel therapeutic lead compounds. There is a need in the industry for technologies that better characterize targets, enabling a more careful selection for subsequent screening approaches, and aid the screening of novel targets that do not belong to the classical well-studied target families,” says Michael Blind, Ph.D., CSO of NascaCell Technologies (

A number of speakers will be talking about new screening approaches and technologies at the upcoming “Society for Biomolecular Sciences” (SBS) conference in Seattle.

Aptamers are short, single-stranded oligonucleotides that fold into defined 3-D conformations, enabling them to specifically bind to target molecules with high affinity by complementary-shape interactions.

Aptamers: The Next Generation

NascaCell Technologies provides aptamer technology to support customers in target validation and drug discovery. “In contrast to the recombinant production of proteinaceous biomolecules, the chemical synthesis of aptamers allows an almost unlimited number of modifications, which can be introduced at defined sites. Since aptamers not only bind to targets but also are drug-like inhibitors of target function, they are suited for target-validation approaches acting on the protein level and are a good complement to siRNA validation technologies. Aptamers offer a unique advantage to bridge target validation and drug discovery since they either can be used to identify functionally equivalent small molecule lead compounds in HTS programs or can even be turned into drugs themselves,” explains Dr. Blind.

Dr. Blind will be discussing parallel integration of postgenomic technologies, covering an approach that was initiated in cooperation between NascaCell Technologies and five European partners under the project name PONT (Parallel Optimization of New Technologies for the post-genomics drug discovery).

“Under this approach, highly disease-deregulated genes identified in human tissues will be interrogated. Parallel-informatics analysis will select target subsets with properties consistent with those expected for effective and safe therapeutics. These target subsets will be further examined for protein-expression profiles, the protein structures will be determined at atomic resolution, functions will be evaluated with in vitro and in vivo models, and small molecules binding to specific targets will be identified with aptamer-based high-throughput screening assays. Resultant leads will be optimized with structural analysis of targets in complex with binding molecules. The combination of these technologies creates a unique multidisciplinary meta-platform that can likely reduce early-stage drug development times by two years or more,” says Dr. Blind.

Electronic Sensing of Cells

ACEA biosciences ( will be presenting on using cytological profiling as a measure of target specificity for identifying antimitotic agents. The seminar will describe the company’s real-time cell electronic-sensing device (RT-CES) and its application for cytological profiling for antimitotic compounds.

“The concept behind this type of screening is the use of electronics to conduct cell assays under label-free conditions. The device measures cell kinetics in real-time and allows cell sampling throughout the duration of the assay,” says Yama Abassi, Ph.D., director of cell biology and assay development at ACEA Biosciences.

ACEA manufactures 96-well plates that have an interdigitated microarray coating of electrodes in each well. Cell growth in the wells creates impedance changes, which is measured by the RT-CES system. The multicomponent system has a plate-handling station, which is placed inside the tissue-culture incubator and connected to an electronic-signal analyzer. The analyzer monitors the signal generated by cells from the 96-well E-Plate and feeds it into software that analyzes the data in real-time.

“Our current system can handle six 96-well plates. We recently introduced the real-time cell invasion and migration (RT-CIM) assay capability with our 96-well plates. We plan to unveil a 384-well plate format at the upcoming SBS meeting,” says Dr. Abassi. “Advantages of using RT-CES for cell assays are real-time data collection and responses that can be measured at the exact time of drug addition.”

The use of cytological-profiling data obtained by using RT-CES or RT-CIM systems is a tool to identity agents acting at the mitotic phase of cell cycle. “When cells are subject to drug treatments, each drug gives a characteristic cytological profile. Similar drugs give similar cytological profiles. An example of one such profile is comprised of viability, morphology, and adhesion outputs. This type of cytological profiling is valuable in screening for inhibitor molecules whose mechanism is unknown. We have demonstrated proof of concept with a study on human cancer cell lines using both small molecule inhibitors of known mitotic targets as well as siRNA. We have also observed similar signature-activity profiles for DNA damage, kinase inhibitors, and actin-disrupting agents,” adds Dr. Abassi.

Enzyme-Fragment Complementation in Bioengineered Cells

“The use of cell-based assays is becoming more of the norm in screening. Cell-based assays provide useful information on bioavailability and toxicology resulting in good-quality hits. They are widely used in GPCR studies and are being increasingly used for other target classes as well,” says Keith Olson, Ph.D., vp of product and market development at DiscoverRx (

A key DiscoverRx technology is the use of enzyme fragment complementation technology, PathHunter™, in bioengineered cell lines. A truncated b-galactosidase gene is engineered into host cells. Peptide tags that carry the truncated part are fused to a protein of interest. If this protein is translocated, for instance to the cell nucleus, the truncated b-galactosidase gene is activated due to the resulting complementation. Output is measured as a chemiluminescent signal that is generated upon cleavage of exogenously provided substrate by the fully functional b-galactosidase.

“This technology has applications in three major areas: Cell-signaling pathway profiling and screening, protein-expression analysis, and the study of protein-protein interactions, such as GPCRs and b-arrestin. With our technology we are able to measure translocation directly, resulting in a faster assay that is not dependent on a transcriptionally active target. Other advantages include no dependence on antibodies or fluorescent proteins to measure target activity and the ability to look at proteins expressed at very low levels due to our signal-amplification scheme,” says Dr. Olson.

Cells as Reagents

Zhong Zhong, Ph.D., vp of drug discovery technologies at Cell and Molecular Technologies (, will be discussing cells as reagents for high-throughput screening and assay development, focusing on uncoupling cell-production process from the cell-assay screening process.

“Our company provides tools and cells as reagents to support cell-based assays. Maintaining cell production in-house can be resource-intensive and result in a lot of waste if cells are not used because robots are down. Uncoupling cell production from cell-assay screening allows one to focus on executing good assays rather than dealing with cell variabilities. We offer a variety of division-arrested and transiently transfected cell banks that are assay-ready and supplied to our customers in a cryopreserved format. The cryopreserved cells can be thawed and used within four hours for a cell assay. Use of assay-ready cells, validated in reporter, GPCR, ion-channel, and imaging assays, can improve the consistency of assays and is price-competitive to in-house cell production,” says Dr. Zhong.

An advantage of division-arrested cells is that these cells are “frozen” in time and space without affecting signaling activity. These cells can thus be used in assays involving a large number of plates without the worries of cell-number variances from plate 1 to plate 100.

GPCR Targets

Ralf Heilker, Ph.D., team leader for HTS in lead discovery at Boehringer Ingelheim Pharma (, will be discussing the impact of high-content screening on GPCR-target drug discovery.

“We implemented high-content screening to support lead generation and selection. One of the focal points for our high-content screening assays is GPCR-targets. Some of our assays include GPCR desensitization, GPCR-induced cell signaling, and parallel detection of putative compound liabilities such as cytotoxicity. Receptor-desensitization assays help predict compound-tolerance effects for potential drugs, signaling assays enable us to identify receptor pathways that are relevant in disease conditions, and cytotoxicity assays help us assess adverse effects of candidate compounds,” explains Dr. Heilker.

These high-content screening assays are performed using a variety of fluorescently labeled primary cells or cell lines that endogenously or recombinantly express targets of interest. The technology for these assays is based on automated fluorescence-based microscopic imaging coupled to automated image-analysis software. Multiplexing based on different-colored fluorophores generates information about primary and secondary effects of molecular-targeted agents in a cellular environment.

Some of the benefits of doing high-content screening are novel assay formats and the ability to multiplex and record protein trafficking like arrestin redistribution, actin filamentation, and nuclear translocation. Example studies demonstrating tetramethyl rhodamine-endothelin 1 internalization, ETAR-red fluorescent protein internalization, VSVG-b2AR internalization, and chemokine receptor signaling will be presented.

Natural Screening Approach

Analyticon Discovery ( is adopting a nature-inspired approach to aid drug discovery screening. “Biology-oriented synthesis is a synthesis strategy based on natural building blocks to yield more meaningful biological molecules as opposed to diversity-oriented synthesis. We believe that natural products have been assembled for a biological purpose and that we can find in this purpose the molecular basis that is the key to successful drug discovery,” says Matthias Hess, Ph.D., global head of marketing, sales, and business development at Analyticon Discovery.

The company provides a natural-product drug discovery product suite ranging from supply of molecules to delivery of all necessary medicinal chemistry to develop hits until entering the clinical phases. The company markets MEGAbolite® natural product small molecule libraries and NatDiverse™ synthetic small molecule libraries. MEGAbolite is made of pure natural products isolated from biosources such as plants and microorganisms, resulting in compounds with well-analyzed chemical entities. NatDiverse provides access to new chemical spaces that are not always available with pure synthetic approaches, according to Dr. Hess. The NatDiverse line currently comprises 12,000 compounds based on over 45 different motifs (templates).

Two case studies that demonstrate the potential of BIOS-based libraries for identifying lipooxygenase and phosphatases inhibitors will be highlighted at SBS.

NMR-based Fragment Screening

Combinature Biopharm ( provides a fragment-based screening strategy service that is supported by NMR technology for drug discovery. Markus Schade, Ph.D., vp of NMR drug discovery, says, “Our fragment-based approach by NMR screens low molecular weight compounds (designated fragments) for drug activity. This typically results in lead candidates that possess superior ADMET properties and ones that can access novel chemical spaces for competitive targets.

“In contrast to crystallography-based fragment screening, the NMR technology is high-throughput, with the ability to screen for 5,000–20,000 compounds in a short time. While NMR-based screening is broadly applicable, it is particularly useful for screening difficult targets, such as protein-protein interactions,” explains Dr. Schade.

Dr. Schade’s talk at SBS will highlight a successful case study on a challenging protein-protein interaction target that was not achievable by standard screening methodologies. “The study validated the NMR screening technology and resulted in novel fragment-like lead compounds (such as 5-aryl-2-thioxo-4-thiazolidinones and related chemical entities) against postsynaptic density/discs large/zona occludens-1 (PDZ) domains. These PDZ domain inhibitors have the potential to translate into lead candidates for several human diseases,” says Dr. Schade.

Targeting Malaria

In April, Genzyme ( reported that it established a program to participate in efforts to discover and advance novel treatments for neglected diseases affecting the developing world. The company’s new Humanitarian Assistance for Neglected Diseases initiative (HAND) will serve as a vehicle to identify, evaluate, and manage scientific projects and partnerships focused on diseases that collectively affect hundreds of millions of people. These could include malaria, tuberculosis, leishmaniasis, Chagas disease, sleeping sickness, and other diseases.

Under the program, the company earlier this year began collaborating with the Broad Institute of MIT and Harvard University to discover and advance new therapeutic candidates for malaria. Approximately 300–500 million cases of malaria occur each year, killing more than one million people annually, with young children and pregnant women being the most vulnerable. The majority of malaria cases occur in sub-Saharan Africa, but the disease also remains a significant health problem in Asia and South America.

According to Roger Wiegand, Ph.D., director of infectious diseases at the Broad Institute, almost all of the drugs currently in clinical trials for malaria were discovered 30 or more years ago. Artemisinin, the most promising new drug, was first isolated from the Chinese medicinal plant in 1972, he notes.

“Until recently there has been relatively little application of the newest technologies to antimalarial drug discovery,” says Dr. Wiegand, adding that the Broad Institute has advanced drug discovery capabilities that are being employed in this project. These include high-throughput screening, chemical genomics, sequencing and genetic mapping, and genome annotation.

“With respect to high-throughput screening, we use an assay in which we monitor the growth of parasites in infected red blood cells,” explains Ted Sybertz, Ph.D., senior vp of scientific affairs at Genzyme.

“Through work at the Broad, we have access to several different strains of parasite with different drug sensitivity profiles. The Broad has identified the gene sequence on some of these. By looking at patterns of drug sensitivity across different strains and attempting to match drug sensitivity with known genetic differences in the strains, we hope to identify new targets of drug action. In addition, we can utilize techniques of affinity labeling and proteomics to define targets of unknown drugs.”

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