June 15, 2005 (Vol. 25, No. 12)

Finding Potential Therapeutics More Efficiently

The various methods used in target validation were the focus of discussion earlier this month at two separate meetings held in Boston and a third meeting in Singapore. The Boston meetings included the “IBC USA Early Efficacy Assessment Meeting” and the “Cambridge Healthtech Protein Kinase Targets Meeting;” the meeting in Singapore was “IBC’s Drug Discovery and Development in Asia-Pacific.”

Target validation involves proving that DNA, RNA, or a protein molecule is directly involved in a disease process and can be a suitable target for development of a new therapeutic drug. Pharmaceutical industry experts and analysts agree that drug target validation is the most critical challenge facing large pharmaceutical companies.

There appears to be a paradigm shift in the pharmaceutical industry with the emphasis on validation of targets in animal disease models before beginning lead screening. Since genomics has delivered a huge number of potential drug targets, it has become imperative to identify the targets that will most likely be suitable for therapeutic intervention. Thus, target validation technologies are in high demand.

Target validation is the functional study of the target that has been identified in genomic or proteomic investigations. The inhibition of the protein’s function in cellular models followed by the investigation of the resulting phenotype is the focus of target validation, said Michael Blind, Ph.D., CSO, Nascacell (Munich, Germany).

A target is validated if its inhibition reverts a disease phenotype in a model system that is as close to the disease as possible.

Functional Validation

The main strategies involved in target validation are gene knock-out studies, knock-down by antisense RNA or RNA interference, and direct inhibition of the protein by small molecules, peptides, antibodies, aptamers, or any other class of inhibitors.

There are nuances to the target validation issue that include functional validation as well clinical context validation, said Eric Kaldjian, senior scientific director, GeneLogic (Gaithersburg, MD), who presented his research at the “IBC USA Early Efficacy Assessment” meeting. Functional validation is required to show that the inhibition of the target protein results in a desirable biological end point in terms of disease progression.

For example, introduction of a drug into cell culture might cause apoptosis. Some of this functional validation information may be already available in the literature. Clinical context target validation is required to show the significance of the target in the clinical population. If the target is present only in a subset of patients, then it is important to define that subset clinically for a detectable activity signal in patient studies.

Clinical context target validation therefore depends on both the presence of the target protein as well the intensity of expression, said Dr. Kaldjian. Also, a target protein might be valid in one context but not in another depending upon whether redundant elements or alternative pathways are present. Therefore, an understanding of the pathways the target protein is involved in is necessary.

GeneLogic makes use of its large database of tumor and normal gene expression to generate a short list of targets that are preferentially expressed in disease situations. The targets in this list can then be screened for functional target validation. The use of the database provides the investigator with a differential expression pattern of the target as well as its expression levels in different tissue and disease types.

The database also allows the researcher to investigate the expression levels of other proteins known to be involved in certain pathways.

The database as the starting point provides a list of targets that can be studied further with a higher degree of confidence. By being aware of target presence, clinical development strategies can also be poised to monitor toxicities that otherwise might not be expected, said Dr. Kaldjian.

RNAi Technology

Intradigm (Rockville, MD) makes use of RNA interference (RNAi) technology in which small interfering RNA (siRNA) oligos are used as gene inhibitors via the RNA-induced silencing complex (RISC) to degrade homologous mRNA with high specificity and potency. Intradigm focuses on an angiogenesis pathway shown to be important in cancer, inflammation, autoimmune, and other diseases.

The ability to perform in vivo target validation with efficacious, clinically viable siRNA delivery provides high value information to understand the role of a particular gene or protein in the disease process, multiple genes of the same pathway, as well as the role of the pathway in the disease.

This information is not only critical to the drug discovery process but also important for potential therapeutic siRNA development.

Patrick Lu, Ph.D., executive vp, Intradigm, who presented the company’s technology approach at the “IBC Drug Discovery and Development Asia Pacific” meeting, pointed out that the use of siRNA in vivo to down-regulate the expression of a specific gene requires knowledge of target sequence accessibility, target tissue deliverability, and siRNA stability in both extracellular and intracellular environments.

It has been recognized that in vivo drug target validation performed in animal disease models is superior to in vitro validation performed in cell culture assays. Earlier studies that used cell culture assays to validate targets had a low success rate when the studies were taken to the animal models.

Dr. Lu emphasized that they have the capability to provide both systemic and local delivery of siRNA depending upon the disease of interest. The dual targeted ligand-directed nanoparticle allows them to deliver siRNA systemically with tissue-specific and gene-specific targeting. This process results in the release RNAi in the cytoplasm of the cells by taking advantage of receptor-mediated endocytosis.

More potent tumor inhibition can be achieved by using up to three different siRNA in the same transfection cocktail. This is unique to RNAi as it is difficult to use mixtures of antibodies to the target protein in vivo, said Dr. Lu. Cocktails of siRNA will provide better understanding of the entire pathway involved in disease progression, he added.

Immusol (San Diego) recently launched a proprietary technology that allows fast and efficient in vivo target validation for efficacy and safety in multiple disease models using siRNA vectors.

According to Henry Li, Ph.D., director of oncology, Immusol, who presented his company’s research at the “IBC USA Early Efficacy Assessment” meeting, this technology represents a major breakthrough in disease target validation in vivo, providing critical information prior to the lengthy and expensive high throughput compound screening and lead development, and can potentially save millions of dollars and years in drug discovery cost.

Immusol has inducible RNAi vectors that can be stably introduced into cultured tumor cells or cell lines. Induction results in the expression of RNAi then can be used for target validation. The inducible vector can also be studied in a mouse xenograft tumor model.

In this case, human tumor cells that have been transduced with the inducible vector are grafted on nude mice, creating stably silenced cells in vitro and establishment of tumor in vivo in the absence of an inducer. Upon induction, tumor response to gene inactivation in vivo can be measured, thus allowing target validation and also rapid advance through the discovery process.

Simply by varying the timing of induction, the expression of the target can be modulated at any stage of tumor progression. This model can also be used for the study of two targets at the same time. Dr. Li added that the Immusol vector does not necessitate single cell cloning, which makes it faster and more efficient to obtain cells containing the vector.

There seems to be agreement among researchers that target validation in vivo with siRNA with efficacious, clinically viable siRNA provides a lot of information that helps understand the role of a particular gene in a disease, multiple genes in the pathway, as well as the role of the pathway in the disease.

Dr. Li announced that Immusol is in the process of generating transgenic mice with RNAi.


Nascacell works with aptamers, which are synthetic nucleic acid ligands, for target validation as well as screening. Aptamers mimic the effect of a small molecule drug in terms of binding the active site and inactivating a distinct functional epitope on a protein, leaving the rest of the molecule untouched.

Additional advantages of aptamers are that they can be used to inactivate stable protein with a physiological turnover rate and they can distinguish between different post-translational modifications or conformations.

They therefore provide a valuable, complementary approach to existing genetic knockout and siRNA technologies. In particular, aptamers can recognize different protein domains, subdomains, catalytic centers, or post-translational modifications within the same protein, which allows direct analysis of functional sub-domains at the molecular level.

At the “Protein Kinase Targets” meeting, Dr. Blind highlighted the development of specific aptamers against several kinases, and presented the specificity of inhibiting the target kinase without affecting a panel of control kinases, and different modes of inhibition, e.g., via the ATP binding pocket or regulatory domains.

He also talked about the advantage of the same aptamers that were used in target validation being used in high throughput screening assays to identify small molecule leads thus streamlining the connection between target validation and drug development.

Aptamers can be isolated rapidly against almost any protein target by automated in vitro selection protocols in only a few weeks, exhibit high affinity and specificity for their target protein, have a high inhibitory potential, are intrinsically not toxic or immunogenic, and can be easily produced and modified by chemical synthesis.

The combination of the above features makes aptamers one of the most efficient technologies among the inhibitor-based ap-proaches, said Dr. Blind.

The enormous expense of bringing a drug to market necessitates that the selection and validation of drug targets be performed with the utmost rigor and that is the direction these technologies are leading the pharmaceutical companies. In fact the high cost of target identification and validation is giving rise to new interest in drug repositioning.

GeneLogic has a dedicated group that is using target validation technologies to reposition candidate drugs that were previously shown to be nontoxic but failed the efficacy test. They use a combination of technologies to identify other diseases or tissues in which the failed drug can be useful.

And then they have to perform target validation analyses to confirm the validity of the new target. Pharmaceutical companies are hoping that these target validation technologies will provide them with targets that have been more rigorously tested and also that these technologies can be ported to the high throughput screening step.

Previous articleIntegrated Workflow for Development Testing
Next articleAmerican Thoracic Society – Gene Express