Pushing Frontiers in High Throughput Screening (HTS)

Drug discovery efforts by the pharmaceutical industry today employ cell based, High Throughput Screening (HTS) technologies to identify small molecule drugs that interact with a range of molecular targets including G Protein Coupled Receptors (GPCRs), protein kinases and other enzymes. This process is iterative, involving the screening of chemically diverse small molecule libraries and then optimizing the "hits" for specificity, toxicity and other druggable characteristics, prior to advancing compounds to in vivo and eventually clinical testing.

Generally, cell based assays employ either immortalized cell lines in which the molecular target has been recombinantly expressed. These cells have the advantage of possessing essentially a null background to screen against a molecular target. More importantly, they provide an unlimited homogeneous source of cells for multiple screening campaigns.

Technology in Progress

Although these tumors and immortalized cells have been employed for years in HTS against multiple targets, technological advances are leading the drug discovery industry to begin adapting their HTS screening programs. This is for the use of cells with more natural characteristics, similar to those found in vivo. Such cells include primary and stem cells.

The shift to focus on primary and stem cells for drug discovery is due to a variety of reasons. While HTS that use immortalized cells are efficient in detecting "hits," many of the compounds that are identified do not act in a predicted manner in vivo, due to the highly artificial nature of immortalized cells.

In many cases, the molecular targets under study are over-expressed at levels that are found to be much higher than in natural tissue. In some cases, the host immortalized cells that are used in drug screening against these targets lack these endogenous factors which can affect the efficacy of the compounds discovered from in vitro screening when tested in vivo.

The “artificial” systems that have been used over the last two decades, have contributed to the low efficiency of success in drug development. This may explain why only a small percentage of drugs that have been discovered in vitro, reach clinical development or get approved for marketing.

These potential disparities between the physiological environment of screening systems using recombinant tumor cell lines compared to natural tissue, has led to a growing interest in the use of primary mammalian cells and human stem cells. In these cells, the endogenous molecular targets are tacitly assumed to be expressed in an environment that closely resembles that which is found in a patient. Consequently, novel drugs that are characterized using these cell systems, particularly human stem cells, are presumed to act in a more predictable fashion in clinical evaluation than those that are characterized in tumor cells.

Primary cells, comprised of cells that are derived from embryonic tissue including neuronal and cardiomyocyte cultures, as well as those from adult tissue (such as pituitary cells or hepatocytes), play a major role in the preclinical drug development process. Primary cells from various organs including liver, heart, pituitary, pancreas, and brain can be cultured and even cryopreserved for later use.

Key Benefits

Primary cells have a number of advantages over the use of immortalized cells. First, the properties of important functional proteins such as cell surface receptors and their signaling pathways, have more similarities to the same cells in vivo compared to immortalized cells.

As a result, researchers are able to monitor drug effects on the primary cells because the responses are more predictive. Second, primary neurons and cardiomyocytes maintain their firing activity, indicating that the cells are more physiologically relevant than most tumor cell lines. Additionally, most primary cells in culture do not proliferate. This has the advantage of testing the prolonged effects of drugs on primary cell activity. Primary cells can be transfected with recombinant

Deoxyribonucleic Acid (DNA) and genes, using viral vectors or transfection reagents with markers for selective functions. These markers are used for studying phenotypic responses of drugs on primary cell activity. More importantly, primary cells can also be derived from transgenic mice, either expressing probes that are useful in studying molecular targets or with disease causing modifications in specific cell populations, that could be useful in drug discovery.

However, primary cells also have a number of disadvantages when compared to immortalized cells. Primary cell numbers are limited since they generally do not proliferate. This can be problematic for standard HTS assays that are used by the pharmaceutical companies that require larger cell volumes. Second, their use can be expensive due to the number of animals that are needed to generate the cultured cells.

Third, in most cases, primary cells are heterogeneous, creating difficulties in linking a drug’s effect to a response in a specific cell population. Finally, primary cells are only derived from animals – which can be useful, but may also be viewed as a problem in linking a response to potential clinical benefit in humans. Embryonic Stem (ES) cells give rise to all of the differentiated cells and tissue in the body. Much work has been performed over the years on ES cells to determine how essential they are for the development of organisms. For this reason, there is a major industry effort underway to employ these cells for drug discovery.

Developments in Research

A scientific discovery concluded that adult cells can be reprogrammed to become induced Pluripotent Stem (iPS) cells. iPS cells have many of the biochemical properties of ES cells, and when implanted in mice, generate tumors expressing different germ cell layers.

This is significant for researchers because it demonstrates that the mouse iPS cells are able to generate an entire mouse. Adult human iPS (hiPS) cells have a likely future for cell therapeutics because they retain the same immunogenicity as the host tissue and therefore reduce the possibility of rejection when implanted in a patient.

In addition to the potential for cell therapeutics, hiPS cells provide the means to generate populations of cells for tissue selective drug discovery. From a practical standpoint, this allows researchers to screen drugs for the potential toxicity against different human tissue, such as liver, brain, kidney, and heart. Screening these cells in vitro may provide a better predictor of clinical safety than the current standard preclinical methods.

Developing hiPS cells can create the so-called "disease in a test-tube" where hiPS cells from individuals with different diseases could be used for disease modeling and profiling. The disease modeling characteristics provide major advantages for the use of such cells for drug discovery as compared to primary cells. The hiPS cells can be grown in unlimited amounts making them ideal for HTS assays. As a result, a disease process can be grown in larger quantities.

By comparing drug effects against other cells in the body that are not affected by disease, one can identify possible therapeutic treatments on the desired cells. This improves the efficacy and minimizes the potential of reaching toxic levels. Third, hiPS cells can be obtained from adult humans of almost any age and can be sampled from a patient at different times and stages of their disease, such as in pre-symtpomatic, onset and as the disease progresses.

This is useful in identifying drugs that have the ability to prevent the continued progression of a particular disease. Rather than targeting drugs to affect one molecular component of a disease pathway, drugs can be tested throughout the entire disease pathway at a cellular level.

Considering Challenges

However, there are still a number of considerable hurdles in using stem cells for drug discovery. While technologies have advanced to make mouse ES cells propagate and retain their pluripotency, self-renewal of human ES cells is still difficult to achieve. Unlike in mice, human ES cells generate multiple cell types when they differentiate. While culturing conditions and growth factors can target the generation of specific cell types, a mixture of cells are still being produced.

Therefore, the utility of these cells for drug discovery may only be fully realized when approaches are developed to derive relatively pure populations of differentiated cells from the original ES cells. In the case of iPS cells, induction is primarily caused by the transfection of differentiated cells with a series of genes encoding transcription factors.

Even today, it is still not clear how such genetic transformations can alter the cells and affect the phenotype, and subsequently alter the iPS cells. However, the industry is making significant strides to overcome these challenges, and in the near future it may be possible for both ES and hiPS cells to be the principal cells that are used in drug discovery.

Today, one of the most widely adopted approaches in phenotypic assays involves cellular imaging tools, to measure changes in primary cell activity in response to drug treatments. Cellular imaging involves the use of fluorescent microscopy to detect luminescent probes either applied to the cells or expressed in the target cell. For HTS assays, cell imaging not only requires fluorescent microscopes, but also automated systems to allow for the movement of plates of cells, as well as sophisticated software for data analysis.

While primary cells can be employed for many purposes in drug discovery, they are most widely used to detect and measure the potential risks that are associated with the possibility of drug induced toxicity in humans. With the limited number of primary cells that are available for HTS and drug screening, sophisticated technologies have been developed to overcome this hurdle, allowing the screening process to be carried out with a limited amount of cells.

Looking Ahead

These optical and label free technologies can employ microfluidics to concentrate reactants to use a small quantity of cells in a single well or chip. As technology continues to evolve, researchers will be able to screen for primary cells from human and animal samples, allowing the pharmaceutical industry to easily use ES and iPS cells for drug discovery research.

The reality of using human stem cells for drug discovery is still at a nascent level of development. A number of pharmaceutical companies and academic researchers have used HTS formats to identify small molecule compounds that maintain the ability of pluripotent stem cells to self-renew, allowing them to expand and grow in unlimited quantities. Today, some efforts have been made to use stem cells to generate disease models using adult hiPS cells to develop in vitro models for neurological diseases.

A number of companies including Pfizer, Merck, Galapagos and Life Technologies have employed ES cells for Ribonucleic Acid interference (RNAi) HTS for target discovery and validation. This has also been accomplished by Scripps, the Harvard Stem Cell Institute and NIH. These types of screening efforts are important for identifying mechanisms that are involved in pluripotency and differentiation, which is critical in being able to use these cells for therapeutics and in vitro drug screening.

This could provide alternative targets for drug discovery rather than the obvious gene mutations involved in the disease. Additionally, efforts have been made to employ ES cells for toxicology assessment and these models have been adapted by European regulatory agencies in evaluating the toxicity potential of drugs for marketing approval.

ES cells have the potential to be useful as models for the discovery of drugs targeting specific diseases and mapping to the cells that are critically affected by the disease. They can be transfected with disease causing genes to simulate the disease, thereby making it a less expensive and less labor intensive process.