LEAD DISCOVERY PROCESS

Figure 2.1. A prototypical early drug discovery process and the key activities in lead discovery.

chemical leads against targets of interest. Each component of the lead discovery process is critical to the overall success and impact of a screening campaign. The disease relevance and "druggability" of the biological target and the availability of technologies to supply and test vast arrays of compounds are key factors for success.

The following sections of this chapter de- scribe the generic process of lead discovery and are followed by a more detailed analysis of some of the important functions involved in its execution.

but overlapping activities that are essential for effective, scalable lead discovery in the modern pharmaceutical industry (Fig. 2.1).

Discovering and validating a target of interest

Designing a bioassay to measure biological activity

Constructing a high-throughput screen Selecting screening decks and screening to find hits

Profiling hits and selecting candidates for optimization

3 LEAD DISCOVERY PROCESS

Of course, the drug discovery process is a In contrast to the highly defined and strictly

regulated process of drug development and manufacture, the initial discovery of drug can- didates can be described as somewhat ad

and ill defined. Each pharmaceutical com- pany, although agreeing on the ultimate goal, has developed a surprisingly wide array of technologies and approaches to achieve the same endpoint

However, in this prototypical early discov-

e r y process, we can describe several distinct

multi-faceted, complex process with numer- ous iterative sequences and feedback loops, which cannot be fully captured in a chapter such as this. Therefore, we will keep

throughput screening, the array of ancillary technologies, and processes as the central theme throughout the following section.

Throughout the chapter, we will endeavor to highlight the process by which these dis- tinct activities are brought together in an in- tegrated process.

High-Throughput Screening for Lead Discovery

3.1 Target Discovery and Validation

The drug discovery process starts with the identification, or growing evidence of, biologi- cal targets that are believed to be connected to a particular disease state or pathology. Infor- mation supporting the role of these targets in disease modulation can come from a variety of sources. Traditionally, the targets have been researched and largely discovered in academic laboratories, and to a lesser extent in the lab- oratories of pharmaceutical and biotechnology companies. Basic research into understanding the fundamental, essential processes for sig- naling within and between cells and their per- turbation in disease states has been the basic approach for establishing potential targets suitable for drug intervention. By pharmaco- logically modifying these intra- and

lar events, it is hoped that particular disease mechanisms and their ensuing pathologies can be modified. This type of approach has generated a number of significant and unique biological targets. More recently,

genomics and proteomics techniques combined with the huge output from the human genome sequencing project, are helping to identify thousands of potential tar- gets.

This is a significant change to past ap- proaches where the majority of the medicines developed by the pharmaceutical industry had been targeted at only 500 known human tar- gets The emphasis has now changed from finding new drugs that exploit the same

validated targets to finding drugs against po- tentially new and innovative biological mech- anisms connected to disease. The tough questions that need to be answered at this stage of the process are related to which tar- gets to focus on among the many thousands of potential choices.

Clearly, one of the more important steps in the process of developing a novel pipeline of drugs is the identification of novel genes and assessing their expression and role in various physiological and pathological states. The hu- man genome project has generated a wealth of data around human DNA sequences and gene mapping. There are now billions of base pair DNA sequence data and an estimated 30,000 human genes to be searched and investigated

as potential drug discovery start points. A host of genomics technologies are now available for discovery scientists to find and validate biolog- ical targets. Rapid gene sequencing, single cleotide polymorphism identification, differential display analysis, and

formatics data mining tools are just some of the now standard techniques that are avail- able to help identify and analyze novel genes. The ability to search the billions of data points available through the various human

related databases has rapidly evolved with the development of bioinformatics tools. Powerful computers and search algorithms allow poten- tial genes to be identified within stretches of DNA sequence. Their DNA coding sequences can be aligned and compared with other known DNA sequences stored in databases to allow comparison with known genes. Pro- grams such as basic local alignment search tool (BLAST) are used for this type of similar- ity searching. Other methodologies can also be used to identify genes such as using

netic analysis to place the novel gene in con- text with other genes by molecular evolution. Cloning, expression, and distribution of the gene of interest are all-important next steps in qualifying the target for further discovery work. A gene that is uniquely expressed in a particular type of cell or tissue will trigger fur- ther evaluation. For instance, brain-specific expression of a gene engages neuroscientists in the same way as specific expression of a novel gene in an immune cell would spike the attention of inflammation experts.

Of course, cell and tissue distribution does not solely validate a target, but provides useful circumstantial evidence that this target could be worth investigating in disease models. In fact, differential expression of a candidate gene in disease and non-disease systems helps to focus the interest considerably.

An essential next step in the process is at- tributing functionality to an unknown gene. The elucidation of gene function and the un- derstanding of its role in the activity of other genes can be critically important.

There are a number of critical methods for determining gene function some of that are similar to those used to initially identify genes.

3 Lead Discovery Process

3.1 Gene Function by Homology to Other Defined Genes. By aligning the sequence of an unknown gene and comparing it with sequences genes, homologies are identified that allow a tentative potential function to be as- signed. These homologies can be as general as identifying the unknown gene as a target in- volved in metabolism or the homology could be as specific as identifying a specific function such as fatty acid biosynthesis (27-28).

3.1.2 Gene Function by Gene Subtraction. When a gene function cannot be identified by direct homology methods or when you want further proof of the importance of a particular candidate gene, it is possible to determine function by deleting the gene (gene knockout) in an in model and describing the result- ing phenotype An example would be the use of gene knockout studies to establish whether protein phosphatase IB is an important target for anti-diabetic drugs Certain animal models such as worms,

and mice also have well-characterized pheno- types that will infer function. Yeast models can also be used for this type of analysis. A number of biotechnology companies offer this as a valuable service both for in and in

systems Lexicon, Sequitur).

3.1.3 Gene Function by Expression Analysis. Genes specific for a particular tissue can be expressed differently under a variety of differ- ent metabolic conditions or stresses within the cell or organism. and protein expression as different indicators of gene ac- tivity in normal and disease tissue is now one of the more important methods for gene vali- dation. This type of analysis involves exten- sive use of DNA microarrays and related chip technologies

By using this extensive array of genomics techniques, the discovery scientist can quickly generate a list of novel, tissue-specific genes showing differential expression in disease and non-disease systems. Direct homology or knockout experimentation may have also elu- cidated their function. These targets may also be members of a known "druggable" target class such as or ion channels, which suggests they can be readily integrated into a drug discovery process If the

property position on this target is also clear, then this target could rise to the top of the prioritized list for entry into the pipeline. Building a prioritized portfolio of disease targets and subsequently deploying resources is critical in the discovery process. Which dis- ease areas are the foci for the organization? In those disease areas of interest, what target choices are available for drug intervention? What is known about the relationship of this target to disease? All the answers to these questions and more determine the or- der by which targets may be worked on or not. The fact that a target is known and well vali- dated reduces the discovery and development risk but raises the chances that your competi- tors are also working on this target.

Selecting a novel target with just the earli- est indications of validation, but no real clini- cal proof, may keep you clear of the competi- tion, but raises the risk of discovery and development failure as the target and drug de- velop through the pipeline. Entering a biolog- ical target into the discovery process is an ex- tremely critical step that can affect the successful discovery of leads and the ultimate success of the drug development process. The greater the knowledge of the biological sys- tems and disease pathologies involved, the greater the chances are that the right type of targets will be selected. Overall, target disease portfolio decision-making processes are not within the scope of this chapter, but clearly play a direct role in the potential suc- cess or failure of any lead discovery program.

By the end of this initial phase of the

process, a list of targets will have been selected and passed into the lead discovery phase. At this next stage, the ability to measure the ac- tivity and function of the biological target and find compounds that modulate the activity is critical.

3.2 Bioassay Design and Screen Construction After the identification of a biological target of interest, the next challenge begins with the conversion of the target into a bioassay that can give a readout of biological activity. The range of potential targets is large, from en- zymes and receptors to cellular systems that represent an entire biochemical pathway or a disease process. Consequently, the range of

High-Throughput Screening for Lead Discovery

say design techniques and types of assay avail- able have to be correspondingly comprehen- sive.

Once an assay has been developed that measures the biological activity of the target, by some direct or indirect means, then com- pounds can be tested in the bioassay to see if inhibit, enhance, or do nothing to this activity.

This approach is the basis of all compound testing for HTS and structure-activity rela- tionship (SAR) studies in drug discovery pro- grams.

A variety of techniques can be applied to bioassay design depending on the nature of the biological activity being measured. Mea- surement of product accumulation, measure- ment of enzyme substrate use, measurement of receptor mediated signaling, receptor an- tagonism and agonism, cell death, and cell pro- liferation are just a few of the activities that could be assessed in a bioassay. Each of these bioactivities can be directly or indirectly mea- sured using reporter signals and detection sys- tems such as radioisotopes and scintillation counters, fluorescence and fluorometers, lu- minescence and luminometers, or voltage changes and patch clamp.

Regardless of the biological activity being measured and the detection system being used, the assay will be optimized against an array of multifactorial parameters to provide an in vitro milieu for the bioassay, which most closely resembles the physiological "normal" for the functional activity. In this assay opti- mization process, factors such as pH,

and a whole host of other significant parame- ters are modified to provide the most appropri- ate bioassay.

If the bioassay is the basis of a high- throughput screen, then other crucial factors are assessed before screen construction. The stability and scalability of the assay's core re- agents are just two of the more important fac- tors. An assay format designed to use scarce and time-dependent labile reagents will not make the best design for a HTS environment. Here the reagents have to be made at bulk levels to allow testing of thousands of com- pounds in conditions where they may have to be stable for hours on an automated screening system.

The process from bioassay design to HTS construction can significantly change the de- sign and final format of an assay. It is impor- tant that assay to screen reproducibility is monitored and maintained to ensure consis- tent results from the different laboratories in- volved in the discovery process.

3.2.1 Assay Design. Ideally, in the plan- ning phase, a team of medicinal chemists, therapeutic area biologists, and HTS design- ers define the important objectives for a high- throughput screening campaign. These could include criteria that would initiate a medicinal chemistry program, such as the desired data from the screen, a definition on how to assess the value of the screening hits, etc. The HTS assay designer's responsibility is to select the correct assay methodology to meet the screen- ing objectives. Usually there are numerous po- tential assay options, and it is often necessary to create a decision matrix to compare the po- tential methods. A typical matrix of questions is outlined below.

1. What compound characteristics are re- quired, antagonists or agonist?

2. Do you have the relevant expertise avail- able to build the type of assay? Modification of an existing screening protocol is the nat- ural starting point, especially if one is con- sidering screening a gene family like nases or nuclear hormone receptors. If at all possible you would like to avoid de novo assay design unless there is potential for more targets within this class and the in- vestment can have future impact.

3. Are there any restrictions on the use of the assay technology? For example, are there patent restrictions on the use of any re- agents or the actual biochemical technique itself.

4. Does the proposed HTS assay provide the desired information to progress com- pounds along the drug discovery pipeline? Will the hits need to be assessed in addi- tional assays before progression into me- dicinal chemistry? For example, if the screen needs to find receptor agonists, there are potential cellular functional as- says that directly measure in agonist