Figure - Three main phases of the creation of a new drug: discovery, development and commercialization.

DRUG DISCOVERY PROCESS

Process Overview

Target Identification and Validation The process of drug discovery starts long before the screening of compound libraries for molecules that bind to and affect the action of a protein associated with disease. Drug discovery starts with the identification of the disease associated protein or molecular target. In the past, identification of molecular targets was a painstakingly slow process, generally carried out by investigators who could spend entire careers studying one pathway or one protein that is involved in some aspect of human development, metabolism, or disease. In the course of defining the role of a given protein in the cellular life cycle, the direct relevance of this protein to a disease state would often become clear. By removing or altering the target’s function from the cell, the target would then be validated as a suitable target for drug screening. In recent times, methods to identify panels of targets that are associated with particular pathways or disease states have emerged, and the acceleration of target identification has led to a bottleneck in target validation. Methods described in this chapter are being deployed to reduce the bottleneck and increase the availability of new, useful, disease-associated molecular targets for development of new classes of effective therapeutic compounds.

Screening for Hits Once a disease-associated molecular target has been identified and validated in disease models, screening for a selective and potent inhibitor (or activator) of the target is the next step. Libraries of compounds that are either synthetic chemicals, peptides, natural or engineered proteins, or antibodies are exposed to the target in a manner that will detect and isolate those members of the library that interact with and, preferably, have an effect on the target. The compounds selected are called hits. Initially, screening can be performed by searching for compounds that bind to the target, but binding is not sufficient for therapeutic activity. More recent screening procedures include an activity-based readout as part of the initial screening assay. For example, if the goal is to inhibit a protein that is involved in activating the expression of a particular gene or set of genes, the assay can include a readout to determine if the expression of the gene is reduced by the compound. Such assays can be cell-based, but more often they are enzymatic assays that can be performed in a high-throughput manner.

Lead Optimization Once the initial screening is performed, a large collection of hits is obtained. These hits are then evaluated and the best ones identified in a process known as lead optimization. During this stage, scientists determine which, if any, of the hits selected has the appropriate properties to justify continued expenditure of resources on the development of the compound as a clinical candidate. These properties include ease of synthesis; adherence to the Lipinsky rules, which describe chemical characteristics that are predictive of biodistribution and in vivo activity; specificity for the target; and efficacy in the disease. The hits discovered during the screening process are therefore characterized by a variety of biochemical, biophysical, and biological methods to narrow the set down to a handful of compounds. Assays that measure each compound’s activity directly are a useful first step at determining potency and can enable the most effective compounds to be identified. The resulting smaller set of hits, or leads, is then tested in more rigorous models of disease, including either cell-based or animal models of the

disease.

Pharmacology and Toxicology Once one or more potential lead compounds emerge from the lead optimization efforts, the compound must be evaluated in multiple disease models, and if possible, compared to existing therapies for the same disease. Further, properties of the compound are studied when delivered into a living organism. The classic set of properties that must be appropriate include Absorption (through the intended route of administration), distribution (what organs does it end up in?), metabolism (what are the by-products of cellular metabolism of the compound, and what potential effects do these compounds have on the organism and disease?), and excretion (how is it processed and eliminated from the body?). Collectively known as ADME, this set of properties is essential to evaluate in multiple species, in addition to measuring efficacy in sophisticated disease models. Design of ADME and efficacy experiments is a crucial activity in drug discovery and development. All drugs that are intended for human clinical applications must be tested for toxicity using the same material that will be used in humans, manufactured by the same process. Toxicology testing is a highly regulated process that is governed by regulatory authorities such as the U.S. Food and Drug Administration (FDA). Developing a manufacturing process for a drug (which is outside the scope of this chapter) and executing the necessary toxicology program are very expensive and are therefore usually performed on only one lead compound identified during lead optimization and subsequent efficacy testing. Only if the compound fails at this stage will another compound from the same screening (if possible) be advanced into preclinical development. Different companies, and in fact countries, have different standards for which products move into human clinical testing, but in all cases the product’s safety must be adequately demonstrated before regulatory approval can be obtained for advancing the product into humans.

Clinical Trials Human clinical testing follows an established process in most regulated countries worldwide. If possible, the initial administration of a compound to a human subject is performed on people who do not have the disease indication for which the product is designed. The compound is administered at a dose that is significantly lower than the intended therapeutic dose, and the trial subjects are monitored for any signs of toxicity. Most often, these adverse events or side effects consist of headache, fever, nausea, or other discomforts. If the trial subjects receiving the initial low dose have no or minimal side effects, the next cohort of subjects is treated with a higher dose. Using this dose escalation method, the aim is to determine the maximum tolerated dose (MTD) or to determine the safety of a dose level that is known to generate therapeutic benefit. Depending on the disease indication, the tolerance for adverse reactions is different. For example, almost all chemotherapeutic agents for cancer are significantly toxic, but many cancer patients are willing to suffer these effects in order to have a chance to overcome their disease and continue to live without cancer. Due to their known toxicity, oncology drugs are therefore rarely tested in normal volunteers, but in fact are often tested initially in patients who have failed all other chemotherapy or other treatment regimens and have no other option for survival. Chronic, nonfatal diseases, however, are not usually treated with agents that have severe side effects because the benefit of the drug does not outweigh the risks and discomfort of the side effects. Once the MTD has been determined, drugs usually move into phase II testing, in which the drug’s efficacy is determined in a small select group of patients who have the disease for which the drug was developed. These trials can also include a range of doses and dosing regimens (modes of delivery, frequency, etc.), in order to measure the clinical efficacy of different doses. Designing phase II trials and choosing endpoints or objectives for the therapy that will accurately reflect the compound’s efficacy is a very demanding process, and many compounds can fail at this stage. Only 60% of drugs that enter phase II successfully complete this stage and move on to the pivotal phase III trial.

Phase III is considered pivotal because the drug is administered to a much larger group of patients and is evaluated for efficacy with greater rigor than in phase II. These trials can cost millions of dollars and therefore are attempted only when the phase II results are highly convincing. There are regulatory hurdles that must be crossed to enter phase III. The drug product that is used in phase III must be manufactured exactly as the first commercial product will be made. The facility or reactor that is used, the process, the analytical methods, and the formulation and vialing are all identical to the intended final product that will be made and launched upon product approval. Following the successful completion of phase III clinical testing, the owner of the compound then fi les an application with regulatory authorities in various countries, such as the U.S. FDA, for permission to sell the compound for the intended therapeutic indication. The FDA or other agency must then grant a license to market the compound, and the new drug is launched onto the market.

Motivation for Improvement

The drug discovery and development process described above follows a logical and linear path from target identification through completion of clinical trial and submission of an application to the regulatory authorities seeking approval to market a drug. However, this process is lengthy, cumbersome, and most important, generates compounds that are more likely than not to fail during clinical testing. The major hurdles in drug discovery and development today are:

• Time. From the initial discovery and validation of a molecular target to final marketing of an effective drug can take 10 to 15 years.

• Efficiency. a significant portion of the high cost of drug development is due to the very high attrition rate of compounds that enter the clinic; only 12% succeed.

• Expense. Drug development can cost up to $800 million per successful compound.

Therefore, improved clinical outcomes of therapeutic candidates at all stages of development would contribute significantly to reducing development costs and improving the number of candidates that succeed in obtaining approval for commercialization. New methods that enable better target identification and validation, a deeper understanding of total systems biology and the implications for any specific drug or target in development, and methods to understand the nature of molecular targets to enable more effective screening and lead identification would be extremely valuable in meeting the needs for cost-effective development of new and useful therapeutics for human disease. The emerging technologies described in this chapter aim to address the many aspects of drug development and to enable more effective understanding of targets and compounds through high-throughput high-content analysis.

Target Identification

The initial stage of the drug discovery process is the identification of a molecular target, almost always a protein, which can be shown to be associated with disease. To accelerate this, more proteins need to be identified and characterized. Although the set of human genes is by now quite well understood, this is not as true for all the proteins in the human proteome. Because of splice variants and posttranslational modifications, the number of proteins far exceeds the number of genes. As of today, a great many human proteins have yet to be fully identified and characterized. The new high-throughput technologies of proteomics are helping to close this knowledge gap in a number of ways:

1. Identify large numbers of novel proteins in the search for new drug targets. High throughput separation, purification, and identification methods are being used for this. 2DPAGE is often used for separation, even though it has some drawbacks. HPLC is a common separation and purification technique, and is particularly adapted for high-throughput use when coupled directly to tandem MS for identification. These procedures produce peptide sequences that must then be compared to possible homologs in other organisms (or paralogs in humans) using informatics. Edman sequencing may be needed for sequence verification.

2. Perform initial functional characterization of novel proteins. Initially, assign them to functional areas that might implicate some of them in disease pathology:

a. Metabolic pathways

b. Transcriptional regulatory networks

c. Cell cycle regulatory networks

d. Signal transduction pathways

Computational sequence homology analysis can help putatively assign a novel protein to a known pathway or network that its homolog participates in. Sequence analysis also helps identify known motifs and functional domains that the new protein shares with known proteins. This information can provisionally assign membership in a protein family to the new protein.

3. Screen a compound with known therapeutic effect against a large number of human proteins to identify the exact target. It is not uncommon to have a compound with known but perhaps limited or suboptimal therapeutic effect against a disease, yet not know the molecular target for this compound. A proteome-scale search can pinpoint a possible target, once binding has been identified. The case study of the development of LAF389, presented at the end of this section, describes a successful example of this in detail.

4. Perform high-throughput differential protein expression profiling, comparing diseased with normal tissue samples, to identify the biomarker proteins that are possible contributors to the disease by their over- or underexpression. This is one of the most promising areas of application of proteomics in target identification. Several proteomics technologies can be used for expression profiling. Two-dimensional PAGE has been used, although comparing two different gels (one for normal, one for disease state) can be problematic, due to the replicability issue discussed earlier. A variant of this called two-dimensional differential in-gel electrophoresis (2D-DIGE) overcomes this drawback. 2D-DIGE has been used successfully to identify biomarkers that could be potential drug targets. As noted previously, protein chips have perhaps the greatest potential for high-throughput simultaneous differential protein expression profiling. Several successful attempts to use protein arrays for target identification have been reported. However, the technology is still relatively immature and is therefore further out on the horizon as far as general industry acceptance for target identification.

Example A recent study showed how an integrated approach involving proteomics, bioinformatics, and molecular imaging was used to identify and characterize disease-tissue specific signature proteins displayed by endothelial cells in the organism. Working with blood vessels in normal lungs and in lung tumors in rats, the researchers used several high-throughput affinity-based separation procedures, followed by MS and database analysis to identify and map the proteins displayed by endothelial cells that line the blood vessels. With this approach they identified proteins that are displayed only on solid tumor blood vessel walls. They then demonstrated that radioisotope-labeled antibodies can recognize these tumor-specific proteins, allowing them to be imaged. The radioisotope labeling itself also resulted in significant remission of solid lung tumors, demonstrating tissue-targeted therapeutic potential. Although individual endothelial proteins had been identified and targeted in previous studies, this is the first time a proteomics approach had been used to move toward a complete tissue-specific mapping of the proteins displayed on the blood-exposed surfaces of blood vessels. This demonstrates a new approach for identifying potential novel targets for therapy.

Target Validation

At this point in the drug discovery process, one or more potentially disease-related protein targets have been identified. For now, let us assume one. The next step is to validate the target. Primarily, this means that the target’s relevance to disease pathology must be determined unambiguously. This involves more detailed functional characterization, more evidence for the pathway or network assignment, and modulating the protein’s activity to determine its relationship to the disease phenotype. Some determination of tractability may be done in this stage as well. Proteomics can assist in several ways:

1. Determine in what tissues and cell components it appears and in which developmental stages. High-throughput techniques such as protein chips and 2D-DIGE can be used for proteome-scale expression studies comparing different tissue types and developmental stages. These add evidence that the putative target is found in a disease-related tissue and at the expected developmental stage. Interaction studies can help determine subcellular localization by showing binding to proteins or phospholipids in the cell that have known location. Further, sequence analysis can identify known location-specifying signal peptides on the protein. Finally, posttranslational modification analysis can identify certain PTMs that determine the destination of the protein.

2. Understand when and for how long the target gets expressed, and when degraded.

High-throughput protein expression studies can be done in multiple runs over time, and then compared. In this case, expression patterns are assayed and then compared across multiple time points rather than normal vs. disease states. For additional evidence it would be very informative to do a time-based expression study first of normal tissue, then another one of diseased tissue, and compare the behavior over time of normal vs. disease tissues. This would provide a multidimensional target validation.

3. Verify the target protein’s specific role within the protein family and the pathway

or network identified in the previous stage. Initial putative functional assignments for the new protein were made in the target identification stage. In target validation, high throughput protein–protein interaction studies can be used to strengthen the evidence for the protein family, network, or pathway involvement. Protein–phospholipid interaction assays can determine whether the new protein is membrane associated. Technologies such as protein chips are beginning to be used for these interaction studies and have great potential in this area. Other techniques such as Y2H, phage display, and tandem affinity purification have also been used with success in this area.

Posttranslational modifications to the new protein can also be identified by methods discussed earlier. Knowledge of the binding partners and posttranslational modifications of a new protein goes a long way to help characterize it functionally, to solidify its assignment to a pathway, and so on. Since the association of a disease and a particular function or pathway is often already known, a solid assignment of the new protein to such a function or pathway implicates the protein in the disease. This adds evidence toward validating the target.

4. Determine the effect of inhibiting the putative target.

a. Does target inhibition disrupt a disease-related pathway?

b. Does target inhibition slow or stop uncontrolled growth? Have other effects?

c. Does this effect validate the target as unambiguously related to the disease pathology?

Several methods can be used to answer these questions. Gene knockout studies in mice have been an effective tool for some time, but knockouts cannot be done with all putative target proteins. It is in any case a slow, laborious process to breed the knockout strain correctly and reliably and may not result in viable mice to study.

One recent alternative method has garnered a great deal of attention and is currently achieving rapid adoption in the industry due to its relative ease, rapidity, effectiveness, and lower cost. It is called RNA interference (RNAi). This technique has roots in both genomics and proteomics. Small interfering RNAs (siRNA) are synthetic 19- to 23-nucleotide RNA strands that fold in hairpin configuration. They elicit strong and specific suppression of gene expression; this is called gene knockdown or gene silencing. RNAi works by triggering the degradation of the mRNA transcript for the target’s gene before the protein can be formed. RNAi is done in vitro in the lab to verify disease relevance of a putative target in several possible ways:

• If the target is believed to cause pathology by overexpression, investigators can knock down the gene for the target and observe whether the disease pathology is reduced.

• If underexpression is assumed to contribute to disease, the gene can be knocked down in healthy tissue samples to see whether this elicits the same disease phenotype.

• The pathway in which the target is believed to participate can itself be validated as disease-related by performing knockdowns of some or all of the genes in the pathway.

Then the putative target gene can be knocked down, and the effect of this on the pathway’s function can be observed. This both validates disease relevance and verifies the functional assignment of the target to the pathway. Note that RNAi is also being investigated for its therapeutic potential. Although there are major ADME issues to be addressed, RNAi molecules, with their high specificity and efficacy in gene suppression, may themselves hold great promise as drug candidates.

Screening for Hits

Now we have one or more possible targets. For each target we need to screen many compounds to look for drug candidates that show activity against the target (i.e., hits). This can be like searching for a needle in a haystack, so any techniques that can help accelerate and focus this search are of great value. The following techniques are fairly new but are in current use.

1. Develop sets of compounds to screen for activity against the target. The first order of business is to construct focused sets of compounds to screen against the target. Structural proteomics and combinatorial chemistry can play major roles at this stage. As mentioned before, an x-ray crystallographic structure for the protein provides the golden standard for three-dimensional structure. Given such structural information, it is possible to develop much more focused compound sets for screening libraries than would otherwise be possible. Combinatorial chemistry can then be used to design the libraries of such compounds. These are numerous small modifications of a basic small molecule or side group that is likely to fit the known binding pocket of the target protein based on the structural information. Computational chemistry is also adding to this effort. It is being used to generate large virtual compound libraries as a part of structure-based drug design. Like the combinatorial compound libraries, these are sets of compounds that are likely to fit well with the target’s binding site. However, these compounds are all in silico, in the computer only.

2. Screen for compounds that affect the target. (In most cases, inhibition is aimed for. Most drug targets are enzymes, and most of these are overactive, either by being overabundant or by being stuck in an active state. Therefore, most drug candidates attempt to inhibit or shut off the enzyme by binding to its active site.)

High-throughput laboratory screening can now proceed. This technique uses 96- or 384-well plates to combine the target protein with each of the screening compounds, one per well. In this way, the compounds are individually tested for activity against the target. Once activity is detected, even if it is only moderate, that compound is designated as a hit. Chemists can later attempt to increase its activity by modifying the compound and retesting.

Virtual screening is another, newer technique that is showing some promise. Using the virtual compound libraries, virtual screening uses elaborate computational chemistry techniques to determine in silico the fi t between each virtual compound model and the binding site of the protein model derived from the x-ray crystallographic structure. This involves computing the chemical affinity, the steric fi t, and the energetics of the compound in the binding pocket. When certain thresholds are reached, a hit is declared, and reserved for further optimization.

3. Structure-based drug design. As mentioned above, virtual screening entails the computational identification of a drug candidate from the ground up. This is called structure based or rational drug design. Structural proteomics provides the core information needed to achieve this. There are two main approaches used in structure-based drug design:

a. Building up an optimized ligand from a known inhibitor molecule. If structural information is available for a known inhibitor ligand, this can be modeled and used as a starting point. Placed computationally into the target protein’s binding site, it can be manipulated on the computer by chemical changes or the addition, moving, or subtraction of chemical groups or even atoms until its fi t is considered strong. Some docking programs first decompose the known ligand into fragments before the user docks them appropriately in the binding pocket and begins optimizing.

b. De novo ligand design and/or docking into the binding pocket model. If no known inhibitor exists, or there is no structural information on one, a ligand can be built up from scratch. A base fragment is initially placed in the binding site, then additional fragments or atoms are added according to sets of rules derived from many known

protein–ligand structures.

Biochemical and cell-based assays

There is a wide range of assays formats that can be deployed in the drug discovery arena, although they broadly fall into two categories: bio­chemical and cell-based.

Biochemical assays (Figure) involve the use of cell-free in-vitro systems to model the biochemistry of a subset of cellular processes. The assay systems vary from simple interactions, such as enzyme/substrate reactions, receptor binding or protein–protein interactions, to more complex models such as in-vitro transcription systems. In contrast to cell-based assays, biochemical assays give direct infor­mation regarding the nature of the molecular interaction (e.g. kinetic data) and tend to have increased solvent tolerance compared to cellular assays, thereby permitting the use of higher compound screening concentration if required. However, biochemical assays lack the cellular context, and are insensitive to properties such as mem­brane permeability, which determine the effects of com­pounds on intact cells.

Figure - Types of biochemical assay.

Unlike biochemical assays, cell-based assays (Figure) mimic more closely the in-vivo situation and can be adapted for targets that are unsuitable for screening in biochemical assays, such as those involving signal trans­duction pathways, membrane transport, cell division, cytotoxicity or antibacterial actions. Parameters measured in cell-based assays range from growth, transcriptional activity, changes in cell metabolism or morphology, to changes in the level of an intracellular messenger such as cAMP, intracellular calcium concentration and changes in membrane potential for ion channels. Importantly, cell-based assays are able to distin­guish between receptor antagonists, agonists, inverse ago­nists and allosteric modulators which cannot be done by measuring binding affinity in a biochemical assay.

Many cell-based assays have quite complex protocols, for example removing cell culture media, washing cells, adding compounds to be tested, prolonged incubation at 37°C, and, finally, reading the cellular response. There­fore, screening with cell-based assays requires a sophisti­cated infrastructure in the screening laboratory (including cell cultivation facilities, and robotic systems equipped to maintain physiological conditions during the assay proce­dure) and the throughput is generally lower.

Cell-based assays frequently lead to higher hit rates, because of non-specific and ‘off-target’ effects of test com­pounds that affect the readout. Primary hits therefore need to be assessed by means of secondary assays such as non- or control-transfected cells in order to determine the mechanism of the effect. Although cell-based assays are generally more time-consuming than cell-free assays to set up and run in high-throughput mode, there are many situations in which they are needed. For example, assays involving G-protein coupled receptors (GPCRs), membrane transporters and ion channels generally require intact cells if the functional­ity of the test compound is to be understood, or at least membranes prepared from intact cells for determining compound binding. In other cases, the production of bio­chemical targets such as enzymes in sufficient quantities for screening may be difficult or costly compared to cell-based assays directed at the same targets. The main pros and cons of cell-based assays are summarized in Table.

Figure - Types of cell-based assay.

Radiological Ligand binding assays

Assays to determine direct interaction of the test com­pound with the target of interest through the use of radiolabelled compounds are sensitive and robust and are widely used for ligand-binding assays. The assay is based on measuring the ability of the test compound to inhibit the binding of a radiolabelled ligand to the target, and requires that the assay can distinguish between bound and free forms of the radioligand. This can be done by physical separation of bound from unbound ligand (heterogeneous format) by filtration, adsorption or centrifugation. The need for several washing steps makes it unsuitable for fully automated HTS, and generates large volumes of radioactive waste, raising safety and cost concerns over storage and disposal. Such assays are mainly restricted to 96-well format due to limitations of available multiwell filter plates and achieving consistent filtration when using higher density formats. Filtration systems do provide the advan­tage that they allow accurate determination of maximal binding levels and ligand affinities at sufficient throughput for support of hit-to-lead and lead optimization activities. In the HTS arena, filtration assays have been superseded by homogeneous formats for radioactive assays. These have reduced overall reaction volume and eliminate the need for separation steps, largely eliminating the problem of waste disposal and provide increased throughput.

The majority of homogenous radioactive assay types are based on the scintillation proximity principle. This relies on the excitation of a scintillant incorporated in a matrix, in the form of either microbeads (’SPA’) or microplates (Flashplates™, Perkin Elmer Life and Analytical Sciences), to the surface of which the target molecule is also attached (Figure). Binding of the radioligand to the target brings it into close proximity to the scintillant, resulting in light emission, which can be quantified. Free radioactive ligand is too distant from the scintillant and no excitation takes place. Isotopes such as 3H or 125I are typically used, as they produce low-energy particles that are absorbed over short distances. Test compounds that bind to the target compete with the radioligand, and thus reduce the signal.

With bead technology, polymer beads of ~5 μm diameter are coated with antibodies, streptavidin, receptors or enzymes to which the radioligand can bind. Ninety-six- or 384-well plates can be used. The emission wavelength of the scintillant is in the range of 420 nm and is subject to limitations in the sensitivity due to both colour quench by yellow test compounds, and the variable efficiency of scintillation counting, due to sedimentation of the beads. The homogeneous platforms are also still subject to limitations in throughput associated with the detection technology via multiple photomultiplier tube-based detection instruments, with a 384-well plate taking in the order of 15 minutes to read.

The drive for increased throughput for radioactive assays led to development of scinitillants, containing europium yttrium oxide or europium polystyrene, contained in beads or multiwell plates with an emission wavlength shifted towards the red end of the visible light spectrum (~560 nm) and suited to detection on charge-coupled device (CCD) cameras. The two most widely adopted instruments in this area are LEADseeker™ (GE Healthcare) and Viewlux™ (Perkin Elmer), using quantitative imaging to scan the whole plate, resulting in a higher throughput and increased sensitivity. Imaging instruments provide a read time typically in the order of a few minutes or less for the whole plate irrespective of density, representing a significant improvement in throughput, along with increased sensitivity. The problem of compound colour quench effect remains, although blue compounds now provide false hits rather than yellow. As CCD detection is independent of plate density, the use of imaging based radioactive assays has been adopted widely in HTS and adapted to 1536-well format and higher.

In the microplate form of scintillation proximity assays the target protein (e.g. an antibody or receptor) is coated on to the floor of a plate well to which the radioligand and test compounds are added. The bound radioligand causes a microplate surface scintillation effect. FlashPlate™ has been used in the investiga­tion of protein–protein (e.g. radioimmunoassay) and receptor–ligand (i.e. radioreceptor assay) interactions, and in enzymatic (e.g. kinase) assays.

Due to the level of sensitivity provided by radioactive assays they are still widely adopted within the HTS setting. However, environmental, safety and local legislative considerations have led to the necessary development of alternative formats, in particular those utilizing fluorescent-ligands. Through careful placement of a suitable fluorophore in the ligand via a suitable linker, the advantages of radioligand binding assays in terms of sensitivity can be realized without the obvious drawbacks associated with the use of radioiso­topes. The use of fluorescence-based technologies is dis­cussed in more detail in the following section.

Fluorescence technologies

The application of fluorescence technologies is wide­spread, covering multiple formats and yet in the simplest form involves excitation of a sample with light at one wavelength and measurement of the emission at a different wavelength. The difference between the absorbed wavelength and the emitted wave­length is called the Stokes shift, the magnitude of which depends on how much energy is lost in the fluorescence process. A large Stokes shift is advanta­geous as it reduces optical crosstalk between photons from the excitation light and emitted photons.

Fluorescence techniques currently applied for HTS can be grouped into six major categories:

• Fluorescence intensity

• Fluorescence resonance energy transfer

• Time-resolved fluorescence

• Fluorescence polarization

• Fluorescence correlation

• AlphaScreen™ (amplified luminescence proximity homogeneous assay).

Fluorescence intensity

In fluorescence intensity assays, the change of total light output is monitored and used to quantify a biochemical reaction or binding event. This type of readout is fre­quently used in enzymatic assays (e.g. proteases, lipases). There are two variants: fluorogenic assays and fluorescence quench assays. In the former type the reactants are not fluorescent, but the reaction products are, and their forma­tion can be monitored by an increase in fluorescence intensity.

In fluorescence quench assays a fluorescent group is covalently linked to a substrate. In this state, its fluores­cence is quenched. Upon cleavage, the fluorescent group is released, producing an increase in fluorescence intensity.

Fluorescence intensity measurements are easy to run and cheap. However, they are sensitive to fluorescent inter­ference resulting from the colour of test compounds, organic fluorophores in assay buffers and even fluores­cence of the microplate itself.

Fluorescence resonance energy transfer (FRET)

In this type of assay a donor fluorophore is excited and most of the energy is transferred to an acceptor fluorophore or a quenching group; this results in measurable photon emission by the acceptor. In simple terms, the amount of energy transfer from donor to acceptor depends on the fluorescent lifetime of the donor, the spatial distance between donor and acceptor (10–100 Å), and the dipole orientation between donor and acceptor. The transfer effi­ciency for a given pair of fluorophores can be calculated using the equation of Förster.

Usually the emission wavelengths of donor and accep­tor are different, and FRET can be determined either by the quenching of the donor fluorescence by the acceptor (as shown in Figure) or by the fluorescence of the acceptor itself. Typical applications are for protease assays based on quenching of the uncleaved substrate, although FRET has also been applied for detecting changes in membrane potential in cell-based assays for ion channels. With simple FRET techniques interfer­ence from background fluorescence is often a problem, which is largely overcome by the use of time-resolved fluo­rescence techniques, described below.

Time resolved fluorescence (TRF)

TRF techniques use lanthanide chelates (samarium, europium, terbium and dysprosium) that give an intense and long-lived fluorescence emission (>1000 μs). Fluorescence emission is elicited by a pulse of excitation light and measured after the end of the pulse, by which time short-lived fluorescence has subsided. This makes it possible to eliminate short-lived autofluorescence and reagent background, and thereby enhance the signal-to-noise ratio. Lanthanides emit fluorescence with a large Stokes shift when they coordinate to specific ligands.

Typically, the complexes are excited by UV light, and emit light of wavelength longer than 500 nm. Europium (Eu3+) chelates have been used in immu­noassays by means of a technology called DELFIA (dissociation-enhanced lanthanide fluoroimmuno assay). DELFIA is a heterogeneous time-resolved fluorometric assay based on dissociative fluorescence enhancement. Cell-and membrane-based assays are particularly well suited to the DELFIA system because of its broad detection range and extremely high sensitivity. High sensitivity – to a limit of about 10−17 moles/well – is achieved by applying the dissociative enhancement principle. After separation of the bound from the free label, a reagent is added to the bound label which causes the weakly fluorescent lanthanide chelate to dissociate and form a new highly fluorescent chelate inside a protective micelle. Though robust and very sensitive, DELFIA assays are not ideal for HTS, as the process involves several binding, incubation and washing steps.

The need for homogeneous (‘mix and measure’) assays led to the development of LANCETM (Perkin Elmer Life Sciences) and HTRF® (Homogeneous Time-Resolved Fluo­rescence; Cisbio). LANCETM, like DELFIA®, is based on chelates of lanthanide ions, but in a homogeneous format. The chelates used in LANCETM can be measured directly without the need for a dissociation step, however in an aqueous environment the complexed ion can spontane­ously dissociate and increase background fluorescence.

In HTRF® (Figure) these limitations are overcome by the use of a cryptate molecule, which has a cage-like struc­ture, to protect the central ion (e.g. Eu+) from dissociation. HTRF® uses two separate labels, the donor (Eu)K and the acceptor APC/XL665 (a modified allophycocyanine from red algae) and such assays can be adapted for use in plates up to 1536-well format. In both LANCETM and HTRF®, measurement of the ratio of donor and acceptor fluorophore emission can be applied to compensate for non-specific quenching of assay reagents. As a result, the applications of both technologies are widespread, covering detection of kinase enzyme activ­ity , protease activity, second messengers such as cAMP and inositiol tri-phosphate (InsP3) and numerous biomarkers such as interleukin 1β (IL-1β) and tumour necrosis factor alpha (TNFα).

Fluorescence polarization (FP)

When a stationary molecule is excited with plane-polarized light it will fluoresce in the same plane. If it is tumbling rapidly, in free solution, so that it changes its orientation between excitation and emission, the emission signal will be depolarized. Binding to a larger molecule reduces the mobility of the fluorophore so that the emission signal remains polarized, and so the ratio of polarized to depolarized emission can be used to determine the extent of binding of a labelled ligand. The rotational relaxation speed depends on the size of the molecule, the ambient temperature and the viscosity of the solvent, which usually remain constant during an assay. The method requires a significant difference in size between labelled ligand and target, which is a major restriction to its application and the reliance on a single, non-time resolved fluorescence output makes the choice of fluorphore important to minimize compound interference effects. FP-based assays can be used in 96-well up to 1536-well formats.

Fluorescence correlation methods

Although an uncommon technique in most HTS depart­ments, due the requirement for specific and dedicated instrumentation, this group of fluorescence technologies provide highly sensitive metrics using very low levels of detection reagents and are very amendable to ultra-high throughput screening (uHTS). The most widely applied readout technology, fluorescence cor­relation spectroscopy, allows molecular interactions to be studied at the single-molecule level in real time. Other proprietary technologies such as fluorescence intensity dis­tribution analysis (FIDA and 2-dimensional FIDA also fall into this grouping, sharing the common theme of the analysis of biomolecules at extremely low concentrations. In contrast to other fluores­cence techniques, the parameter of interest is not the emis­sion intensity itself, but rather intensity fluctuations. By confining measurements to a very small detection volume (achieved by the use of confocal optics) and low reagent concentrations, the number of molecules monitored is kept small and the statistical fluctuations of the number contributing to the fluorescence signal at any instant become measurable. Analysis of the frequency compo­nents of such fluctuations can be used to obtain informa­tion about the kinetics of binding reactions.

With help of the confocal microscopy technique and laser technologies, it has become possible to measure molecular interactions at the single molecule level. Single molecule detection (SMD) technologies provide a number of advantages: significant reduction of signal-to-noise ratio, high sensitivity and time-resolution. Furthermore, they enable the simultaneous readout of various fluorescence parameters at the molecular level. SMD readouts include fluorescence intensity, translational diffusion (fluorescence correlation spectroscopy, FCS), rotational motion (fluores­cence polarization), fluorescence resonance energy trans­fer, and time-resolved fluorescence. SMD technologies are ideal for miniaturization and have become amenable to automation. Further advantages include very low reagent consumption and broad applica­bility to a variety of biochemical and cell-based assays.

Single molecular events are analysed by means of confo­cal optics with a detection volume of approximately 1 fL, allowing miniaturization of HTS assays to 1 μL or below. The probability is that, at any given time, the detection volume will have a finite number of molecular events (movement, intensity, change in anisotropy), which can be measured and computed. The signal-to-noise ratio typi­cally achieved by these methods is high, while interference from scattered laser light and background fluorescence are largely eliminated.

Fluorescence lifetime analysis is a relatively straightforward assay methodology that over­comes many of the potential compound intereference effects achieved through the use of TRF, but without the requirement for expensive fluorophores. The technique utilizes the intrinsic lifetime of a fluorophore, correspond­ing to the time the molecule spends in the excited state. This time is altered upon binding of the fluorophore to a com­pound or protein and can be measured to develop robust assays that are liable to minimum compound intereference using appropriate detection instrumentation.

AlphaScreen™ Technology

The proprietary bead-based technology from Perkin Elmer is a proximity-based format utilizing a donor bead which, when excited by light at a wavelength of 680 nm, releases singlet oxygen that is absorbed by an acceptor bead, and assuming it is in sufficiently close proximity (<200 nm) this results in the emission of light between 520 and 620 nm (Figure). This phenomenon is unusual in that the wavlength of the emitted light is shorter and therefore has higher energy than the excitation wavelength. This is of significance since it reduces the potential for compound inner filter effects; however, reactive functionality may still inhibit the energy transfer.

As with other bead-based technologies the donor and acceptor beads are available with a range of surface treat­ments to enable the immobilization or capture of a range of analytes. The range of immobolization formats and the distance over which the singlet oxygen can pass to excite the donor bead provide a suitable format for developing homogeneous antibody-based assays similar to enzyme-linked immunosorbent assays (ELISA) which are generally avoided in the HTS setting due to multiple wash, addition and incubation steps. These bead-based ELISA, such as AlphaLISA™ (Perkin Elmer), provide the required sensitiv­ity for detection of biomarkers in low concentration and can be configured to low volume 384-well format without loss of signal window.

AlphaLISA 

AlphaLISA is a further development of the AlphaScreen technology that relies on the same donor beads but uses a different type of acceptor beads. In AlphaLISA beads, anthracene and rubrene are substituted by europium chelates. Excited europium emits light at 615 nm with a much narrower wavelength bandwidth than AlphaScreen (fig.). Hence, AlphaLISA emission is less susceptible to compound interference and can be employed for the detection of analytes in biological samples like cell culture supernatants, cell lysates, serum, and plasma.

AlphaLISA allows the quantification of secreted, intracellular, or cell membrane proteins. For biomarker detection, AlphaLISA is mainly employed as a sandwich immunoassay. A biotinylated anti-analyte antibody binds the streptavidin donor bead while a second anti-analyte antibody is conjugated to AlphaLISA acceptor beads. In the presence of the analyte, beads come into close proximity. Donor bead excitation releases singlet oxygen molecules that transfer energy to the acceptor beads with light emission at 615 nm (fig.). Alternatively, competition immunoassays can also be adapted.

Cell-based assays

Here described five cell-based readout technologies that have found general application in many types of assay, namely fluorometric methods, reporter gene assays, yeast complementation assays, high-throughput electrophysiology assays and more recently label free detection platforms.

Fluorometric assays

Fluorometric assays are widely used to monitor changes in the intracellular concentration of ions or other constitu­ents such as cAMP. A range of fluorescent dyes has been developed which have the property of forming reversible complexes with ions such as Ca2+ or Tl+ (as a surrogate for K+). Their fluorescent emission intensity changes when the complex is formed, thereby allowing changes in the free intracellular ion concentration to be monitored, for example in response to activation or block of membrane receptors or ion channels, Other membrane-bound dyes are available whose fluorescence signal varies according to the cytoplasmic or mitochondrial membrane potential. Membrane-impermeable dyes which bind to intracellular structures can be used to monitor cell death, as only dying cells with leaky membranes are stained. In addition to dyes, ion-sensitive proteins such as the jellyfish photo-protein aequorin (see below), which emits a strong fluores­cent signal when complexed with Ca2+, can also be used to monitor changes in [Ca2+]i. Cell lines can be engineered to express this protein, or it can be introduced by electro­poration. Such methods find many applications in cell biology, particularly when coupled with confocal microscopy to achieve a high level of spatial resolution. For HTS applications, the development of the Fluorescence Imaging Plate Reader (FLIPR™, Molecular Devices Inc.,), allowing the simultaneous application of reagents and test compounds to multiwell plates and the capture of the fluorescence signal from each well was a key advance in allowing cel­lular assays to be utilized in the HTS arena. Early instru­ments employed an argon laser to deliver the excitation light source with the emission measured using a CCD imaging device. In more recent models the laser has been replaced with an LED light source (www.moleculardevices.com) and overcomes some of the logistical considerations for deploying these instruments in some laboratories. Repeated measurements can be made at intervals of less than 1 s, to determine the kinetics of the cellular response, such as changes in [Ca2+]i or membrane potential, which are often short-lasting, so that monitoring the time profile rather than taking a single snapshot measurement is essential.

Reporter gene assays

Gene expression in transfected eukaryotic cells can be quantified by linking a promoter sequence to a reporter gene, whose level of expression is readily monitored, and reflects the degree of activation or inhibition of the pro­moter. Compounds activating or inhibit­ing the promoter itself, or interfering with a signal pathway connected to that promoter, can thus be detected. By using two different reporter constructs e.g. firefly and Renilla luciferase, different targets can be screened simultaneously. The principle of a reporter gene assay for GPCR activity, based on luciferase, is shown in Figure. Reporter readouts can also be duplexed with more immediate readouts of cell signalling, such as calcium sensitive dyes, to reduce the false positive liability associ­ated with using a single assay readout. Commonly used reporter genes are CAT (chlorampheni­col acetyltransferase), GAL (β-galactosidase), LAC (β-lactamase), LUC (luciferase) and GFP (green fluorescence protein), usually employing a colorimetric or fluorescent readout and each having relative merits. The number of reporter genes is dwarfed compared to the range of promoters that can be employed in this format, covering a diversity of signalling events. Whilst having been widely deployed in the drug discov­ery process there are several limitations of reporter gene technology, not least because of the measurement of a response distal to the ligand interaction and the longer compound incubation times required, increasing the potential for cytotoxic events.

Yeast complementation assay

Yeast is a well-characterized organism for investigating mammalian systems, and the yeast two-hybrid assay is a powerful genetic screening technique for measuring the protein–protein and protein–DNA interactions that under­lie many cellular control mechanisms. Widely applied in cell and systems biology to study the binding of transcription factors at the sequence level, it can also be used to screen small molecules for their interference with specific protein–protein and protein–DNA interactions, and has recently been adapted for other types of drug–target interactions. Conventional in vitro measurements, such as immunoprecipitation or chromatographic co-precipitation, require the interacting proteins in pure form and at high concentra­tions, and therefore are often of limited use.

The yeast two-hybrid system uses two separated peptide domains of transcription factors: a DNA-specific binding part (DNB) and a transcription activation domain (AD). The DNB moiety is coupled to one protein (the ‘bait’), and the AD moiety to another (the ‘prey’). If the prey protein binds to the bait protein, the AD moiety is brought into close association with the reporter gene, which is thereby activated, producing a product (e.g. GAL or LAC, as described above, or an enzyme which allows the yeast to grow in the presence of cycloheximide). The addition of a test compound that blocks the specific protein–protein interaction prevents activation of the reporter gene. The bait and prey proteins contained domains of two different channel subunits which need to associate to form a functional channel.

High throughput electrophysiology assays

The progression of ion channels, and in particular voltage-gated ion channels, as druggable targets using screening approaches was, until recently, severely limited by the throughput of conventional electrophysiology techniques and lack of suitable higher throughput assay platforms. Although fluorescence methods using membrane poten­tial sensitive dyes such as DiBAC4(3) and the FLIPR™ vari­ants of this and the FRET based voltage sensor probes were widely used, the meth­odology could not provide accurate voltage control and the temporal resolution of the evoked responses was poor. The introduction of planar patch-clamp instruments, par­ticularly systems such as IonWorks Quattro which record using multihole planar substrate consumables has to a certain extent overcome the throughput hurdle. The operating principle of this instrument is shown in Figure and whilst the data point generation is not as high throughput so as to compete with fluorescence methods (a maximum of approximately 3000 data points per day per instrument compared with > 20 000 per day for a FLIPR™) it is suffi­cient for screening of targeted libraries, diverse compound decks up to around 100 000 compounds and the confirma­tion of a large number of hits identified in less physiologi­cally relevant platforms.

Label free detection platforms

The current drive for the drug discovery process is to move towards as physiologically relevant systems as possible and away from target overexpression in heterologous expres­sion systems and, in the case of G-protein coupled recep­tors, to avoid the use of promiscuous G-proteins where possible. The downside to this is that endogenous receptor expression levels tend to be lower and therefore more sensitive detection methods are required. Also, for the study of targets where the signalling mechanism is unknown, e.g. orphan GPCRs, multiple assay systems would need to be developed which would be time con­suming and costly. Consequently, the application of assay platforms which detect gross cellular responses, usually cell morphology due to actin cytoskeleton remodelling, to physiological stimuli have been developed. These fall into two broad categories, those that detect changes in impedance through cellular dielectric spectroscopy, e.g. CellKey™, Molecular Devices Corporation; xCelligence, Roche Diagnostics or the use of optical biosensors, e.g. Epic™, Corning Inc; or Octect, Fortebio. The application of these platforms in the HTS arena is still in its infancy largely limited by throughput and relatively high cost per data point com­pared to established methods. However, the assay develop­ment time is quite short, a single assay may cover a broad spectrum of cell signalling events and these methods are considered to be more sensitive than many existing methods enabling the use of endogenous receptor expres­sion and even the use of primary cells in many instances.

High content screening

High content screening (HCS) is a further development of cell-based screening in which multiple fluorescence read­outs are measured simultaneously in intact cells by means of imaging techniques. Repetitive scanning provides tem­porally and spatially resolved visualization of cellular events. HCS is suitable for monitoring such events as nuclear translocation, apoptosis, GPCR activation, recep­tor internalization, changes in [Ca2+]i, nitric oxide produc­tion, apoptosis, gene expression, neurite outgrowth and cell viability. The aim is to quantify and correlate drug effects on cel­lular events or targets by simultaneously measuring mul­tiple signals from the same cell population, yielding data with a higher content of biological information than is provided by single-target screens. Current instrumentation is based on automated digital microscopy and flow cytometry in combination with hard and software systems for the analysis of data. Within the configuration a fluorescence-based laser scanning plate reader (96, 384- or 1536-well format), able to detect fluo­rescent structures against a less fluorescent background, acquires multicolour fluorescence image datasets of cells at a preselected spatial resolution. The spatial resolution is largely defined by the instrument specification and whether it is optical confocal or widefield. Confocal imaging enables the generation of high-resolution images by sampling from a thin cellular section and rejection of out of focus light; thus giving rise to improved signal : noise compared to the more commonly applied epi-fluorescence microscopy. There is a powerful advantage in confocal imaging for applications where subcellular localization or membrane translocation needs to be measured. However, for many biological assays, confocal imaging is not ideal e.g. where there are phototoxicity issues or the applica­tions have a need for a larger focal depth. HCS relies heavily on powerful image pattern recogni­tion software in order to provide rapid, automated and unbiased assessment of experiments. The concept of gathering all the necessary information about a compound at one go has obvious attractions, but the very sophisticated instrumentation and software produce problems of reliability. Furthermore, the principle of ‘measure everything and sort it out afterwards’ has its drawbacks: interpretation of such complex datasets often requires complex algorithms and significant data storage capacity. Whilst the complexity of the analysis may seem daunting, high content screening allows the study of complex signalling events and the use of phenotypic rea­douts in highly disease relevant systems. However, such analysis is not feasible for large number of compounds and unless the technology is the only option for screening in most instances HCS is utilized for more detailed study of lead compounds once they have been identified.

Biophysical methods in high-throughput screening

Conventional bioassay-based screening remains a main­stream approach for lead discovery. However, during recent years alternative biophysical methods such as nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) and X-ray crystallography have been developed and/or adapted for drug discovery. Usually in assays whose main purpose is the detection of low-affinity low-molecular-weight compounds in a different approach to high-throughput screening, namely fragment-based screening. Hits from HTS usually already have drug-like properties, e.g. a molecular weight of ~ 300 Da. During the following lead optimization synthesis programme an increase in molecular weight is very likely, leading to poorer drug-like properties with respect to solubility, absorption or clearance. Therefore, it may be more effec­tive to screen small sets of molecular fragments (<10 000) of lower molecular weight (100–250 Da) which can then be chemically linked to generate high-affinity drug-like compounds. Typically, such fragments have much weaker binding affinities than drug-like compounds and are outside the sensitivity range of a conventional HTS assay. NMR-, SPR- or X-ray crystallography-based assays are better suited for the identification of weak binders as these methodologies lend themselves well to the area of frag­ment based screening. As the compound libraries screened are generally of limited size throughput is less important than sensitive detection of low-affinity interactions. Once the biophysical interactions are determined, further X-ray protein crystallographic studies can be undertaken to understand the binding mode of the fragments and this information can then be used to rapidly drive the fragment-to-hit.

Pharmacology

Typically, when a molecular target has been selected, and lead com­pounds have been identified which act on it selectively, and which are judged to have ‘drug-like’ chemical attributes (including suitable pharmacokinetic properties), the next stage is a detailed pharmacological evaluation. This means investigation of the effects, usually of a small number of compounds, on a range of test systems, up to and includ­ing whole animals, to determine which, if any, is the most suitable for further development (i.e. for nomination as a drug candidate). Pharmacological evaluation typically involves the following:

• Selectivity screening, consisting of in vitro tests on a broad range of possible drug targets to determine whether the compound is sufficiently selective for the chosen target to merit further investigation

• Pharmacological profiling, aimed at evaluating in isolated tissues or normal animals the range of effects of the test compound that might be relevant in the clinical situation. Some authorities distinguish between primary pharmacodynamic studies, concerning effects related to the selected therapeutic target (i.e. therapeutically relevant effects), and secondary pharmacodynamic studies, on effects not related to the target (i.e. side effects). At the laboratory level the two are often not clearly distinguishable, and the borderline between secondary pharmacodynamic and safety pharmacology studies (see below) is also uncertain. Nevertheless, for the purposes of formal documentation, the distinction may be useful

• Testing in animal models of disease to determine whether the compound is likely to produce therapeutic benefit

• Safety pharmacology, consisting of a series of standardized animal tests aimed at revealing undesirable side effects, which may be unrelated to the primary action of the drug.

The pharmacological evaluation of lead compounds does not in general follow a clearly defined path, and often it has no clearcut endpoint but will vary greatly in its extent, depending on the nature of the compound, the questions that need to be addressed and the inclinations of the project team. Directing this phase of the drug dis­covery project efficiently, and keeping it focused on the overall objective of putting a compound into develop­ment, is one of the trickier management tasks. It often happens that unexpected, scientifically interesting data are obtained which beg for further investigation even though they may be peripheral to the main aims of the project. From the scientists’ perspective, the prospect of opening up a new avenue of research is highly alluring, whether the work contributes directly to the drug discovery aims or not. In this context, project managers need to bear in mind the question: Who needs the data and why? – a question which may seem irritatingly silly to a scientist in academia but totally obvious to the commercial mind. The same principles apply, of course, to all parts of a drug discovery and development project, but it tends to be at the stage of pharmacological evaluation that conflicts first arise between scientific aspiration and commercial need.

An important principle in pharmacological evaluation is the use of a hierarchy of test methods, covering the range from the most reductionist tests on isolated molecular targets to much more elaborate tests of integrated physi­ological function. Establishing and validating such a series of tests appropriate to the particular target and indication being addressed is one of the most important functions of pharmacologists in the drug discovery team. In general, assays become more complicated, slow and expensive, and more demanding of specialist skills as one moves up this hierarchy.

The strengths and weaknesses of these test systems are summarized in Table. Pharmacological characterization of a candidate com­pound often has to take into account active metabolites, based on information from drug metabolism and phar­macokinetics (DMPK) studies. If a major active metabolite is identified, it will be necessary to syn­thesize and test it in the same way as the parent compound in order to determine which effects (both wanted and unwanted) relate to each. Particular problems may arise if the metabolic fate of the compound shows marked species differences, making it difficult to predict from animal studies what will happen in humans.

Although most of the work involved in pharmacological characterization of a candidate drug takes place before clinical studies begin, it does not normally end there. Both ongoing toxicological studies and early trials in man may reveal unpredicted effects that need to be investigated pharmacologically, and so the discovery team needs to remain actively involved and be able to perform experi­ments well into the phase of clinical development. They cannot simply wave the compound goodbye once the dis­covery phase is completed.

SCREENING FOR SELECTIVITY

The selectivity of a compound for the chosen molecular target needs to be assessed at an early stage. Compounds selected for their potency, for example on a given amine receptor, protease, kinase, transporter or ion channel, are very likely to bind also to related – or even unrelated – molecular targets, and thereby cause unwanted side effects. Selectivity is, therefore, as important as potency in choos­ing potential development candidates, and a ‘selectivity screen’ is usually included early in the project. The range of targets included in such a screen depends very much on the type of compound and the intended clinical indica­tion. Ligands for monoamine receptors and transporters form a large and important group of drugs, and several contract research organizations (e.g. CEREP, MDL) offer a battery of assays – mainly binding assays, but also a range of functional assays – designed to detect affinity for a wide range of receptors, transporters and channels. In the field of monoamine receptors, for example, it is usually important to avoid compounds that block or activate peripheral muscarinic receptors, adrenergic receptors or histamine (particularly H1) receptors, because of the side effects that are associated with these actions, and a standard selectivity test battery allows such problems to be discovered early. Recently, several psychotropic and anti-infective drugs have been withdrawn because of sudden cardiac deaths, probably associated with their ability to block a particular type of potassium channel (known as the hERG channel) in myocar­dial cells. This activity can be detected by electrophysio­logical measurements on isolated myocardial cells, and such a test is now usually performed at an early stage of development of drugs of the classes implicated in this type of adverse reaction.

Interpretation of binding assays

Binding assays, generally with membrane preparations made from intact tissues or receptor-expressing cell lines, are widely used in drug discovery projects because of their simplicity and ease of automation. Detailed technical manuals describing the methods used for performing and analysing drug binding experiments are available (Keen, 1999; Vogel, 2002). Generally, the aim of the assay is to determine the dissociation constant, KD, of the test com­pound, as a measure of its affinity for the receptor. In most cases, the assay (often called a displacement assay) measures the ability of the test compound to inhibit the binding of a high-affinity radioligand which combines selectively with the receptor in question, correction being made for ‘non-specific’ binding of the radioligand. In the simplest theoretical case, where the radioligand and the test compound bind reversibly and competitively to a homogeneous population of binding sites, the effect of the test ligand on the amount of the radioligand specifi­cally bound is described by the simple mass-action equation:

 (1)

where B = the amount of radioligand bound, after correct­ing for non-specific binding, Bmax = the maximal amount of radioligand bound, i.e. when sites are saturated, [A] = radioligand concentration, KA = dissociation constant for the radioligand, [L] = test ligand concentration, and KL = dissociation constant for the test ligand.

By testing several concentrations of L at a single concentration of A, the concentration, [L]50, needed for 50% inhibition of binding can be estimated. By rearranging equation 1, KL is given by:

 (2)

This is often known as the Cheng–Prusoff equation, and is widely used to calculate KL when [L]50, [A] and KA are known. It is important to realize that the Cheng–Prusoff equation applies only (a) at equilibrium, (b) when the interaction between A and L is strictly competitive, and (c) when neither ligand binds cooperatively. However, an [L]50 value can be measured for any test compound that inhibits the binding of the radioligand by whatever mechanism, irrespective of whether equilibrium has been reached. Applying the Cheng–Prusoff equation if these conditions are not met can yield estimates of KL that are quite mean­ingless, and so it should strictly be used only if the condi­tions have been shown experimentally to be satisfied – a fairly laborious process. Nevertheless, Cheng–Prusoff estimates of ligand affinity constants are often quoted without such checks having been performed. In most cases it would be more satisfactory to use the experimentally determined [L]50 value as an operational measure of potency. A further important caveat that applies to binding studies is that they are often performed under conditions of low ionic strength, in which the sodium and calcium concentrations are much lower than the physiological range. This is done for technical reasons, as low [Na+] commonly increases both the affinity and the Bmax of the radioligand, and omitting [Ca2+] avoids clumping of the membrane fragments. Partly for this reason, ligand affini­ties estimated from binding studies are often considerably higher than estimates obtained from functional assays (Hall, 1992), although the effect is not consistent, presum­ably because ionic bonding, which will be favoured by the low ionic strength medium, contributes unequally to the binding of different ligands. Consequently, the correlation between data from binding assays and functional assays is often rather poor (see below). Figure  shows data obtained independently on 5HT3 and 5HT4 receptors; in both cases the estimated KD values for binding are on average about 10 times lower than estimates from func­tional assays, and the correlation is very poor.

Figure - Correlation of binding and functional data for 5HT receptor ligands. (A) 5HT3 receptors, (B) 5HT4 receptors.

Pharmacological profiling

Pharmacological profiling aims to determine the pharma­codynamic effects of the new compound – or more often of a small family of compounds – on in vitro model systems, e.g. cell lines or isolated tissues, normal animals, and animal models of disease. The last of these is particu­larly important, as it is intended to give the first real pointer to therapeutic efficacy as distinct from pharmaco­dynamic activity. It is valuable to assess the activity of the compounds in a series of assays representing increasingly complex levels of organization. The choice of test systems depends, of course, on the nature of the target. For example, characterization of a novel antagonist of a typical G-protein-coupled receptor might involve the following:

• Ligand-binding assay on membrane fragments from a cell line expressing the cloned receptor

• Inhibition of agonist activity in a cell line, based on a functional readout (e.g. raised intracellular calcium)

• Antagonism of a selective agonist in an isolated tissue (e.g. smooth muscle, cardiac muscle). Such assays will normally be performed with non-human tissue, and so interspecies differences in the receptor need to be taken into account. Sometimes specific questions have to be asked about effects on human tissues for particular compounds and then collecting viable tissues to use becomes a major challenge

• Antagonism of the response (e.g. bronchoconstriction, vasoconstriction, increased heart rate) to a selective receptor agonist in vivo. Prior knowledge about species specificity of the agonist and antagonist is important at this stage.

Pharmacological profiling is designed as a hypothesis-driven programme of work, based on the knowledge previously gained about the activity of the compound on its specific target or targets. In this respect it differs from safety pharmacology (see below), which is an open-minded exercise designed to detect unforeseen effects. The aim of pharmacological profiling is to answer the follow­ing questions:

• Do the molecular and cellular effects measured in screening assays actually give rise to the predicted pharmacological effects in intact tissues and whole animals?

• Does the compound produce effects in intact tissues or whole animals not associated with actions on its principal molecular target?

• Is there correspondence between the potency of the compound at the molecular level, the tissue level and the whole animal level?

• Do the in vivo potency and duration of action match up with the pharmacokinetic properties of the compound? What happens if the drug is given continuously or repeatedly to an animal over the course of days or weeks?

• Does it lose its effectiveness, or reveal effects not seen with acute administration? Is there any kind of ‘rebound’ after effect when it is stopped?

In vitro profiling

Measurements on isolated tissues

Studies on isolated tissues have been a mainstay of phar­macological methodology ever since the introduction of the isolated organ bath by Magnus early in the 20th century. The technique is extremely versatile and applica­ble to studies on smooth muscle (e.g. gastrointestinal tract, airways, blood vessels, urinary tract, uterus, biliary tract, etc.) as well as cardiac and striated muscle, secretory epithelia, endocrine glands, brain slices, liver slices, and many other functional systems. In most cases the tissue is removed from a freshly killed or anaesthetized animal and suspended in a chamber containing warmed oxygen­ated physiological salt solution. With smooth muscle preparations the readout is usually mechanical (i.e. tension, recorded with a simple strain gauge). For other types of preparation, various electrophysiological or bio­chemical readouts are often used.

Studies of this kind have the advantage that they are performed on intact normal tissues, as distinct from iso­lated enzymes or other proteins. The recognition mole­cules, signal transduction machinery and the mechanical or biochemical readout are assumed to be a reasonable approximation to the normal functioning of the tissue. There is abundant evidence to show that tissue responses to GPCR activation, for example, depend on many factors, including the level of expression of the receptor, the type and abundance of the G proteins present in the cell, the presence of associated proteins such as receptor activity-modifying proteins (RAMPs), the state of phosphorylation of various constituent proteins in the signal transduction cascade, and so on. For com­pounds acting on intracellular targets, functional activity depends on permeation through the membrane, as well as affinity for the target. For these reasons – and probably also for others that are not understood – the results of assays on isolated tissues often differ significantly from results found with primary screening assays. The discrep­ancy may simply be a quantitative one, such that the potency of the ligand does not agree in the two systems, or it may be more basic. For example, the pharmacological efficacy of a receptor ligand, i.e. the property that deter­mines whether it is a full agonist, a partial agonist, or an antagonist, often depends on the type of assay used, and this may have an important bearing on the selection of possible development compounds. Examples that illustrate the poor correlation that may exist between measurements of target affinity in cell-free assay systems, and functional activity in intact cell systems, are shown in Figures. Figure shows the relationship between binding and functional assay data for 5HT3 and 5HT4 receptor antagonists. In both cases, binding assays overestimate the potency in functional assays by a factor of about 10 (see above), but more impor­tantly, the correlation is poor, despite the fact that the receptors are extracellular, and so membrane penetration is not a factor. Figure shows data on tyrosine kinase inhibitors, in which activity against the isolated enzyme is plotted against inhibition of tyrosine phosphorylation in intact cells, and inhibition of cell proliferation for a large series of compounds. Differences in membrane penetra­tion can account for part of the discrepancy between enzyme and cell-based data, but the correlation between intracellular kinase inhibition and blocking of cell prolif­eration is also weak, which must reflect other factors.

It is worth noting that these examples come from very successful drug discovery projects. The quantitative dis­crepancies that we have emphasized, though worrying to pharmacologists, should not therefore be a serious distrac­tion in the context of a drug discovery project. A very wide range of physiological responses can be addressed by studies on isolated tissues, including meas­urements of membrane excitability, synaptic function, muscle contraction, cell motility, secretion and release of mediators, transmembrane ion fluxes, vascular resistance and permeability, and epithelial transport and permeabil­ity. This versatility and the relative technical simplicity of many such methods are useful attributes for drug discov­ery. Additional advantages are that concentration–effect relationships can be accurately measured, and the design of the experiments is highly flexible, allowing rates of onset and recovery of drug effects to be determined, as well as measurements of synergy and antagonism by other compounds, desensitization effects, etc.  The main shortcomings of isolated tissue pharmacology are (a) that tissues normally have to be obtained from small laboratory animals, rather than humans or other primates; and (b) that preparations rarely survive for more than a day, so that only short-term experiments are feasible.

Figure Correlation of cellular activity of EGFR receptor kinase inhibitors with enzyme inhibition.

In vivo profiling

As already mentioned, experiments on animals have several drawbacks. They are generally time-consuming, technically demanding and expensive. They are subject to considerable ethical and legal constraints, and in some countries face vigorous public opposition. For all these reasons, the number of experiments is kept to a bare minimum, and experimental variability is consequently often a problem. Animal experiments must, therefore, be used very selectively and must be carefully planned and designed so as to produce the information needed as effi­ciently as possible. In the past, before target-directed approaches were the norm, routine in vivo testing was often used as a screen at a very early stage in the drug discovery process, and many important drugs (e.g. thiazide diuretics, benzodiazepines, ciclosporin) were discovered on the basis of their effects in vivo. Nowadays, the use of in vivo methods is much more limited, and will probably decline further in response to the pressures on time and costs, as alternative in vitro and in silico methods are developed, and as public attitudes to animal experimenta­tion harden. An additional difficulty is the decreasing number of pharmacologists trained to perform in vivo studies1.

Imaging technologies are increasingly being used for pharma­cological studies on whole animals. Useful techniques include magnetic resonance imaging (MRI), ultrasound imaging, X-ray densitometry tomography, positron emis­sion tomography (PET) and others. They are proving highly versatile for both structural measurements (e.g. cardiac hypertrophy, tumour growth) and functional measurements (e.g. blood flow, tissue oxygenation). Used in conjunction with radio-active probes, PET can be used for studies on receptors and other targets in vivo. Many of these techniques can also be applied to humans, providing an important bridge between animal and human pharma­cology. Apart from the special facilities and equipment needed, currently the main drawback of imaging tech­niques is the time taken to capture the data, during which the animal must stay still, usually necessitating anaesthe­sia. With MRI and PET, which are currently the most ver­satile imaging techniques, data capture normally takes a few minutes, so they cannot be used for quick ‘snapshots’ of rapidly changing events.

A particularly important role for in vivo experiments is to evaluate the effects of long-term drug administration on the intact organism. ‘Adaptive’ and ‘rebound’ effects (e.g. tolerance, dependence, rebound hypertension, delayed endocrine effects, etc.) are often produced when drugs are given continuously for days or weeks. Generally, such effects, which involve complex physiological interactions, are evident in the intact functioning organism but are not predictable from in vitro experiments.

The programme of in vivo profiling studies for charac­terization of a candidate drug depends very much on the drug target and therapeutic indication. A comprehensive catalogue of established in vivo assay methods appropriate to different types of pharmacological effect is given by Vogel (2002). Charting the appropriate course through the plethora of possible studies that might be performed to characterize a particular drug can be difficult.

A typical example of pharmacological profiling is sum­marized in Box. The studies were carried out as part of the recent development of a cardiovascular drug, berap­rost. Beraprost is a stable analogue of prostaglandin I2 (PGI2) which acts on PGI2 receptors of platelets and blood vessels, thereby inhibiting platelet aggregation (and hence thrombosis) and dilating blood vessels. It is directed at two therapeutic targets, namely occlusive peripheral vascular disease and pulmonary hypertension (a serious complication of various types of cardiovascular disease, drug treatment or infectious dis­eases), resulting in hypertrophy and often contractile failure of the right ventricle. The animal studies were, therefore, directed at measuring changes (reduction in blood flow, histological changes in vessel wall) associated with peripheral vascular disease, and with pulmonary hypertension. As these are progressive chronic conditions, it was important to establish that long-term systemic administration of beraprost was effective in retarding the development of the experimental lesions, as well as moni­toring the acute pharmacodynamic effects of the drug.

Species differences

It is important to take species differences into account at all stages of pharmacological profiling. For projects based on a defined molecular target – nowadays the majority – the initial screening assay will normally involve the human isoform. The same target in different species will generally differ in its pharmacological specificity; commonly, there will be fairly small quantitative differences, which can be allowed for in interpreting pharmacological data in experi­mental animals, but occasionally the differences are large, so that a given class of compounds is active in one species but not in another. An example is shown in Figure, which compares the activities of a series of bradykinin receptor antagonists on cloned human and rat receptors. The complete lack of correlation means that, for these compounds, tests of functional activity in the rat cannot be used to predict activity in man.

Species differences are, in fact, a major complicating factor at all stages of drug discovery and preclinical devel­opment. The physiology of disease processes such as inflammation, septic shock, obesity, atherosclerosis, etc., differs markedly in different species. Most importantly (see Chapter 10), drug metabolism often differs, affecting the duration of action, as well as the pattern of metabo­lites, which can in turn affect the observed pharmacology and toxicity.

Species differences are, of course, one of the main argu­ments used by animal rights activists in opposing the use of animals for the purpose of drug discovery. Their claim – misleading when examined critically (see Under­standing Animal Research website) – is that animal data actually represent disinformation in this context. While being aware of the pitfalls, we should not lose sight of the fact that non-human data, including in vivo experiments, have actually been an essential part of every major drug discovery project to date. The growing use of transgenic animal models has led to an increase, rather than a decrease, in animal experimentation, as even breeding such animals is counted as an experiment for statistical purposes.

Figure - Species differences in bradykinin B2 receptors.

ANIMAL MODELS OF DISEASE

The animal models discussed earlier were used to investi­gate the pharmacodynamic effects of the drug and to answer the question: How do the effects observed at the molecular and cellular levels of organization translate into physiological effects in the whole animal?

The next, crucial, question is: Can these physiological effects result in therapeutic benefit? Animal experiments can never answer this conclusively – only clinical trials can do that – but the use of animal models of human disease provides a valuable link in the chain of evidence, and there is strong pressure on drug discovery teams to produce data of this sort as a basis for the important deci­sion to test a new compound in man. Despite the immense range and diversity of animal models that have been described, this is often the most problematic aspect of a drug discovery project, particularly where a novel target or mechanism is involved, so that there is no mechanistic precedent among established drugs. The magnitude of the difficulties varies considerably among different therapeu­tic areas. Many inflammatory conditions, for example, are straightforward to model in animals, as are some cancers. Animal models of hypertension generally predict very well the ability of compounds to lower blood pressure in man. Endocrine disorders involving over- or under secretion of particular hormones can also be simply modelled in animals. Psychiatric disorders are much more difficult, as the symptoms that characterize them are not observable in animals. In most therapeutic areas there are certain disorders, such as migraine, temporal lobe epilepsy, asthma or irritable bowel syndrome, for which animal models, if they exist at all, are far from satisfactory in predicting clinical efficacy.

Here we consider, with a few selected examples, the main experimental approaches to generating animal models, and the criteria against which their ‘validity’ as models of human disease need to be assessed.

Types of animal model

Animal models of disease can be divided broadly into acute and chronic physiological and pharmacological models, and genetic models.

Acute physiological and pharmacological models are intended to mimic certain aspects of the clinical disorder. There are many examples, including:

• Seizures induced by electrical stimulation of the brain as a model for epilepsy (see below)

• Histamine-induced bronchoconstriction as a model for asthma

• The hotplate test for analgesic drugs as a model for pain

• Injection of lipopolysaccharide (LPS) and cytokines as a model for septic shock

• The elevated maze test as a model for testing anxiolytic drugs.

Chronic physiological or pharmacological models involve the use of drugs or physical interventions to induce an ongoing abnormality similar to the clinical condition. Examples include:

• The use of alloxan to inhibit insulin secretion as a model for Type I diabetes

• Procedures for inducing brain or coronary ischaemia as models for stroke and ischaemic heart disease

‘Kindling’ and other procedures for inducing ongoing seizures as models for epilepsy

• Self-administration of opiates, nicotine or other drugs as a model for drug-dependence

• Cholesterol-fed rabbits as a model for hypercholesterolaemia and atherosclerosis

• Immunization with myelin basic protein as a model for multiple sclerosis

• Administration of the neurotoxin MPTP, causing degeneration of basal ganglia neurons as a model of Parkinson’s disease

• Transplantation of malignant cells into immunodeficient animals to produce progressive tumours as a model for certain types of cancer.

Details of these and many other examples of physiologi­cal and pharmacological models can be found in Vogel (2002). As discussed above, species differences need to be taken into account in the selection of animal models, and in the interpretation of results. In septic shock, for example, rodents show a much larger elevation of nitric oxide (NO) metabolites than do humans, and respond well to NO synthesis inhibitors, which humans do not. Rodents and rabbits transgenically engineered to favour cholesterol deposition nevertheless develop atherosclerosis only when fed high-cholesterol diets, whereas humans often do so even on low-cholesterol diets. Genetically obese mice are deficient in the hormone leptin and lose weight when treated with it, whereas obese humans frequently have high circulating leptin concentrations and do not respond to treatment with it. It is often not clear whether such discrepancies reflect inherent species differences, or simply failure of the model to replicate satisfactorily the predomi­nant human disease state.

Genetic models

There are many examples of spontaneously occurring animal strains that show abnormalities phenotypically resembling human disease. In addition, much effort is going into producing transgenic strains with deletion or over-expression of specific genes, which also exhibit disease-like phenotypes.

Long before genetic mapping became possible, it was realized that certain inbred strains of laboratory animal were prone to particular disorders, examples being spon­taneously hypertensive rats, seizure-prone dogs, rats insensitive to antidiuretic hormone (a model for diabetes insipidus), obese mice and mouse strains exhibiting a range of specific neurological deficits. Many such strains have been characterized (Jackson Laboratory website, www.jaxmice.jax.org) and are commercially available, and are widely used as models for testing drugs.

The development of transgenic technology has allowed inbred strains to be produced that over- or under-express particular genes. In the simplest types, the gene abnormal­ity is present throughout the animal’s life, from early development onwards, and throughout the body. More recent technical developments allow much more control over the timing and location of the transgene effect. For reviews of transgenic technology and its uses in drug dis­covery.

The genetic analysis of disease-prone animal strains, or of human families affected by certain diseases, has in many cases revealed the particular mutation or mutations responsible, thus pointing the way to new transgenic models. Several diseases associated with single-gene mutations, such as cystic fibrosis and Duchenne muscular dystrophy, have been replicated in trans­genic mouse strains. Analysis of the obese mouse strain led to the identification of the leptin gene, which is mutated in the ob/ob mouse strain, causing the production of an inactive form of the hormone and overeating by the mouse. Transgenic animals closely resembling ob/ob mice have been produced by targeted inactivation of the gene for leptin or its receptor. Another example is the discovery that a rare familial type of Alzheimer’s disease is associated with mutations of the amyloid precursor protein (APP). Transgenic mice expressing this mutation show amyloid plaque formation characteristic of the human disease. This and other transgenic models of Alzheimer’s disease rep­resent an important tool for drug discovery, as there had hitherto been no animal model reflecting the pathogenesis of this disorder.

The number of transgenic animal models, mainly mouse, that have been produced is already large and is growing rapidly. Creating and validating a new disease model is, however, a slow business. Although the method­ology for generating transgenic mice is now reliable and relatively straightforward, it is both time-consuming and labour-intensive. The first generation of transgenic animals are normally hybrids, as different strains are used for the donor and the recipient, and it is necessary to breed several generations by repeated back-crossings to create animals with a uniform genetic background. This takes 1–2 years, and is essential for consistent results. Analysis of the phe­notypic changes resulting from the transgene can also be difficult and time-consuming, as the effects may be numerous and subtle, as well as being slow to develop as the animal matures. Despite these difficulties, there is no doubt that transgenic disease models are playing an increasing part in drug testing, and many biotechnology companies have moved into the business of developing and providing them for this purpose. The fields in which transgenic models have so far had the most impact are cancer, atherosclerosis and neurodegenerative diseases, but their importance as drug discovery tools extends to all areas.

Producing transgenic rat strains proved impossible until recently, as embryonic stem (ES) cells cannot be obtained from rats. Success in producing gene knockout strains by an alternative method has now been achieved, and the use of transgenic rats is increasing, this being the favoured species for pharmacological and physi­ological studies in many laboratories.

The choice of model

Naturally occurring diseases produce a variety of structural biochemical abnormalities, and these are often displayed separately in animal models. For example, human allergic asthma involves: (a) an immune response; (b) increased airways resistance; (c) bronchial hyperreactivity; (d) lung inflammation; and (e) structural remodelling of the airways. Animal models, mainly based on guinea pigs, whose airways behave similarly to those of humans, can replicate each of these features, but no single model reproduces the whole spectrum. The choice of animal model for drug discovery purposes, therefore, depends on the therapeutic effect that is being sought. In the case of asthma, existing bronchodilator drugs effectively target the increased airways resistance, and steroids reduce the inflammation, and so it is the other components for which new drugs are particularly being sought. A similar need for a range of animal models covering a range of therapeutic targets applies in many disease areas.

Validity criteria

Obviously an animal model produced in a laboratory can never replicate exactly a spontaneous human disease state, so on what basis can we assess its ‘validity’ in the context of drug discovery?

Three types of validity criteria were originally proposed by Willner (1984) in connection with animal models of depression. These are:

• Face validity

• Construct validity

• Predictive validity.

Face validity refers to the accuracy with which the model reproduces the phenomena (symptoms, clinical signs and pathological changes) characterizing the human disease.

Construct validity refers to the theoretical rationale on which the model is based, i.e. the extent to which the aetiology of the human disease is reflected in the model. A transgenic animal model in which a human disease-producing mutation is replicated will have, in general, good construct validity, even if the manifestations of the human disorder are not well reproduced (i.e. it has poor face validity).

Predictive validity refers to the extent to which the effect of manipulations (e.g. drug treatment) in the model is predictive of effects in the human disorder. It is the most pragmatic of the three and the most directly relevant to the issue of predicting therapeutic efficacy, but also the most limited in its applicability, for two main reasons. First, data on therapeutic efficacy are often sparse or non-existent, because no truly effective drugs are known (e.g. for Alzheimer’s disease, septic shock). Second, the model may focus on a specific pharmacological mechanism, thus successfully predicting the efficacy of drugs that work by that mechanism but failing with drugs that might prove effective through other mechanisms. The knowledge that the first generation of antipsychotic drugs act as dopamine receptor antagonists enabled new drugs to be identified by animal tests reflecting dopamine antagonism, but these tests cannot be relied upon to recognize possible ‘break­through’ compounds that might be effective by other mechanisms. Thus, predictive validity, relying as it does on existing therapeutic knowledge, may not be a good basis for judging animal models where the drug discovery team’s aim is to produce a mechanistically novel drug. The basis on which predictive validity is judged carries an inevitable bias, as the drugs that proceed to clinical trials will nor­mally have proved effective in the model, whereas drugs that are ineffective in the model are unlikely to have been developed. As a result, there are many examples of tests giving ‘false positive’ expectations, but very few false nega­tives, giving rise to a commonly held view that conclusions from pharmacological tests tend to be overoptimistic.

Some examples

We conclude this discussion of the very broad field of animal models of disease by considering three disease areas, namely epilepsy, psychiatric disorders and stroke. Epilepsy-like seizures can be produced in laboratory animals in many different ways. Many models have been described and used successfully to discover new anti-epileptic drugs (AEDs). Although the models may lack construct validity and are weak on face validity, their pre­dictive validity has proved to be very good. With models of psychiatric disorders, face validity and construct validity are very uncertain, as human symptoms are not generally observable in animals and because we are largely ignorant of the cause and pathophysiology of these disorders; nev­ertheless, the predictive validity of available models of depression, anxiety and schizophrenia has proved to be good, and such models have proved their worth in drug discovery. In contrast, the many available models of stroke are generally convincing in terms of construct and face validity, but have proved very unreliable as predictors of clinical efficacy. Researchers in this field are ruefully aware that despite many impressive effects in laboratory animals, clinical successes have been negligible.

Epilepsy models

The development of antiepileptic drugs, from the pioneer­ing work of Merritt and Putnam, who in 1937 developed phenytoin, to the present day, has been highly dependent on animal models involving experimentally induced sei­zures, with relatively little reliance on knowledge of the underlying physiological, cellular or molecular basis of the human disorder. Although existing drugs have significant limitations, they have brought major benefits to sufferers from this common and disabling condition – testimony to the usefulness of animal models in drug discovery.

Human epilepsy is a chronic condition with many underlying causes, including head injury, infections, tumours and genetic factors. Epileptic seizures in humans take many forms, depending mainly on where the neural discharge begins and how it spreads.

Some of the widely used animal models used in drug discovery are summarized in Table. The earliest models, namely the maximal electroshock (MES) test and the pentylenetetrazol-induced seizure (PTZ) test, which are based on acutely induced seizures in normal animals, are still commonly used. They model the seizure, but without distinguishing its localization and spread, and do not address either the chronicity of human epilepsy or its aetiology (i.e. they score low on face validity and construct validity). But, importantly, their predictive validity for con­ventional antiepileptic drugs in man is very good, and the drugs developed on this basis, taken regularly to reduce the frequency of seizures or eliminate them altogether, are of proven therapeutic value. Following on from these acute seizure models, attempts have been made to replicate the processes by which human epilepsy develops and contin­ues as a chronic condition with spontaneous seizures, i.e. to model epileptogenesis by the use of models that show greater construct and face validity. This has been accomplished in a variety of ways (Table) in the hope that such models would be helpful in developing drugs capable of preventing epi­lepsy. Such models have thrown considerable light on the pathogenesis of epilepsy, but have not so far contributed significantly to the development of improved antiepileptic drugs. Because there are currently no drugs known to prevent epilepsy from progressing, the predictive validity of epileptogenesis models remains uncertain.

Psychiatric disorders

Animal models of psychiatric disorders are in general problematic, because in many cases the disorders are defined by symptoms and behavioural changes unique to humans, rather than by measurable physiological, biochemical or structural abnormalities. This is true in conditions such as schizophrenia, Tourette’s syndrome and autism, making face validity difficult to achieve. Depressive symptoms, in contrast, can be reproduced to

some extent in animal models, and face validity is therefore stronger. The aetiology of most psychiatric conditions is largely unknown2, making construct validity questionable.

Models are therefore chosen largely on the basis of pre­dictive validity, and suffer from the shortcomings men­tioned above. Nonetheless, models for some disorders, particularly depression, have proved very valuable in the discovery of new drugs. Other disorders, such as autism and Tourette’s syndrome, have proved impossible to model so far, whereas models for others, such as schizophrenia, have been described but are of doubtful validity. The best predic­tion of antipsychotic drug efficacy comes from pharmaco­dynamic models reflecting blockade of dopamine and other monoamine receptors, rather than from putative disease models, with the result that drug discovery has so far failed to break out of this mechanistic straitjacket.

Stroke

Many experimental procedures have been devised to produce acute cerebral ischaemia in laboratory animals, resulting in long-lasting neurological deficits that resem­ble the sequelae of strokes in humans. Interest in this area has been intense, reflecting the fact that strokes are among the commonest causes of death and disability in developed countries, and that there are currently no drugs that significantly improve the recovery process. Studies with animal models have greatly advanced our understanding of the pathophysiological events. Stroke is no longer seen as simple anoxic death of neurons, but rather as a complex series of events involving neuronal depolarization, activation of ion channels, release of excitatory transmitters, disturbed calcium homeostasis leading to calcium overload, release of inflammatory mediators and nitric oxide, generation of reactive oxygen species, disturbance of the blood–brain barrier and cerebral oedema. Glial cells, as well as neurons, play an important role in the process. Irreversible loss of neurons takes place gradually as this cascade builds up, leading to the hope that inter­vention after the primary event – usually thrombosis – could be beneficial. Moreover, the biochemical and cellular events involve well-understood signalling mecha­nisms, offering many potential drug targets, such as calcium channels, glutamate receptors, scavenging of reac­tive oxygen species and many others. Ten years ago, on the basis of various animal models with apparently good con­struct and face validity and a range of accessible drug targets, the stage seemed to be set for major therapeutic advances. Drugs of many types, including glutamate antag­onists, calcium and sodium channel blocking drugs, anti-inflammatory drugs, free radical scavengers and others, produced convincing degrees of neuroprotection in animal models, even when given up to several hours after the ischaemic event. Many clinical trials were undertaken, with uniformly negative results. The only drug currently known to have a beneficial – albeit small – effect is the biopharmaceutical ‘clot-buster’ tissue plasminogen activator (TPA), widely used to treat heart attacks. Stroke models thus represent approaches that have revealed much about pathophysiology and have stimu­lated intense efforts in drug discovery, but whose predic­tive validity has proved to be extremely poor, as the drug sensitivity of the animal models seems to be much greater than that of the human condition. Surprisingly, it appears that whole-brain ischaemia models show better predictive validity (i.e. poor drug responsiveness) than focal ischae­mia models, even though the latter are more similar to human strokes.

Zebrafish

The Zebrafish is a vertebrate animal model with high genetic homology to humans that gathers all these benefits simultaneously. They are often seen as a bridge between in vitro and in vivo models. Specifically, Zebrafish larvae offer a viable and cost-effective model that supports High Content Screening Assays without the ethical concerns typically associated with in vivo studies, as they are not classified as such when used under 5-6 days post-fertilization (dpf). Given these attributes, Zebrafish are increasingly becoming valuable in the Drug Discovery and Development process.

Mouse

Among mammals, the mouse is the most suitable for genetic analysis, which is facilitated by the availability of its complete genome sequence. Although the technical difficulties in studying mouse genetics (compared, for example, to the genetics of yeasts or Drosophila) are formidable, many mutations affecting mouse development have been identified. Most important, recent advances in molecular biology have enabled the production of genetically engineered mice in which specific mutant genes have been introduced into the mouse germ line, allowing the functions of these genes to be studied in the context of the whole animal. The suitability of the mouse as a model for human development is indicated not only by the similarity of the mouse and human genomes but also by the fact that mutations in homologous genes result in similar developmental defects in both species; piebaldism is a striking example.

Ex Vivo studies

Ex vivo, meaning out of the living in Latin, refers to experiments or procedures performed on tissues or organs extracted from a living organism and conducted outside the original biological context. These experiments are conducted in a laboratory setting, where the biological material is maintained in conditions that mimic those found within the organism as closely as possible.

The architecture, including cell-extracellular matrix (ECM) and cell-cell connections, is maintained, granting that metabolic processes are closer to the in vivo state than an in vitro model. 

Ex vivo models are frequently used for disease modeling, Drug Discovery and Development, regenerative medicine, and tissue engineering. Ex vivo and in vitro models represent an excellent alternative to animal testing. 

There are three critical differences between ex vivo and in vitro: 

·        Ex vivo approaches maintain a higher level of biological complexity by keeping the organ or tissue structure intact, whereas in vitro experiments are typically conducted with isolated cell(s).

·        In vitro experiments offer more control over external variables and are less complex and cost-effective, making them suitable for studying specific molecular or cellular mechanisms. These characteristics make them indispensable for high-throughput screening (HTS) of large libraries of compounds to identify potential Drug candidates, even Drug repositioning.  Ex vivo experiments, while still controlled, introduce more variables due to the complexity of tissue or organ systems.

·        Ex vivo methods offer more physiologically relevant insights into the whole organism than in vitro studies because they maintain some native interactions with tissues or organs. 

Note : Ex-vitro studies - In plant tissue culture, for example, ex vitro refers to the transition of plants or tissues from an artificial, sterile laboratory environment to a natural or greenhouse environment. 

Ex Vivo Models

Ex vivo in science refers to experimentation done in or on tissues obtained from an organism and maintained under optimum conditions mimicking the natural condition. The main advantage of ex vivo models lies in the fact that there are controlled conditions at all times, i.e., minimum alteration and variation in experimental models; secondly, tests and measurements that were not possible to be conducted in living subjects due to ethical issues could be easily carried out. The ex vivo model and experimentation includes assays, measurement of physical, thermal, electrical, mechanical, optical, and other tissue properties in varied conditions along with determining treatment against cancerous tissues with the help of various imaging techniques, etc.

Ex vivo models used for peptide studies mainly involves intestinal studies conducted to either measure their bioavailability, or secretion of mucus from goblet cells, or as simple as contraction of peptides present in intestine. This may be since intestine is the first main organ from where the basic metabolism, absorption, and distribution begins and thus is the main site of action for all nutrients in general. Peptides, proteins, and dairy hydrolysates alter the mucin secretion by changing the expression and number of goblet cells and thus influence the dynamics of mucus, for example, casein hydrolysates and β-casomorphin 7 increased mucin secretion in ex vivo preparations of rat jejunum (Claustre et al., 2002; Trompette et al., 2003). Another example is that of casoxin D (YVPFPPF), which showed opioid antagonistic action in a field-stimulated guinea pig ileum preparation (ileum contraction).

The other most widely used area of ex vivo models included role of peptide in cancer treatment and drug delivery. The studies have been conducted using endothelial cells. Systemic delivery of macromolecular proteins and peptide through pulmonary route could not be accessed accurately. For this purpose ex vivo model of isolated perfused lung has been used in various studies. It provided an accurate observation and separate analysis of lung absorption and nonabsorptive clearance, i.e., phagocytosis and/or metabolism, mucociliary clearance, etc. Such models have also been successfully used for pulmonary insulin studies in isolated perfused rat lung.

Another research area where ex vivo models are extensively exhausted involves assessment of phagocytosis and lymphocyte proliferation response to determine immunomodulatory response. For phagocytotic activity, macrophages isolated from peritoneal fluid are cocultured with pathogen along with BAPs to observe the enhancement or suppression in their activity as was seen in a study conducted in which β-casomorphin increased sheep RBCs’ phagocytosis by murine peritoneal macrophages elucidating their role as immunobooster. Thus ex vivo models play a pivotal role in research related to efficacy studies in ways mentioned previously.

One widely performed ex vivo study is the chickchorioallantoic membrane (CAM) assay. In this assay, angiogenesis is promoted on the CAM membrane of a chicken embryo outside the organism (chicken).

Ex vivo tibia model

In addition to primary cell isolation and cell lines, ex vivo models such as freshly-dissected and cultured long bones have proven useful in the study of the mechanical response of bone cells. For example, the activation of actin contractions by load-induced Ca^2 + signaling was confirmed through axial compressive loading on an ex vivo tibia observed via fluorescent microscopy. This system preserves the native bone environment, allowing both cell level and tissue level factors to play into early biochemical responses. Furthermore, this system can serve as a baseline method of analysis to address numerous biological alterations made in transgenic mice, for example. Of note, however, there are limitations in study duration of this ex vivo system since cells cannot be kept alive indefinitely, and there is also of course the absence of other organ systems influences (Fig.).

Ex vivo model - Cardiovascular Activity

PURPOSE AND RATIONALE

Prolonged administration of coronary drugs has been shown to increase the number and size of interarterial collaterals of dogs and pigs after coronary occlusion. An increased rate of development of collateral arteries was observed after physical exercise in dogs, as well as after chronic administration of coronary dilating drugs. An even more effective stimulus for collateral development is an acute or gradual occlusion of one or several major coronary branches. Filling the arterial coronary bed with a plastic provides the possibility to make the collaterals visible and to quantify them.

PROCEDURE

Dogs weighing 10–15 kg are anesthetized with pentobarbital sodium 30 mg/kg i.v. They are respirated artificially and the thorax is opened. After opening of the pericard, Ameroid cuffs are placed around major coronary branches. Gradual swelling of the plastic material occludes the lumen within 3–4weeks. The dogs are treated daily with the test drug or placebo. After 1 week recovery period they are submitted to exercise on a treadmill ergometer. After 6weeks treatment, the animals are sacrificed, the heart removed and the coronary bed flushed with saline. The liquid plastic Araldite is used to fill the whole coronary tree from the bulbus aortae. The aortic valves are glued together in order to prevent filling of the left ventricle. Red colored Araldite is used to fill the arterial tree. The venous part of the coronary vasculature can be filled with blue colored Araldite from the venous sinus. The uniformity of the filling pressure, the filling time, and the viscosity of the material are important. Polymerization is complete after several hours. Then, the tissue is digested with 35% potassium hydroxide. The method gives stable preparations which can be preserved for a long time.

EVALUATION

Plastic casts from drug treated animals are compared with casts from dogs submitted to the same procedure without drug treatment.

CRITICAL ASSESSMENT OF THE METHOD

The procedure allows impressive demonstration of the formation of arterial collaterals. The results of post mortem Araldit impletion agree with the functional results of experimental coronary occlusion.

Good laboratory practice (GLP) compliance in pharmacological studies

GLP comprises adherence to a set of formal, internation­ally agreed guidelines established by regulatory authori­ties, aimed at ensuring the reliability of results obtained in the laboratory. The rules (GLP Pocketbook, 1999; EEC directives 87/18/EEC, 88/320/EEC, available online: pharmacos.eudra.org/F2/eudralex/vol-7/A/7AG4a.pdf) cover all stages of an experimental study, from planning and experimental design to documentation, reporting and archiving. They require, among other things, the assign­ment of specific GLP-compliant laboratories, certification of staff training to agreed standards, certified instrument calibration, written standard operating procedures cover­ing all parts of the work, specified standards of experimen­tal records, reports, notebooks and archives, and much else. Standards are thoroughly and regularly monitored by an official inspectorate, which can halt studies or require changes in laboratory practice if the standards are thought not to be adequately enforced. Adherence to GLP stand­ards carries a substantial administrative overhead and increases both the time and cost of laboratory studies, as well as limiting their flexibility. The regulations are designed primarily to minimize the risk of errors in studies that relate to safety. They are, therefore, not generally applied to pharmacological profiling as described in this chapter. They are obligatory for toxicological studies that are required in submissions for regulatory approval. Though not formally required for safety pharmacology studies, most companies and con­tract research organizations choose to do such work under GLP conditions.

Lead Optimization

At this point, one or more promising drug candidate hits have been identified and are ready to be promoted to lead status. This entails verifying or optimizing the previously mentioned qualities necessary for the drug candidate to be pursued: ease of synthesis, adherence to the Lipinsky rules, target specificity, and efficacy against the disease.

Lead optimization Process

The lead optimization (modification) process involves improvement of pharmacokinetic (ADME) properties. The lead optimization process attempts to increase the desired therapeutic potency (therapeutic index) and to decrease toxicity of the parent molecule. The lead optimization process also offers specificity optimization and molecular modification of agonist (lead) to antagonist and vice versa. The lead optimization process involves the identification of the pharmacophore and the auxophore. The functional features of molecules responsible for the drug-receptor interaction and biological response is known as a pharmacophore. The molecular features responsible for the conformational change and molecular functions are considered as pharmacophoric part. The remaining molecular features (atoms and groups) maintain the molecular integrity and are called as auxophore. The removal of such groups abolishes the activity. Similarly, the molecular features responsible for altered (increase or decrease) potency refers to an auxophoric group.

Lead Optimization Approaches

Chemical manipulation of molecules enhances their therapeutic function. The structural geometry (size, shape and hydrogen bonding capacity) determines the structural complementary and the receptor binding. The molecular groups are responsible for the absorption, distribution and excretion pattern of the compound. Different lead modification approaches widely applied are listed below.

1. Functional group modification

2. Structure-activity relationship

3. Molecular modification

4. Molecular hybridization

5. Bio-isosterism

6. Stereochemical aspects

1. FUNCTIONAL GROUP MODIFICATION

In general, the polarity and ionization of compounds can be altered through functional group modification. The antibacterial agent, carbutamide was found to have anti-diabetic activity (side effect). Functional group modifications were utilized in the improvement of the anti-diabetic potential and to remove the antibacterial activity. The replacement of the amino group of carbutamide with the methyl group retained the anti-diabetic function and removed antibacterial potential. The molecule with methyl substitution is named as tolbutamide. The change of methyl group with chloro group and shortening of liphophilic alkyl side chain (butyl to propyl) generated another drug namely chlorpropamide. The chlorpropamide has extended half life and six fold increase in activity.

2. STRUCTURE ACTIVITY RELATIONSHIP

The size and shape of the molecules significantly contribute to biological activity. In most instances the therapeutic effect of molecules depend on their structure. This process involves collection and analysis of structural activity relationship (SAR) data. The incorporation and replacement of functional groups generates analogues with different size and shape, e.g., zolpidem.

The structural modifications produce newer analogues with altered pharmacokinetic as well as pharmacodynamic properties. Based on the structural dependence for the intended biological functions, drugs are classified into structurally specific and structurally non-specific drugs.

• Structurally specific drugs: The activity and potency of structurally specific drugs are very sensitive to even small change in chemical structure. The N-methyl derivative of morphine (natural alkaloid) produces skeletal muscle relaxant effect. Morphine has analgesic potential. The molecular function variation suggests the importance of structure and receptor specificity.

Structurally non-specific drugs: The drugs such as gaseous anaesthetics do not depend on their structures for their function. The activity of these molecules depends on their physicochemical properties. The gaseous anaesthetics (halothane, isoflurane and enflurane) produce their anaesthetic function by altering the membrane electrochemical gradient sensing potential.

Structural Flexibility and Rigidity: The molecules having several rotatable bonds adopt different conformations and influence the biological activity. The introduction of rigidity or flexibility in the molecular structure is the most common lead modification approach. The molecular conformation can be locked by connecting certain atoms/groups (cyclization). The altered conformation may enhance (agonist) or remove (antagonist) the biological activity of parent molecule. The conformation responsible for the desired pharmacological function is known as a bio-active conformation. The oxo-burimamide, oxo-analogue of thioburimamide shows no anti-ulcer function. The inter-molecular hydrogen bonding (rigidity) of oxoburimamide is reason for no anti-ulcer activity. The thio group (exchange for oxo group of oxo-burimamide) of thioburimamide would not establish hydrogen bond, hence elicits anti-ulcer activity.

The exchange of N,N-dimethylamino groups (acyclic) of chlorpheniramine into pyrrolidine (cyclic) group generated triprolidine. In this case, the rigid (cyclic) as  well as flexible (acyclic) molecules exhibit anti-histaminergic functions. The transformation of acyclic structural component into cyclic analogue may not necessarily affect the molecular function.

3. MOLECULAR MODIFICATION

Homologation: A process of increasing the molecular chain length of molecule by a constant unit (e.g., methylene). The increase or decrease in the alkyl group size (methylene shuffle strategy) influences the therapeutic potential. The molecular modification either increases or decreases the biological activity of molecules. The increase in carbon length in turn increases the molecular lipophilicity (membrane penetration) and the bioavailability. This offers enhanced therapeutic effect. The carbon chain length beyond certain level disturbs the optimal balance between lipophilicity and hydrophilicity. The imbalance in molecular properties decreases the bioavailability and therapeutic activity, (e.g., local anaesthetics).

In the development of second generation sildenafil derivatives with increased drug selectivity, the propyl group attached to the pyrazolo portion is exchanged with ethyl group. Similarly sildenafil was modified into vardenafil.

Chain branching: The branched alkyl chains have more lipophilic character than the corresponding straight alkyl chain (lower Log P). The chain branching also interferes with bioactive conformation of the molecules and receptor binding. The straight alkyl bridge in phenothiazine derivative of chlorpromazine produces antipsychotic function. In contrast, promethazine with branched alkyl bridge feature offers anti-histaminergic function.

4. MOLECULAR HYBRIDIZATION

The association of two individual molecules generate hybrid molecules (molecular hybridization). In several cases, this improves pharmacokinetic and pharmacodynamic functions compared with the individual effects of two drugs. For example, the hybridization of aspirin and paracetamol produced acetaminosalol. The hybrid molecules activate different targets and increase the therapeutic index. The association of identical pharmacophoric entities (drug duplication, triplication and tetraplication) produces more selective and potent therapeutic effects. This process is known as molecular replication and the example is di-aspirin.

5. BIO-ISOSTERISM

Replacement of atom or group of atoms in a molecule by another group with similar electronic and steric properties is an important lead optimization process. Two molecules or molecular fragments having identical number and arrangement of electrons are termed as isosteres.

CO and N

2

…….CO = 6 + 8 = 14;

N₂ = 7 + 7 = 14

These isosteres will have similar physicochemical properties in most instances. It induces modifications in size, shape, electronic distribution, chemical reactivity, liphophilicity and hydrogen bonding capacity of molecules. The existence of such phenomenon is termed as isosterism. The widespread application of the isosterism  concept to modify biological activity has given rise to the term bio-isosterism. It is an important tool in rational drug design (RDD) process. • Langmuir postulation (1919): The atoms or groups with the same number of valence electrons are considered as isosteres and the phenomenon as isosterism.

• Grimm’s hydride displacement law (1925): Grimm hydride displacement law states that the atoms belonging to group 4A, 5A, 6A and 7A in the periodic table change their properties by adding a hydride. These hydride added atoms are known as isoelectronic pseudo atoms. These pseudo atoms may exhibit similar bio-physicochemic properties.

Erlenmeyer concept (1932): Erlenmeyer broadened and refined Grimm’s concept. He proposed isosteres as atoms, ions and molecules with identical number of peripheral layer electrons. The elements in the same column of the periodic table are isosteric.

Bio-isosterism is a special process of lead modification. Bio-isosteric modification led to a fruitful yield of new and improved drug. The bio-isosteric approach requires rigorous and careful analysis.

• Friedman concept (1951): Friedman defined bio-isosteres as compounds similar to that of isosteres and having similar bioactivity.

• Thornber theory (1979): This theory states that the bio-isosteres are subunits, groups and molecules possessing similar physicochemical as well as bioactive properties.

• Alfred Burger hypothesis (1991): The compounds or groups possessing near-equal molecular shape and volumes are considered as isosteres. The similar electronic distribution offers similar physicochemical properties to the isosteres.

Classification

1.     Classic bio-isosteres: The bio-isosteric groups are classified based on their electronic features and valence electrons. (a) Monovalent groups (b) Divalent groups (c) Trivalent groups (d) Tetravalent groups (e) Ring equivalents

2.     Non-classic bio-isosteres (a) Cyclic vs non-cyclic (b) Functional groups (c) Retroisomerism

CLASSIC BIO-ISOSTERISM

Modification of Monovalent Groups

 Hydrogen bonding modification: The similar steric size, spatial arrangement and the hydrogen bond donor/acceptor nature is utilized in the lead modification process.

• Fluorine-hydrogen interchange: Hydrogen and fluorine are sterically similar, but differ in their electronegativity. 5-Fluorouracil (anticancer drug) and uracil (natural substrate for thymidylate synthase) is an example. In 5fluorouracil, the fluoro atom in the fifth position of uracil (in place of hydrogen in uracil) is responsible for the altered thymidylate synthase pathway. The negative inductive effect of fluorine atom, inhibits the formation of thymidine (thymidylate synthase product). This property offers anti-proliferative (anticancer) function to 5-fluorouracil.

Amino-hydroxyl interchange: The replacement of hydroxyl group of folic acid with amino group generates aminopterin (anti-bacterial). Tautomeric nature and hydrogen bonding capability of amino group of aminopterin (compared with hydroxyl group) facilitates the bonding with dihydrofolate reductase. This enzyme is essential for the cell growth function. Thus aminopterin competes with folic acid and inhibits enzyme binding. This enzyme inhibition in turn inhibits the folic acid metabolism and offers antibacterial activity.

Thiol-hydroxyl interchange: In 6-thioguanine (anticancer drug) the keto group of the guanine is exchanged with thio group. This change contributes to cell proliferation inhibition (anticancer).

• Grimm’s concept: The soft metabolic site (methyl group; -CH3) present in SC-58125 reduces their systemic concentration (reduced activity). Hence, the bio-isosteric approach of methyl (-CH3) group replacement with bioisosteric amino (-NH2) group generated celecoxib (selective and potent cyclooxygenase-II inhibitor).

Modification of Divalent Groups The divalent bio-isosteres include methylene (-CH2-), secondary amino (-NH-), ether (-O-) and thioether (-S-) functionalities. It also includes -C=N-, -C=O and -C=S. In all these groups, the bond angle and corresponding molecular conformations are nearly equal. These divalent bio-isosteres were utilized for eliminating the pharmacokinetic difficulties with retention of biological activity.

The bioisosteric replacement of divalent ether (-O-) group of procaine (local anesthetic) with secondary amine group (-NH-) generated procainamide (antiarrthythmic).

Modification of Tetravalent Groups

Replacement of quaternary nitrogen with tertiary carbon is classical example of this type. Carnitine acyl transferase inhibitors include replacement of the hydroxyl group of carnitine with amino and as well as quarternary nitrogen with carbon to get trimethyl ammonium group.

Ring Equivalents

Benzene and thiophene shares similar physicochemical properties and are known as ring equivalents. This inspired medicinal chemists to apply this concept in drug design.

Pyrazole-isoxazole-pyridine exchange: The replacement of pyrazole of celecoxib with isoxazole and pyridine produced more potent cyclooxygense-II inhibitors namely valdecoxib and etoricoxib.

Non-Classical Bio-Isosterism

• Cyclic vs non-cyclic: Local anaesthetic agents lidocaine and mepivacaine are best examples of this kind. The open chain lidocaine and closed ring structure in mepivacaine do not change their biological activity.

Functional groups: Diverse functional groups are known for their bioisosteric relationship. The angiotensin-II antagonist losartan was developed by exchanging carboxylic group of EXP 7711 (lead) with tetrazole group.

The exchange of keto group (-C=O) group of oxo-tolrestat with thio (-C=S) group generated more potent aldose reductase inhibitor namely tolrestat. The oxotolrestat is less active orally.

·       Retroisomerism: It involves the inversion of functional groups. The inversion of amide group of procainamide into anilide group generated more potent local anesthetic drug lidocaine.

6. STEREOCHEMICAL ASPECTS

The relative functional difference (defined structural features) of active molecules provided insight about the receptor. The spatial arrangement of atoms determines the receptor affinity and pharmacological response. The biological receptors (made up of L-amino acids) also exhibit chiral (steric) features and offers specific affinities for the binding. These chiral molecules are optically active molecules (stereoisomers) and include enantiomers, diasteroisomers and geometric isomers. The chiral nature of receptor demands for the chiral specific drugs.

Chirality: An atom attached with four different atoms or groups (substituents) bears asymmetric center (chiral center). The molecules with chiral centers are called as chiral compounds.

Enantiomers: The optical active isomer which rotate plane polarized light in right direction (clock wise) is known as a dextro-rotatory (d-isomer) and left direction (anti-clock wise) is known as a levo-rotatory (l-isomer). These stereoisomers are called as enantiomers, which show altered affinity for receptors, different binding energies and chemical properties. In case of chlorpheniramine (antihistamine), the dextro isomer is more potent than the levo isomer.

·     Eutomer: An active and potent enantiomer is termed as eutomer. e.g., dextrochlorpheniramine.

• Distomer: A less active enantiomer is termed as distomer. e.g., levochlorpheniramine. Eutomer for one pharmacological effect may be distomer for another pharmacological effect. The dextro isomer of propoxyphen (dextropropoxyphen) is analgesic and levo-propoxyphen is anti-tussive. In terms of analgesic function dextro-propoxyphen is eutomer and levopropoxyphen is distomer. Levopropoxyphen is eutomer for anti-tussive function.

• Hybrid drug: An enantiomer having different pharmacological potential through different molecular mechanism are termed as hybrid drug. An S(+) enantiomer of ketoprofen is non-steroidal anti-inflammatory agent, where as R(-) enantiomer is useful in the treatment of periodontal disease.

• Isomeric ballast: The distomer with undesirable effects (toxicity) are termed as isomeric ballast. The S-isomeric form thalidomide (hypnotic and sedative) produces teratogenic effects and is good example for isomeric ballast.

Diastereomers: The non-super imposable stereoisomers with more than one chiral center are called as diastereomers. The b-receptor antagonistic drug labetalol is an example. The R,R isomeric form of the drug shows selective b -receptor blockade effect, but the S,R isoform produce á-receptor blocking effect.

Racemic switch: The introduction of single enantiomeric form of the racemate drug is called as racemic switch (chiral switch). The single enantiomer drugs esomeprazole (S-omeprazole; antiulcer), levocetrizine (R-cetrizine; antihistamine) and levalbuterol (R-salbutamol; anti-asthmatic) are the examples.

Geometrical isomers: The restricted rotation around the double bond produce geometrical isomers (cis and trans; Z and E). The anti-histaminergic drug triprolidine and a synthetic analogue of oestrogen diethylstilbestrol are examples. Triprolidine is active only in Z-configuration (cis, H/Pyridyl). The E-isomeric form of diethylstibestrol produce required response.

Ex vivo ECG

Relative to in vivo ECG recording, ex vivo ECG procedures are very straight-forward; requiring no anesthetics, paralytics or external perfusion. In the absence of pericardial tissue, AgCl wire electrodes can be positioned in very close proximity to the heart and the ECG signal is easily measured using even rudimentary amplifiers (Lin et al. 2015), as illustrated in Fig. 3B. An obvious limitation of the ex vivo ECG is excision of the heart, which can be technically challenging. However, there are surgical procedures for rapid zebrafish heart isolation (Arnaout et al. 2014)(Lin et al. 2015). By approaching the heart from the cranial aspect or from the caudal aspect, these techniques limit the possibility of tissue damage caused by ventral incisions and can be completed in <5 min with minimal training.