Compound toxicity is an issue that is often addressed later in the drug development process than potency. However, because project costs increases rapidly with time, late stage failures caused by unexpected toxicity are undesirably expensive. It would be greatly advantageous to include some measure of compound toxicity earlier in the drug discovery process. Assays that could help rank order "hits" from HTS and early medicinal chemistry efforts would save time and cost derived from pursuing leads with serious cellular toxicity liabilities. In addition, ready access to structure-activity relationships regarding compound-related toxicity would be useful in directing medicinal chemistry efforts to reduce the toxic aspects of a compound as well as improving on other desired properties.
The impairment of critical cellular functions can result in systemic toxicities such as those associated with the neuromuscular system (tremor, cardiac arrhythmia, and paralysis) . renal (microtubule function), and vasculature (leaky blood vessels). A noteworthy example is the acquired long QT syndrome (LQTS) associated with blockade of cardiac ion channels (14-16). LQTS results in cardiac arrhythmias, torsade de pointes, ventricular fibrillation, and can lead to sudden death. One such ion channel is the HERG protein, respon sible for the rapid component of delayed rectifier K+ current in the myocardium (17). Several different mutations in HERG are known to cause inherited LQTS (18). A large number of drugs have been found to elicit adverse side effects through inhibition of the HERG channel, thereby inducing LQTS (19). Recent evidence suggests that two key residues, Y652 and F656, may be responsible for the promiscuity of drug binding by the HERG channel (20). This serious and sometimes fatal cardio-toxicity has led to withdrawal of otherwise promising drugs from the marketplace (21), for example, the antipsychotic agent pimozide and the gastric reflux medication cisapride. Some estimates indicate that up to one-half of all compounds under review for market approval may elicit LQTS side effects through the HERG channel. The FDA now recommends that all drugs be screened against the HERG channel before release to market.
Developing useful assays for screening drug effects against the HERG channel has become extremely important to the pharmaceutical industry. Drug screening in humans or animals is most physiologically relevant. However, this method is not always ethical, it is very low throughput, and it is difficult to identify the molecular target of any drug interactions. The patch-clamp method has proven very powerful in its sensitivity and ability to evaluate channels at the single molecule level. However, patch-clamp technology is expensive, difficult, low throughput, and far removed from a physiologically relevant context. Cell culture-based assays have been developed that use fluorescent dyes sensitive to plasma membrane potential in conjunction with plate readers and flow cytometry. These assays have proven to be high throughput but often suffer from decreased sensitivity or difficulty in maintaining cell lines. The need exists for a sensitive, high-throughput, cost effective, and physiologically relevant HERG assay.
Interaction of compounds with a toxicity target can result in a wide variety of in cell phenotypes (22). Deregulation of gene expression can result in inappropriate cell division (neoplasia, teratogenesis), apoptosis, and protein synthesis (e.g., peroxisome proliferation). Cell death can result from stimuli resulting in apoptosis, which has been defined as pro grammed cell death, and involves a cascade of events, including caspase activation leading to cytochrome C release from the mitochondria and subsequent degradation of chromosomal DNAinto distinct fragments, formation of cy-toplasmic vacuoles, and plasma membrane and nuclear blebbing (23, 24). Apoptotic cells are removed by macrophages in response to the signals such as the exposure of phosphati-dylserine to the outside of the cell before complete loss of plasma membrane integrity. Necrosis is another classical cell death pathway in which cells lose membrane integrity, swell and burst, and spill their contents into the extracellular space. These cellular components often elicit an inflammatory response leading to further damage to surrounding tissues. Cellular factors determining which pathway a cell follows in response to toxic exposure include caspase activity, degree of ATP depletion, extent of intracellular Ca2+ increase, levels of reactive oxygen species, and rate and extent of thiol oxidation (24, 25). Additionally, factors such as cell cycle, cell type, duration of exposure, active efflux mechanisms,and compound metabolism may influence the response of cells to toxic agents. Together, the characteristics of these apoptotic, necrotic, and metabolic pathways and environmental factors form a continuum of morphological and biochemical indicators of cell death. Thus. the term "cytotoxicity" is somewhat imprecise, and it would be more useful to be able to readily characterize the toxicity mechanism for hit and medicinal chemistry compounds than simply to classify cells as "alive" or "dead."
Apoptosis can be initiated through TNF receptor family signaling coupled to caspase activation. Other apoptosis triggers included pharmacological agents (staurosporine, Ca2+ ionophores, thapsigargin), DNA damaging agents, and a variety of chemical toxicants (24). Assays that measure mitochondrial function and cell viability and growth, cell membrane integrity, membrane potential, intracellular Ca2+, ATP, reduced glutathione concentration, and intracellular pH are useful indicators of cell-based toxicity. The tetrazo-lium salts such as XTT, MTT, WTS, and others are reductase substrates that are reduced in the mitochondria of living cells to colored formazan dyes readily detected by light absor-bance. The colored species generated is proportional to the number of viable cells. Cellular DNA synthesis can be determined by tritium-labeled thymidine (radiometric detection) or bromo-deoxyuridine (antibody detection) incorporatio'n and thus is a direct measure of cell proliferation and can be related to toxic and cytostatic effects of test compounds on proliferating cells. Dyes that do not cross the plasma membrane, such as trypan blue and propidium iodide (PI), are excluded by cells with intact membranes and are an indicator of cell viability. Dye exclusion is readily measured by direct cell counting on a hemocy-coulter counter, or flow cytometer.
Flow cytometer-based assays for several other apoptotic indicators have been developed. Changes in intracellular ion levels, in particular Ca2+ and H+, are considered good early indicators of compound-induced cellular toxicity. Elevated Ca2+ levels may be important in apoptosis by activating nuclease activity. Intracellular pH and Ca2+ levels are readily determined with a variety of H+- and Ca2+-sensitive fluorescent probes (26-28). Assays for reactive oxygen species and cellular free glutathione content, both indicators of apoptosis, are available. In apoptosis, as part of the signaling to macrophages, phosphyti-dylserine (PS) "flips" from the inner to outer side of the cell membrane. Annexin V binding to PS on the outer membrane is a characteristic of early stage apoptosis (29).DNAfragmen-assays are also used to discriminate ne-crotic from apoptotic cells (30, 31). End labeling of the fragmented DNA followed by staining yields signal indicative of the characteristic DNA fragmentation pattern (32). An advantage of measuring several cytotoxicity endpoints simultaneously is that dose and mechanistic properties of moderately and highly toxic compounds are discriminated. These properties are best determined in single-cell analysis as described in the next section.
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