Compound toxicity is an issue that is often addressed later in the drug development pro- cess than potency. However, because project costs increases rapidly with time, late stage failures caused by unexpected toxicity are un- desirably expensive. It would be greatly ad- vantageous to include some measure of
pound toxicity earlier in the drug discovery process. Assays that could help rank order "hits" from HTS and early medicinal
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 re- duce the toxic aspects of a compound as well as improving on other desired properties.
The impairment of critical cellular
tions can result in systemic toxicities such as those associated with the neuromuscular sys- tem (tremor, cardiac arrhythmia, and paraly- sis). renal (microtubule function), and
lature (leaky blood vessels). A noteworthy example is the acquired long QT syndrome (LQTS) associated with blockade of cardiac ion channels LQTS results in cardiac arrhythmias, torsade de pointes, ventricular fibrillation, and can lead to sudden death. One such ion channel is the HERG protein,
Rapid, High Content Pharmacology
sible for the rapid component of delayed recti- fier current in the myocardium (17). Sev- eral 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 chan- nel, thereby inducing LQTS (19). Recent evi- dence suggests that two key residues, Y652 and may be responsible for the promis- cuity of drug binding by the HERG channelThis serious and sometimes fatal
toxicity has led to withdrawal of otherwise promising drugs from the marketplace
for example, the antipsychotic agent pimozide and the gastric medication cisapride. Some estimates indicate that up to one-half of all compounds under review for market ap- proval may elicit LQTS side effects through the HERG channel. The FDA now recom- mends 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 pharma- ceutical 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 in- teractions. The patch-clamp method has proven very powerful in its sensitivity and ability to evaluate channels at the single mol- ecule level. However, patch-clamp technology is expensive, difficult, low throughput, and far removed from a physiologically relevant con- text. Cell culture-based assays have been de- veloped 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 or
diffi-
culty in maintaining cell lines. The exists for a sensitive, high-throughput, cost
and physiologically relevant HERG assay. Interaction of compounds with a toxicity target can result in a wide variety of in cell phenotypes Deregulation of gene expres- sion can result in inappropriate cell division (neoplasia, teratogenesis), apoptosis, and pro- tein synthesis peroxisome proliferation). Cell death can result from stimuli resulting in apoptosis, which has been defined as
grammed cell death, and involves a cascade of events, including caspase activation leading to
C release from the mitochondria and subsequent degradation of chromosomal DNA into 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
dylserine to the outside of the cell before com- plete loss of plasma membrane integrity. Ne- crosis is another classical cell death pathway in which cells lose membrane integrity, swell and burst, and spill their contents into the tracellular space. These cellular components often elicit an inflammatory response leading to further damage to surrounding tissues. Cel- lular factors determining which pathway a cell follows in response to toxic exposure include caspase activity, degree of ATP depletion, ex- tent 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 expo- sure, active efflux mechanisms, and compound metabolism may influence the response of cells to toxic agents. Together, the character- istics of these apoptotic, necrotic, and meta- bolic pathways and environmental factors form a continuum of morphological and bio- chemical 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 re- ceptor family signaling coupled to caspase ac- tivation. Other apoptosis triggers included pharmacological agents (staurosporine,
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, in- tracellular ATP, reduced glutathione concentration, and intracellular pH are useful indicators of cell-based toxicity. The
lium salts such as XTT, MTT, WTS, and oth- ers 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 pro- portional to the number of viable cells. Cellu- lar DNA synthesis can be determined by tritium-labeled thymidine (radiometric detec- tion) or bromo-deoxyuridine (antibody detec- tion) incorporatio'n and thus is a direct mea- sure of cell and can be related to toxic and effects of test compounds on proliferating cells. Dyes that do not cross the plasma membrane, such as blue and propidium iodide (PI), are excluded by cells with intact membranes and are an indi- cator of cell viability. Dye exclusion is readily measured by direct cell counting on a
coulter counter, or flow cytometer. Flow cytometer-based assays for several other apoptotic indicators have been devel- oped. Changes in intracellular ion levels, in particular Ca2+ and H+, are considered good early indicators of compound-induced cellular toxicity. Elevated levels may be impor- tant in apoptosis by activating nuclease activ- ity. Intracellular pH and levels are readily determined with a variety of and fluorescent probes As- says for reactive oxygen species and cellular free glutathione content, both indicators of apoptosis, are available. In apoptosis, as part of the signaling to macrophages,
dylserine "flips" from the inner to outer side of the cell membrane. V binding to PS on the outer membrane is a characteris- tic of early stage apoptosis (29).
assays are also used to discriminate ne- crotic from apoptotic cells (30, End labeling of the fragmented DNA followed by staining yields signal indicative of the charac- teristic DNA fragmentation pattern 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 gle-cell analysis as described in the next sec- tion.
6 HT-FLOW
Many of the cell-death assays described above are readily performed by flow cytometry
Rapid, High Content
(FCM), and can be combined to yield multipa- rametric assays that measure several comple- mentary indices of toxicity. The advantage of single cell multiparameteric analyses includes the ability to test mixed cell populations, such as those obtained upon cell differentiation. Si- multaneous determination of mitochondrial membrane potential, reactive oxygen species generation, and intracellular glutathione by FCM were used to show that loss of mitochon- membrane potential and glutathione de- pletion are nearly simultaneous and could not be uncoupled (33). In particular, while not lim- ited to FCM, determination of light scattering properties of cells yields information that is not available in standard 96-well plate reader assays. Forward scatter is commonly used to measure cell size and decreases early in apo- ptosis while side-scattered light is propor- tional to cellular granularity and often in- creases during apoptosis (34). Thus, FCM has advantages over conventional plate-based as- says with regard to multiplexing, readouts such as light scatter, analysis at the single cell level, and high sensitivity. FCM also provides unique advantages for mixtures of cell types. Differential analysis of cellular responses to test compounds is possible because the differ- ent cell types or differentiation states can be tagged with specific cell-surface markers. Neuronal cells can be derived through differ- entiation of precursor cells, and these can be stained for the presence of neural cell adhe- sion molecule. FCM is therefore well suited to measure the multiplicity of events that occur when cells respond to toxic agents and gives a wealth of information that can be used to iden- tify the mechanism of compound action and direct chemical optimization efforts accord- ingly.
Drug discovery and development follow-up to a typical HTS program requires the assay- ing of hundreds to thousands of compounds identified in screening, or cherry picked by structural similarity to original hits and gen- erated by focused combinatorial chemistry around active compounds. One major disad- vantage of FCM is the need to handle each sample manually and the resulting low throughput. We have previously reported a high-throughput sample delivery system for FCM (34). The data were collected on a Cyto-
Wells
Figure 3.3. Each vertical strip in the top pane from a n well of a 96-well plate with fluorescent beads. Two rows, 24 wells, of plate are shown. The bottom panel represents a o dimensional histogram for a single well. Data collected on a Cytomation MoFlo FACS equip] with a Moskito autosampler.
MoFlo FCM equipped with a Mosik autosampler, which is capable of
96-well plate in min. Each vertical
the upper panel of Fig. 3.3 shows the side sc ter for a single well and the lower panel is
histogram for a single sample. throughput compound screening by flow
provides both a novel set of issues assay cost, compound usage, di handling, and data interpretation and no cell pharmacology assays. All of these are under consideration in the developmenl the next generation of instrumen Introduction of compounds to cells in fl provides another dimension to standard fl cytometry. The HTPS system described e lier has been adapted for flow cytometry a increases the information content and pands the repertoire of assays, including hi content toxicology data, available to the ph macologylmedicinal chemistry drug
ment team. Early physiological
such as changes in pH, intracellular calciu and membrane potential can be monitor and this can be coupled to cell-type spec
is ded the ne- in re- sta vel of ow ow ar- ex- igh ar- ses ific
Summary
markers, allowing assays to be performed on mixed cell populations. The HT-flow system has been used to measure cellular responses to test compounds at rates of 3-4 compounds per minute as well as determine the relationship between receptor occupancy and cell response
7 CONTENT METABOLIC ASSAYS