Phenotypic Screening: Probing Efficacy and Mechanism of Action Simultaneously
Because the ultimate effect of drugs are the net result of on-target and less well understood off-target interactions, the assays traditionally employed in drug discovery and development usually suffer from at least one limitation. Due to their readout being so narrow, target-focused biochemical assays have a higher probability of failing to identify efficacious compounds. If a drug does indeed achieve the desired phenotypic result (such as killing a cancer cell) it will still go undiscovered if it operates via a mechanism different than what is being evaluated in the biochemical assay (i.e. when the compound has an off-target activity that is beneficial).
The focused nature of biochemical assays also precludes them from being able to identify detrimental off-target activity. In contrast, cell-based assays are less biased and can therefore effectively identify compounds with the desired phenotypic result, but usually do not provide information about the biochemical mechanism of action (MOA). xCELLigence® real-time cell analysis (RTCA) assays provide the best of both worlds: in addition to efficiently detecting whether a drug has an effect on cell health/behavior, specific features of the cellular impedance traces and kinetics can be predictive of MOA.
Highly Effective Evaluation of Drug Efficacy
RTCA provides a quantitative readout of cell number/proliferation rate, cell size/shape, and cell-substrate attachment quality. Because these physical properties are the product of thousands of different genes/proteins, RTCA casts a very wide net during drug screening. Everything from endothelial barrier function and GPCR antagonism to filopodia dynamics and immune cell-mediated cytolysis have successfully been analyzed on xCELLigence instruments. Despite the breadth of phenomena accessible with RTCA, sensitivity is in no way compromised. The gold microelectrodes used in ACEA’s E-Plates® cover ~75% of the well bottoms, enabling the simultaneous monitoring of very large numbers of cells – which provides exquisite sensitivity.
Beyond sensitivity, the efficacy of drug screening with xCELLigence is further enhanced by the continuous, real-time nature of data recording. Drugs display a broad spectrum in the rate at which they exert their effect on target cells. While some compounds (such as agonists of GPCRs or ion channels) cause immediate biochemical and cellular changes, other compounds display an effect at much later time points. Moreover, some drugs display multiple and kinetically distinct effects. For these reasons, traditional end point assays have a high probability of not identifying key features of drug responses. The fact that xCELLigence systems interrogate cell number, cell size/shape, and cell-substrate attachment quality continuously makes them extremely adept at evaluating drug efficacy. In short, end points are replaced by continuous data traces, so nothing is missed.
Drugs that impinge upon the same protein target or biochemical pathway typically effect similar phenotypic results. Consistent with this, the glucocorticoid receptor agonists hydrocortisone, betamethasone, and clobetasol all produce similar RTCA data traces (Figure 1). The same is true for the family of drugs that interfere with microtubule equilibrium/dynamics. Importantly, even though nocodazole and paclitaxel are structurally unrelated and influence different stages of microtubule formation, their RTCA data traces are nearly identical. This suggests that the unique impedance trace observed for this category of compounds is indicative of a common underlying global cellular response (i.e. the same cellular machinery is involved). Drugs that act as GPCR antagonists or calcium modulators also display RTCA traces similar to one another, yet different from other drug families. The RTCA trends highlighted in Figure 1 apply to many more compounds, and many more drug families, than just those shown here.
The mechanistic specificity of RTCA traces is highlighted by the fact that drugs which produce the same phenotypic outcome via different MOAs can readily be differentiated from one another. Both staurosporine and 5-fluorouracil induce apoptosis, but do so via different mechanisms. Staurosporine promiscuously inhibits protein kinases, while 5-fluorouracil inhibits DNA synthesis indirectly by blocking dTMP synthesis. When analyzed by RTCA these two compounds cause very distinct responses (Figure 2).
The above data suggest that in contrast to traditional screening methodologies that can only reveal whether a drug is efficacious, a single RTCA data traces can be used both to evaluate efficacy and to provide strong evidence for a specific mechanism of action. This capacity for predicting MOA is highlighted in Figure 3. While screening a 2,000 compound library for effects on the health/behavior of A549 cells, dibenzyltrisulfide was found to induce an RTCA data trace similar to that of epothilone B, which induces mitotic arrest by stabilizing microtubules. When subjected to biochemical characterization dibenzyltrisulfide was indeed found to disrupt the microtubule cytoskeleton, and to do this specifically by inhibiting tubulin assembly into microtubules.
Identifying Off-Target Activity
The inability of target-specific drug screening approaches to identify off target activity is a major obstacle and liability. The sensitivity of xCELLigence RTCA combined with the breadth of phenomena that it is able to observe make it possible to identify off target drug activities that previously went undetected. The long term cellular impedance response of A549 cells to s-trityl-cysteine and monastrol are nearly superimposable – which is expected in light of the fact that both compounds act as antimitotics by inhibiting the kinesin Eg5 motor protein (Figure 4). A significant difference between these two compounds is, however, observed shortly after treatment initiation. The monastrol-induced early/rapid drop in impedance resembles what has been observed in the RTCA profiles of compounds that modulate intracellular calcium levels. Subsequent analyses (the details of which can be read about here) verified that, monastrol possesses a previously unknown ability to block calcium uptake through Cav1.2 channels.
Key Benefits of xCELLigence for Phenotypic Screening
- The integrated, global response of cells to treatment is monitored – enabling a very wide net to be cast during drug screening. Cell number, size/shape, and attachment quality are the products of thousands of different genes and proteins. Monitoring these physical parameters simultaneously enables xCELLigence to efficiently identify compounds with biological activity.
- The kinetics of cellular responses can be predictive of compound mechanism of action.
- RTCA impedance traces can provide an early alert to possible off-target or toxic effects of a compound.
- Since real-time impedance assays are non-invasive and conducted under standard tissue culture conditions, at any time during an assay cells can still be harvested and analyzed by orthogonal methods to verify phenotype.