In an attempt to define the molecular basis of high phospho-FADD levels in tumor versus normal tissue (in HNSCC and lung cancer), we developed FKR, a reporter wherein FADD kinase activity, irrespective of the kinase involved, could be quantitatively and non-invasively monitored in living cells. This assay was adapted from our recently described reporter system for Akt also a Ser/Thr kinase,14
except that the Akt kinase substrate sequence was substituted with residues 142–208 of FADD which includes Ser194, the target for all known FADD kinases. We found that inhibition of FADD kinase activity through the use of siRNA or by small molecule resulted in a decrease in levels of phospho-FADD and a corresponding activation of the reporter’s bioluminescence activity in a dose and time dependent manner. The design of the reporter wherein inhibition of kinase activity would result in activation of the reporter (a positive signal) provides for numerous advantages. Firstly, the signal to noise is significantly improved and secondly, false positives are minimized (cytotoxic compounds would appear as false positives in an assay wherein inhibition of a kinase would result in loss of reporter activity). In contrast, cells expressing FKR-mut wherein a serine to alanine substitution was introduced (corresponding to Ser194 of FADD), inhibition of FADD kinase activity, did not result in a significant change in bioluminescence thereby demonstrating the specificity of the reporter.
The luciferase complementation reporter system described here possesses 1/100th
the activity of wild-type luciferase in the absence of complementation and as much as 1/10th
the activity of wild-type luciferase activity in the presence of complementation.16
Since the mutant reporter lacks the target phosphorylation site (Serine mutated to Alanine), it would be constitutively active and therefore possess a level of luciferase activity that is comparable to FKR in response to a FADD kinase inhibitor. In this regard, the mutant reporter possesses 1/10th
the activity of wild-type luciferase. The presence of compounds in the library that stabilize the half life of luciferase and/or enhance expression of the reporter by up-regulating its transcription or translation would appear as positive hits. Since these compounds exert their FADD kinase independent activities equally on the mutant and the wild-type reporter, a secondary screen would identify them as false positives. This makes the mutant reporter a useful tool in the identification of false positives from a high throughput screen.
Further validation of the reporter was provided by studies demonstrating inhibition of JNK resulted in a four-fold increase in bioluminescence and a corresponding decrease in serine phosphorylation of FKR, demonstrating that the reporter itself was a substrate for FADD kinases. Recent studies in our lab have demonstrated that phosphorylation of FADD results in its stabilization while inhibition of FADD phosphorylation results in its ubiquitin dependent degradation. Our studies using MG132, a proteosome inhibitor, have confirmed that a decrease in total FADD levels in response to FADD dephosphorylation (i.e. in the presence of a FADD kinase inhibitor) can be reversed by inhibiting proteosomal degradation. (Supplementary Figure 2
The FADD Kinase Reporter (FKR) allows for noninvasive monitoring of FADD-phosphorylation in live cells quantitatively and dynamically in real time. There are numerous advantages to screening compounds in a live cell assay as compared to a purified biochemical assay. These include the ability to evaluate drug-target interactions in a physiologically relevant context. For example, compounds unable to diffuse to the target site or are cytotoxic, go undetected, thereby leading to their immediate elimination from further investigation. In addition, the bioluminescence read out can be used quantitatively to determine optimal time points to measure the response of interest. While inherent toxicity is not detected in biochemical assays, live cell assays enable the measurement of short and long term responses, which helps rule out off-target effects such as toxicity to cells. Biochemical assays, however, are advantageous for high throughput screening due to parameters such as high sensitivity and the ability to define screening conditions.
Our data demonstrate that the A549 FKR signal to background ratio is independent of cell density within a given range as evidenced by the proportional increase in signal and background with increasing cell density. When optimizing an assay to a high throughput format, it is advantageous to incorporate flexibility into the screening protocol. Parameters including poor cell growth and differential seeding densities can affect assay results and reproducibility. However, our data demonstrates consistency in signal to background ratio independent of cell density and therefore allows for flexibility in cell plating conditions when conducting HTS. Treatment of cells with SP600125 (JNK inhibitor) results in a dose dependent increase in bioluminescence corresponding to a decrease in FADD kinase activity. The pharmacology of the assay can be used to measure day to day assay variability and to rank order compound hits according to potency. These optimization data were derived using the 96-well assay format; however optimization for the 384-well format has been conducted and can be found online as Supplementary Figure 1
The efficacy of FKR to selectively identify inhibitors of FADD kinase activity was demonstrated by the validation screen. The screen resulted in the positive identification of known FADD kinase modulators i.e. inhibitors of the MAPK pathway, previously shown to inhibit FADD phosphorylation in acute lymphocytic leukemia cells.17
Our positive control JNK inhibitor (SP600125), a known FADD kinase inhibitor, was also part of the compound set and demonstrated activity in the assay (). Notably, 10 μM JNK inhibitor as present within the compound collection demonstrated lower bioluminescent activity as compared to the 10 μM JNK inhibitor positive control wells. This is likely due to the compromised compound integrity as a result of multiple freeze thaw cycles. The ability of the assay to identify compounds that impinge on FADD phosphorylation from a diverse set of small molecule kinase inhibitors demonstrates the utility of the assay at selectively identifying compounds that modulate FADD kinase activity. However, the ability of FKR to identify FADD kinase inhibitors as well as compounds involved in upstream signaling events suggests the need for secondary screens.
Our current experience has identified two major sources of false positives against FKR. The first being compounds with inherent luminescence and the second source are compounds that modulate signaling events upstream of the target. The former can be eliminated with the FKR-mut reporter. Resolution of the latter, i.e., how to identify compounds specific to the inhibition of one of the known FADD kinases as opposed to upstream events, would require a purified system. However, the ability of the screen to identify upstream signaling events could be beneficial in identifying signaling hubs that impinge on the target kinase.
Identifying compound hits from a high throughput screen with desirable pharmacological properties requires tedious PK/PD and ADME/Tox studies and is both time consuming and labor intensive. The in vivo results presented here allows for the identification of compounds with desirable pharmacologic properties by rapidly evaluating drug target interactions in vivo thereby providing preliminary insight as to whether the drug is a potential therapeutic agent. The FKR cell-based assay platform will provide rapid identification of lead compounds from HTS, as well as providing in vivo validation of active compounds identified by HTS by rapidly validating drug target interaction and dose and schedule optimization.