A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.


Introduction
A critical component of small molecule drug discovery is determining and understanding ligand-protein interactions (target engagement) at the site of drug action in the cell. For a large number of potential drug targets, "classical" approaches (e.g. monitoring changes to substrate or product generation) are not amenable. The cellular thermal shift assay (CETSA) first described by Martinez Molina et al [1] has become frequently used in target engagement studies. The assay relies on the principle that ligand binding results in thermal stabilization (or sometimes destabilization) of the bound protein. Practically, the CETSA method measures the amount of soluble protein remaining in cells following heating at various temperatures in the absence or presence of a ligand. The classic method [1,2] relies on treating cells with ligand and then heating in suspension at relatively high densities (of the order of 1-3x10 7 /mL) in a thermocycler. Following cell lysis, cell debris as well as aggregated and precipitated proteins are removed and the remaining soluble protein detected by, for a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 example, western blotting or homogenous detection methods (e.g. AlphaScreen, ELISA, referred to as HT-CETSA etc.) [2,3]. As this method does not rely on modification of either the target or an interacting ligand, it can in theory, be applied to any target in any cellular system. A recent advance has seen the application of high-resolution mass spectrometry to the whole proteome enabling not only the measurement of desired on-target effects but also the identification of potential off-target liabilities [4][5][6][7].
For adherent cells, the requirement to heat the cells in suspension at high density is an obvious drawback and the process of trypsinization and resuspension may alter cellular physiology and target pharmacology. Additionally, having to treat cells at high cell densities may result in an underestimation of target engagement potency and make comparisons to downstream pharmacology assays more difficult. As the CETSA method determines the amount of soluble, folded protein remaining, we hypothesized that heating cells growing attached to a 96 well plate (96WP) and determining changes in the amount of target protein still correctly folded by high content immunofluorescent imaging might be a useful adaption of the CETSA method for adherent cells. We have therefore developed a novel cellular target engagement assay in adherent live cells using the principle of ligand-induced changes to protein thermal stability coupled with high-content single cell immunofluorescent imaging in an attempt to mitigate some of these potential liabilities.

Cell lines and cell culture
HT29 and U2OS cells were purchased from the ATCC and grown in DMEM or McCoys 5a media supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin / streptomycin at 37˚C and 5% CO 2 in a humidified incubator. Cell lines were established as a low passage cell bank and then routinely passaged in our laboratory for less than 3 months after resuscitation. Authentication was by STR profiling (LGC Standards).

Plate heating and temperature determination
Temperature changes in 96 well plates or PCR tubes was determined with two K type hermetically sealed thermocouples (TWHSEK00001M0AP7, Sterling Sensors, UK) connected to a 2TC dual-channel thermocouple thermometer WIFI measuring module (Papago). Temperatures were logged using SNMP Data Logger software (AGG Software). The K type thermocouples and water bath temperatures were calibrated with a pre-calibrated Fisher digital thermometer (11719715, Fisher Scientific Traceable).
Plates were heated as follows: (i) sealed with adhesive aluminum PCR plate foil and then floated or submerged in a water bath (Grant Instruments W14) containing water pre-heated to the desired temperature or (ii) 1 L of DPBS containing magnesium and calcium (D1283, Sigma) was placed in a clean water bath (SUB6, Grant Instruments) and heated to the required temperature. The media was removed from the wells of the CellCarrier plate and the plate submersed in the DPBS so that all wells were full of DPBS and the plate was not touching the bottom of the water bath. The plate was incubated in the DPBS for 3 minutes, removed, the hot DPBS removed and replaced with 100 μL room temperature DPBS and then incubated at room temperature for 5 minutes. In some experiments, the DPBS was substituted for live cell imaging solution (LCIS, 20 mM Hepes, 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , pH 7.4).
For temperature determination experiments, the thermocouples were placed in two different wells (one edge, one center of plate) and the hole sealed with blu-tack and parafilm (S1

Reagents and antibodies
Chk1 inhibitors were provided by Vernalis Research or purchased from Selleckchem and prepared as a 20 mM stock in DMSO, aliquoted and stored at -20˚C. The same DMSO stock was used for all experiments. The antibodies and the dilutions used in these studies are listed in Table 1. The selectivity of the Chk1 antibodies was confirmed using an ON-TARGET plus Human CHEK1 SMART pool siRNA (GE Dharmacon) transfected with Lipofectamine RNAi-MAX (Thermo Fisher Scientific).

Single cell immunofluorescence
Single cell immunofluorescent imaging was conducted as previously described [8,9] in Cell-Carrier 96WP or CellCarrier Ultra 96WP (6005550 or 6055300, Perkin Elmer) and imaged on an Operetta high content imager (Perkin Elmer) using a 10, 20 or 40x objective then analyzed using Harmony software (Perkin Elmer).

CETSA by immunofluorescence (HCIF-CETSA)
10 000 cells were plated per well of a CellCarrier Ultra 96WP (6055300, Perkin Elmer) and allowed to adhere overnight. Compounds were serially diluted in DMSO to 200-times the final assay concentration then diluted 1:200 in media (without FCS). The cell media was removed and replaced with 25 μL of compound in media and incubated at 37˚C, 5% CO 2 for 10 minutes. The plate was sealed with an aluminum plate seal and heated by floating in a pre-heated water bath (Grant Instruments W14) for 3 minutes. 75 μL of room temperature media (without FCS or compound) was added and the plate incubated at room temperature for 5 minutes. Cells were fixed by the addition of 100 μL paraformaldehyde (7.4% in PBS) at room temperature for 15 minutes. Cells were washed 3 times with PBS and Chk1 detected with anti-Chk1 rabbit EP691Y monoclonal antibody (Ab40866, Abcam) and an AlexaFluor488 anti-rabbit secondary antibody (A-11034, Thermo Fisher Scientific). Plates were imaged on an Operetta high content imager using a 20x objective and analyzed using Harmony software.

Methods for effectively heating cells in an imaging compatible 96 well plate
The standard CETSA method (CETSA classic) utilizes a thermocycler (or alternatively a water bath) and thin-walled PCR tubes to ensure the rapid, consistent heating and subsequent cooling of the sample. 96WPs suitable for adherent cell growth and high content imaging were incompatible with heating in a thermocycler. We therefore evaluated alternative methods of heating adherent cells in imaging compatible plates. To accurately monitor in well temperature changes, two hermetically sealed, wire k-type thermocouples (approximately 2 x 1mm) coupled to a Papago thermometer WIFI module and data logging software was utilized (S1 Fig).
The relatively small dimension of the thermocouple allowed temperature changes in 200 μL volumes to be measured accurately and reproducibly every approximately 0.5-1 second when coupled with the Papago WIFI thermometer. We initially determined the maximal temperature achievable in a sealed CellCarrier 96WP containing 200 μL PBS per well either submerged or floated on top of a pre-heated water bath. The in well temperature achieved in the submerged plate was equivalent to that of the heating water bath and linear over the range of temperatures tested (R 2 0.999, Fig 1A). In comparison, the temperature achieved in the floating plate was consistently lower (ΔT 1.9-4.7˚C) than that of the heating water bath. Like the submerged plate, the relationship between water bath temperature and in well temperature was linear over the range of temperatures tested (R 2 0.999). With either heating method, there was no difference in the in well temperature between a central and an edge well.
The time required to reach the target heating temperature was subsequently determined in a thin-walled PCR tube floating in a pre-heated water bath. The PBS in the PCR tube heated up rapidly reaching a T max-1˚C in 44-52 s ( Fig 1B and Table 2). The time required to reach T max-1˚C was 3-and 5.5-fold slower in a CellCarrier plate submerged or floating in a preheated water bath. Evaluation of the heating properties of different 96WP types (CellCarrier Ultra, Isoplate or round-bottomed) revealed similarly slower time-to-target temperature than observed with a PCR tube (Table 2). Likewise, heating the plate on an aluminum dri-block did not improve the heating performance compared to water bath heating.
To improve the rate of cell content heating, we evaluated the in well temperature-time response profile of an empty, unsealed CellCarrier plate in a water bath containing pre-heated PBS. In this scenario, the in well temperature increased rapidly from 22˚C to 52˚C in 9.8 ± 1.7 s (n = 4, mean ± SD). The temperature then increased slowly over the next 170 s reaching a temperature approximately 2˚C (56.9 ± 0.9˚C) higher than the water bath set temperature of 55˚C ( Fig 1C). Addition of pre-heated PBS at 55.7˚C to an empty CellCarrier plate at room temperature resulted in a maximal in well temperature of 45.9 ± 0.3˚C (n = 3), a temperature loss of 9.9 ± 0.3˚C.
Finally, we evaluated the effectiveness of a standard domestic microwave (of the kind found in may laboratories used to heat agar, agarose etc.) to heat 200 μL PBS in a CellCarrier plate. The maximum temperature achieved in a given well following various heating times with a low power setting was highly variable and non-linear with time ( Fig 1D). Additionally, the maximum achieved temperature varied considerably between a central and edge well. Microwave heating was deemed to variable and therefore unsuitable for accurate, reproducible plate heating. We found that immersion of the open plate in pre-heated PBS generated a heating profile that most closely resembled the thermocycler / PCR tube though this too was not without issues. Ideally, a thin-walled 96WP that fits in a thermocycler with a thin, flat bottom compatible with imaging applications is required.

V158411 does not increase Chk1 thermostabilization in intact cells following immersion in pre-heated PBS
Previous work has demonstrated that the selective Chk1 inhibitor V158411 [10] stabilizes Chk1 in HT29 and U2OS cells following heating using the CETSA classic method and western blot detection [11]. As CETSA detects the amount of folded, soluble protein remaining following cell heating, we hypothesized that immunofluorescence and high content imaging may be a viable adaptation of the CETSA method for adherent cells. Based on the data obtained in Fig  1 and Table 2, the PBS immersion heating method was selected as the method of cell heating. Heating U2OS cells in 55˚C PBS for 3 minutes followed by room temperature cooling for 5 minutes and subsequent fixation with formaldehyde (PFA) resulted in a dramatic loss (92% reduction) in cells remaining adhered to the plate bottom (Fig 2A). This was not improved by the use of poly-D-lysine coated plates or an alternative heating buffer (LCIS). In comparison, only 12% of the cells were lost when plates were fixed with either PFA followed by 100% methanol or 100% methanol alone.
Chk1 expression was determined by immunofluorescent (IF) imaging with six different anti-Chk1 antibodies ( Fig 2B and Table 1). Chk1 was found to be predominantly nuclear for the majority of antibodies following PFA, PFA then MeOH or MeOH alone staining. Cellular heating to 55˚C by PBS immersion reduced the Chk1 nuclear intensity following IF staining with 2G1D5, G-4, EP691Y and FL-476 antibodies with the greatest differential between heated and unheated cells observed following PFA then MeOH fixation (Fig 2C). PBS immersion heating left the cells fragile with obvious cellular damage (S2 Fig). We have, however, not exhausted all potential heating matrices / buffer and alternatives may prove more suitable.
The mean nuclear Chk1 intensity in U2OS cells was relatively weak. Comparison to HT29 cells revealed an approximately 2-fold greater Chk1 IF intensity in HT29 cells compared to U2OS cells following staining with either the G-4 or EP691Y antibodies ( Fig 3A). To confirm that the G-4 and EP691Y anti-Chk1 antibodies were detecting Chk1 following PFA then MeOH staining, Chk1 was knocked down with a SMARTpool CHEK1 siRNA. Knockdown of Chk1 in HT29 cells with siRNA reduced the mean nuclear Chk1 intensity by 3.8-fold following Chk1 IF detection with EP691Y ( Fig 3B). In comparison, the CHEK1 siRNA had no effect on the mean nuclear Chk1 intensity in HT29 cells stained with G-4 suggesting that this antibody was not detecting Chk1. All future IF studies were therefore conducted using the EP691Y rabbit monoclonal anti-Chk1 antibody.
The TFI 50 (the temperature that reduced the fluorescence intensity to halfway between the baseline and maximum), was 47.1˚C for Chk1 in HT29 cells heated by the PBS immersion method (Fig 3C and Table 3). This was 3.2˚C lower than that determined using the thermocycler heating of HT29 cells in suspension and Chk1 detection by western blotting ( [11], Table 3). Heating HT29 cells to 46, 49, 52 or 55˚C by immersion in pre-heated PBS reduced the mean nuclear Chk1 intensity measured by IF (Fig 3D). Treating the cells with 1-10 μM V158411 for 30 minutes prior to PBS heating did not alter the mean nuclear Chk1 intensity.

Effects of Chk1 activation by hydroxyurea on Chk1 thermostabilization by V158411
Heating HT29 cells to temperatures between 49 and 55˚C by immersion of a sealed CellCarrier 96WP in a pre-heated water bath did not reduce the mean nuclear Chk1 intensity measured by IF (Fig 4A). As the time-to-temperature was substantially longer for the sealed heating method than for PCR tubes, the plates were heated for 8 minutes rather than 3 minutes.  Chk1 kinase is activated in response to replication stress and DNA damage via the ATR dependent phosphorylation of serine 317 and 345 [12]. In the absence of S317/S345 phosphorylation, the ATP binding site of Chk1 is occluded rendering the kinase catalytically inactive [13,14]. Hydroxyurea (HU) inhibits ribonucleotide reductase thereby decreasing the production of deoxyribonucleotides leading to increased replication stress, S-phase arrest and Chk1 activation [15]. Treatment of HT29 cells with 2.5 mM HU for 18 hours increased Chk1 activation as evidenced by increased Chk1 phosphorylation on S296, S317 and S345 (Fig 4B) with 71.4 ± 0.7% (n = 6 wells ± SD) of HU treated cells exhibiting evidence of S-phase arrest (increased nuclear area, DNA content >G1, <G2) compared to 2.5 ± 1.0% of untreated cells (Fig 4C). V158411 also increased Chk1 activation but to a lesser extent than HU treatment. V158411 inhibited Chk1 autophosphorylation in HU treated and untreated cells suggestive of Chk1 target engagement under both treatment regimens.
Activation of Chk1 with HU allowed the evaluation of two additional antibodies that specifically detect active Chk1 [16]. To evaluate the effect of HU treatment on Chk1 thermostability in the absence or presence of V158411, HT29 cells were treated with 2.5 mM HU for 18 hours followed by 10 μM V158411 for 30 minutes then heated by submerging the sealed plate in a pre-heated water bath set to 55˚C. A small change in nuclear levels of pS317 and pS345 detected by IF following HU and V158411 treatment prior to heating were observed. HU treatment induced a 1.9-fold and 1.6-fold increase in nuclear Chk1 pS317 and pS345 respectively ( Fig 4D). Heating the cells did not alter the nuclear levels of Chk1, Chk1 pS317 or pS345 either in the absence and presence of prior HU treatment therefore V158411 treatment had no effect on the thermostabilization of total Chk1 or phosphorylated Chk1 in either HU treated or untreated cells (Fig 4D) after HU treatment and detection by IF.

Revisiting the sealed heating method-V158411 decreases Chk1 nuclear immunofluorescence in HT29 cells heated in a low well volume
CellCarrier Ultra 96 well plates have a very thin bottom (190 μm thick) which should facilitate the rapid heating of cells attached to this surface. In previous experiments, heating cells in 200 μL media resulted in a long time-to-target-temperature in the well and no thermo-destabilization of Chk1. To minimize the effects of convection, we evaluated lower well media volumes (0, 25 or 50 μL) on Chk1 stability following heating. Heating the cells in a lower volume of media resulted in Chk1 destabilization with a TFI 50 of 47.8 and 50.4˚C in 25 and 50 μL volumes respectively (Fig 5A and Table 3). Heating the cells in a completely empty well was not suitable and resulted in loss of nuclear Chk1 fluorescence and cellular damage (Fig 5B and S3A  Fig). Cells heated in low volumes of media were more robust than those heated by the PBS immersion method with formaldehyde fixation sufficient for cells to remain attached to the plate following IF processing. Indeed, formaldehyde fixation produced a greater signal to background window than formaldehyde followed by methanol fixation (Fig 5A).
To minimize the effects of evaporation we evaluated the effect of V158411 on Chk1 thermal stability following a short 10 minute incubation. Pre-incubation of HT29 cells with 0-20 μM V158411 for 10 minutes resulted in a reduction in Chk1 nuclear immunofluorescence with an EC 50 of 1.9, 1.9 or 2.5 μM when heated to 46, 49 or 52˚C respectively (Fig 6A and S3B Fig). This was in direct contrast to the CETSA classic method where V158411 increased Chk1 thermostabilization [11]. The HCIF-CETSA method was relatively robust and reproducible with EC 50 values of 1.9, 1.9 and 1.2 μM (mean 1.7 ± 0.4 μM) determined in three independent experiments (Fig 6B). Likewise, in U2OS cells an EC 50 value of 1.4 ± 0.3 μM was determined (Fig 6C) though the signal to background was much lower in this cell line. In the unheated U2OS cells, V158411 treatment increased the mean nuclear Chk1 fluorescence coupled with a concomitant decrease in the mean cytoplasmic Chk1 fluorescence (S4 Fig). Chk1 can shuttle between the nucleus and cytoplasm [17] including in response to DNA damage. Single agent Chk1 inhibition induces DNA damage and the effect observed may be the shuttling of Chk1 to the nucleus in response to this damage. In addition, the relatively low Chk1 Incubating the cells with V158411 for a longer time (30 minutes) in a larger volume (50 μL, to minimize evaporation effects) had little effect on the HCIF-CETSA EC 50 value with EC 50 values of 1.0 and 1.2 μM obtained following a 10 and 30 minute incubation respectively (Fig 6D). Under identical cellular conditions to the HCIF-CETSA, V158411 reduced Chk1 autophosphorylation (pS296) in HT29 or U2OS cells with an IC 50 value of approximately 0.16 and 0.11 μM respectively (Fig 6E). Inhibiting the proteasome with MG-132 did not block the temperature induced decrease in Chk1 nuclear fluorescence in HT29 cells pre-treated with either DMSO or V158411 nor did it alter the sub-cellular localization in either unheated or heated cells (Fig 7).

Structurally distinct Chk1 inhibitors decrease Chk1 nuclear fluorescence in HT29 cells following low well volume heating
We further determined the propensity of two additional structurally diverse Chk1 inhibitors (GNE-900 [18]and MK-8776 [19], S5 Fig) to increase Chk1 thermal destabilization and inhibit Chk1 autophosphorylation. GNE-900, MK-8776 and V158411 all induced a large decrease in nuclear Chk1 fluorescence following heating to 49˚C with EC 50 values of 1.3 ± 0.3, 0.32 ± 0.1 and 2.0 ± 0.9 μM respectively (Fig 8A). The same compound dilutions were used to determine the effect of the Chk1 inhibitors on Chk1 autophosphorylation under the HCIF-CETSA treatment conditions. All three compounds reduced S296 phosphorylation (Fig 8B) with IC 50 s of 0.50, 0.44 and 1.2 μM for GNE-900, MK-8776 and V158411 suggestive of Chk1-inhibitor target engagement within the 10 minute incubation period.

Discussion
The measurement of drug-protein interactions in living cells and ultimately the target tissue is fundamental to the drug discovery process. The usage of CETSA [1] to measure drug-target interaction has increased dramatically since its first description in 2013. CETSA has the potential to work on most targets in a wide variety of cell types as it requires no protein modification or the generation of tracer molecules, assays the target protein at the endogenous level, requires simple equipment to perform and when western blotting is used for detection, has a wide range of detection antibodies available. However, the assay format described in these papers has several potential drawbacks namely cells have to be heated in suspension, in PBS, at relatively high densities. For adherent cells, heating cells in suspension is an obvious drawback and the process of trypsinization and resuspension may alter cellular physiology and target pharmacology. Additionally, having to treat cells at high cell densities may result in an underestimation of target engagement potency and make comparisons to downstream pharmacology assays more difficult. We have therefore developed a novel cellular target engagement assay in adherent live cells using the principle of ligand-induced changes to protein thermal stability coupled with high-content single cell immunofluorescent imaging in an attempt to mitigate some of these potential liabilities.
Development of this high content platform based target engagement assay was more technically challenging than anticipated. Problems that required overcoming included (i) heating a 96WP, (ii) accurately measuring in well temperature changes, (iii) detection of target protein and (iv) validation of assay methodology. Chk1 kinase and the potent, selective small molecule inhibitor V158411 [9,10] was selected as an assay test system. We have previously demonstrated that using the assay conditions described in Jafari et al, [2] V158411 increased the thermal stabilization of Chk1 in HT29 and U2OS cells [11]. Whilst heating cells by the PBS immersion method appeared to most closely replicate the heating conditions achieved in a PCR tube and resulted in a temperature dependent decrease in nuclear Chk1 immunofluorescence there was no effect of V158411 on Chk1 nuclear immunofluorescence. Heating cells by the PBS immersion method resulted in significant cellular damage (S2 Fig) which may, in part, account for the lack of additional thermal stabilization / destabilization observed in the presence of V158411. Conversely, covering the cells with 200 μL PBS, sealing the plate and then heating by immersion in a pre-heated water bath did not alter Chk1 nuclear fluorescence in the presence or absence of V158411. This was despite the fact that CellCarrier plates have a very thin (0.180 μm) bottom and most likely reflects that heating could only be achieved through the bottom (and top for the submerged plate) and not the well sides with in-well convection therefore reducing the rate of temperature rise. Heating cells by floating in a water bath where the well volume had been reduced to 25 μL resulted in a temperature dependent decrease in nuclear Chk1 fluorescence that was further reduced by the pre-incubation of cells with V158411. The EC 50 values for V158411 determined by this method were approximately equivalent to the EC 50 values determined using the CETSA classic method suggesting a reasonable correlation between the two methods. The HCIF-CETSA method appeared robust with good inter-assay reproducibility. The EC 50 values determined by CETSA classic or HCIF--CETSA were 2.7-23-fold greater than the autophosphorylation IC 50 . The CETSA classic or HCIF-CETSA EC 50 values appeared to tally more closely with changes in Chk1 inhibitor dependent cellular pharmacology and may reflect the cellular biology of Chk1 kinase.
The reasons behind the difference in Chk1 protein stability following binding by V158411 between the CETSA classic (stabilization) and the HCIF-CETSA method (destabilization) were not immediately apparent. However, it should be noted that the assays have very different conditions / formats and have different but related endpoints. CETSA classic determines the amount of soluble protein remaining following heating whilst HCIF-CETSA determines the loss of an antibody epitope. The interpretation of the immunofluorescence results may be complicated by the choice of antibody used. Cell fixation can result in protein denaturation and therefore the antibodies used in immunofluorescence may not recognize a native state protein but an epitope that is revealed following partial denaturation of the protein by the fixation process. The subsequent heat denaturation and unfolding induced during the HCIF--CETSA process may increase the recognition of the target protein by the antibody utilized in the assay. Therefore selecting an antibody that faithfully reports the protein state may be critical for the HCIF-CETSA method. Utilizing alternative protein detection methods (such as those discussed later) may overcome some of these limitations.
This assay format has some distinct advantages over the CETSA classic methodology first described by Molina et al. [1] namely adherent cells can be heated whilst still attached at relatively low cell densities. As the method uses standard format 96 well plates (and potential miniaturization to 384 well plates), it is readily amenable to scale, automation and laboratory workflows. If longer compound incubations are necessary for target engagement to occur, the cells can simply be incubated in a larger well volume for the required time then the media volume reduced and the cells heated. Adaptation of the CETSA classic method to homogenous plate based methodologies (such as AlphaScreen or ELISA [3], referred to as HT-CETSA) has the potential to increase the throughput of this assay format. In the HCIF-CETSA method, it is more difficult to control the cell heating than the thermocycler method. However, by using the same water bath coupled with precise working, it is possible to produce highly reproducible data.
Another drawback of using immunofluorescence for target detection is that the number of good quality, well validated antibodies available is much lower than that for western blotting. The CETSA classic method has been demonstrated to work with reporter tagged proteins for example (i) using Enzyme Fragment Complementation (EFC) between two inactive b-galactosidase fragments (InCELL Hunter Target Engagement Assay, DiscoveRx) or (ii) using a Nanoluc-tagged protein (Promega). This approach has recently been utilized to develop a highthroughput dose-response cellular thermal shift assay (HTDR-CETSA) based on BacMam titratable expression of ePL tagged full-length target proteins [20]. The possibility therefore exists to generate and detect proteins tagged with a fluorescent protein (e.g. EGFP), a fluorescent reporter (e.g. HaloTag or SNAP-tag) or a protein tag for which high quality IF compatible antibodies are available (e.g. HA or FLAG tag). CRISPR technology now allows in principle gene specific protein tagging at either the N-or C-terminus [21,22]. This technology could be used to generate a cell line containing a reporter-protein fusion protein expressed at endogenous levels to measure target engagement.
In summary, the method described here allows the high throughput determination of target engagement in adherent cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding and is therefore a valuable advance in the CETSA method. It should serve as a valuable tool for those engaged in small molecule drug development.