In Situ Target Engagement Studies in Adherent Cells - ACS Chemical

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In situ target engagement studies in adherent cells Hanna Axelsson, Helena Almqvist, Magdalena Otrocka, Michaela Vallin, Sara Lundqvist, Pia Hansson, Ulla Karlsson, Thomas Lundbäck, and Brinton Seashore-Ludlow ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01079 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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ACS Chemical Biology

In situ target engagement studies in adherent cells Hanna Axelsson1,2, Helena Almqvist1,2, Magdalena Otrocka1,2, Michaela Vallin1,2, Sara Lundqvist3, Pia Hansson3, Ulla Karlsson3, Thomas Lundbäck1,2,3, and Brinton Seashore-Ludlow1,4 1

Chemical Biology Consortium Sweden, Science for Life Laboratory, Karolinska Institutet, SE-171 65 Solna, Sweden 2

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 65 Solna, Sweden 3

Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden

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Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, SE-171 76 Stockholm, Sweden

*Correspondence: [email protected]

Hanna Axelsson, [email protected], +46 8 524 81653 Helena Almqvist, [email protected], +46 8 524 86967 Magdalena Otrocka, [email protected], +46 743 12095 Michaela Vallin, [email protected], +46 8 524 86831 Sara Lundqvist, [email protected] Pia Hansson, [email protected] Ulla Karlsson, [email protected] Thomas Lundbäck, [email protected], +46 72 711 66 89 Brinton Seashore-Ludlow, [email protected], +46 8 524 87873

Keywords: CETSA, imaging, target engagement, p38α, HTS, single cell resolution

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Abstract A prerequisite for successful drugs is effective binding of the desired target protein in the complex environment of a living system. Drug–target engagement has typically been difficult to monitor in physiologically relevant models, and with current methods, especially, while maintaining spatial information. One recent technique for quantifying drug–target engagement is the cellular thermal shift assay (CETSA), in which ligand-induced protein stabilization is measured after a heat challenge. Here, we describe a CETSA protocol in live A431 cells for p38α (MAPK14), where remaining soluble protein is detected in situ using high-content imaging in 384-well, microtiter plates. We validate this assay concept using a number of known p38α inhibitors and further demonstrate the potential of this technology for chemical probe and drug discovery purposes by performing a small pilot screen for novel p38α binders. Importantly, this protocol creates a workflow that is amenable to adherent cells in their native state and yields spatially resolved target engagement information measurable at the single-cell level.

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Introduction An effective drug must bind its anticipated target in a living system at the desired site of action, while minimizing off-target mechanisms that lead to intolerable toxicity. In fact failure of a significant proportion of small-molecule drugs in Phase II clinical trials can be attributed to lack of efficacy, implying that the clinical candidates did not reach the target protein at significant levels, or that modulation of the target protein did not result in the desired pharmacological response.1 If drug–target binding is not confirmed in such cases, it often remains unclear whether the initial biological hypothesis linking the target protein to disease setting was even evaluated. Therefore, the development of techniques for measuring target engagement in live cells is essential for next-generation drug development. In particular, methods allowing for sub-cellular localization of the drug-binding event in heterogeneous patient samples and tissues are desirable, especially if multiple readouts can easily be integrated into the assay platform. Not only can such methods aid in target validation by coupling measurements of drug–target engagement to functional readouts exhibiting response,2-4 but they can also be applied in screening campaigns for novel chemical entities and triaging hit lists. Traditionally, direct physical drug–target engagement in live cells has been difficult to observe, but recent advances in this area have led to a number of new methods.5, 6

Many of the techniques rely on modification of either the chemical probe (e.g. use

of a drug tracer7) or the cells (e.g. use of an enzyme fragment complementation strategy8) to facilitate detection. Innovative techniques allowing for drug–target engagement measurements at the single-cell level are particularly attractive, as they allow for studies of sub-populations in heterogeneous samples, for example. Two recent studies have investigated drug occupancy with subcellular, spatial resolution using functionalized drug analogs in competition with the unfunctionalized drug by either polarized microscopy9, 10 or confocal microscopy.11 Both of these methods yield spatial resolution of the target-drug interaction by monitoring concurrent loss of companion probe signal. These examples serve to inspire further technology developments in this direction, while striving to ensure applicability to patientderived or similar samples with small cell numbers and inherent heterogeneity. One of these approaches, i.e. the cellular thermal shift assay (CETSA), quantifies drug–target engagement of the endogenous protein with unmodified drug by monitoring ligand-induced stabilization of the protein in response to a heat challenge.12 This assay is similar to traditional thermal shift assays on isolated

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proteins, which are often used to confirm binding of lead compounds to the target under investigation.13 Experimentally CETSA measures either the ligand-induced shift in aggregation temperature (Tagg) at a fixed ligand concentration or the ligand concentration necessary to stabilize the protein at a single temperature. The second format is often referred to as isothermal dose response fingerprints in CETSA (ITDRFCETSA), as the apparent stabilization derived from such experiments are dependent on the exact conditions and temperature applied. In most of the reported CETSA adaptations remaining levels of soluble protein are detected by quantitative Western blot.14 For proteome-wide compound selectivity measurements mass spectrometry-based proteomics has been implemented.15-17 However, both throughput and requirements for extensive cell numbers limit the application of these techniques to a few compounds or concentrations. Efforts have been made to improve single protein CETSA throughput by miniaturization to microtiter plates and use of AlphaScreen® technology for detection, yielding assay platforms that are compatible with high throughput screening.18-21 However, there is a current lack of CETSA protocols applicable to single-cell quantification with potential for also measuring the subcellular localization of target engagement.22 Furthermore, reported detection methods are applicable to adherent cells only after surface detachment, and are potentially compromised by choices of experimental workflows and conditions.12, 22 These factors include washing of treated cells, with or without maintenance of compound treatment during trypsinization, or subsequent treatment while the trypsinized cells are kept in suspension. Performing CETSA on adherent cells in suspension with concomitant trypsinization alters the native state of the cells, possibly impacting the observed target–engagement profiles.23, 24 Here, we extend the CETSA platform to allow for single-cell quantification of target engagement in situ with the possibility of monitoring subcellular localization using immunofluorescent antibody detection coupled with high content microscopy. As a model system we investigate known inhibitors of human p38α (MAPK14) in A431 cells and perform a pilot screen identifying a novel class of small-molecule binders of this protein. These results are confirmed using a kinase activity assay based on isolated recombinant p38α. Not only is the present method compatible with high throughput screening demands, but, importantly, it allows for quantification of drug– target engagement in adherent cells while preserving their surface attachment, and requires considerably fewer cells per well for reliable signal. Finally, a single plate is

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used from cell seeding through detection, minimizing transfer steps and costs for screening.

Results and Discussion Model system and assay considerations We have previously designed several microtiter-plate-based CETSAs for monitoring target engagement to p38α in suspension cell lines using AlphaScreen®.25, 26 These results have been further corroborated with the original Western blot detection method. Based on this accumulated knowledge, we decided to use p38α to investigate the feasibility of adapting imaging-based readouts to CETSA. At the outset of this project we expected several challenges. First, in the original Western blot protocol the aggregated protein is removed in a series of centrifugation steps, allowing for reliable detection of remaining soluble protein. In AlphaScreen® two antibodies are used to measure the remaining soluble, folded protein in lysed cells after heat challenge. We have shown that by using an antibody pair we can selectively report on the remaining native protein despite a background of denatured and precipitated proteins in the lysate solution. In addition, the signals from epitope-tagged proteins are lost upon transient heating, indicating that the epitopes become inaccessible to detection reagents as a result of thermal unfolding and irreversible aggregation.20 Here, using immunofluorescence we envisioned that a single antibody could be used to quantify target engagement, however the antibody must selectively discriminate between remaining soluble protein and aggregated or denatured protein, which remains after sample fixation. The feasibility of these selectivity requirements for the antibody needed to be explored. Second, integral to achieving reproducible results in CETSA is controlled heating of the micro-well plate. In our previous protocols we relied on PCR plates for efficient and reliable heat transfer to the sample with little well-to-well variation. Here, we needed to investigate heating in formats suitable for imaging readouts. Finally, the previously reported assays are based on 600,000 or 33,000 cells per well in suspension.25, 26 We realized that by moving to an imaging readout, we could significantly reduce the cell numbers. Furthermore, the transition to an imaging

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format allowed the cells to remain adherent throughout the pre-incubation and the transient heating step, ensuring no target engagement is lost as a result of wash procedures or undesirable changes of cell status as a result of trypsinization. Assay set-up and validation Initial experiments were defined to explore feasibility of heating non-treated versus treated samples using an incubator or a PCR flat block, while comparing the levels of remaining soluble p38α using a immunofluorescence protocol with an imaging readout. We screened a panel of primary antibodies (Table 1) in chamber slides as we envisioned heat transfer on slides would be more efficient than flat bottom plates. In parallel we monitored the success of heat transfer with a modified AlphaScreen® protocol26 that could be applied to adherent instead of suspension cells, and visually inspected wells after heating to confirm that cells remained attached. Heating in the incubator was inconsistent, but we were able to confirm successful heat transfer and reproducible data on slides using the flat block (Supplementary Figure 1A).27 One of the primary antibodies, R1, resulted in differential fluorescent signal between the treated and control wells, while the nuclei stain intensity remained constant, suggesting that the antibody was detecting remaining levels of soluble p38α (data not shown). Incidentally, this antibody is also within the pair of antibodies successfully used for AlphaScreen® detection. With these results in hand, we investigated heating in 384-well imaging plates, since chamber slides are not amenable to higher throughput experiments. We performed the antibody screening and titration in 384-well imaging plates using the flat block following the strategy we previously outlined (Supplementary Figures 1B-D).19 Good consistency was observed for ITDRFCETSA experiments with AMG 548 using imaging and the modified AlphaScreen® method for adherent cells (Supplementary Figure 1E). Although we were able to establish a reliable assay window, we were unable to find conditions to obtain an even heating across the entire plate despite considerable efforts to minimize plate edge effects (Supplementary Figures 1F,G). As an alternative heating strategy we examined the use of a water bath. Holes were drilled in the edge of the plate to ensure that no air was trapped under the plate during heating and a thermocouple was used to measure temperature the wells of a dummy plate to monitor both heating and cooling (Supplementary Figures 2A,B). We rescreened the antibody panel with a new twist performing permeabilization either before or after fixation (Figures 1A-C). We reasoned that in the first case the

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remaining soluble protein would leak out from the permeabilized cells prior to the fixation step. Thus, if an antibody were truly selective for the remaining soluble protein in the presence of precipitated protein a loss of signal would be observed. This was observed with antibody R1, which also displayed a reasonable assay window. We titrated the primary and secondary antibodies and examined the effect of heating time on plate edge artifacts (Figure 1D, Supplementary Figures 2 C-F), finally settling on conditions with 1:500 dilution of the primary antibody and 1:1000 dilution for the secondary antibody with a 3 minute heating time (see Supplementary Table 1). We further evaluated the assay by conducting ITDRFCETSA for seven known inhibitors of p38α (Figure 1E). Comparison with the apparent ITDRFCETSA values in HL-60 cell lysates using AlphaScreen® detection showed reasonable consistency in compound ranking between the two assay systems for these known binders (Figure 1F).26 Interestingly using the live cell assay format we could not detect any significant stabilization of p38α by BIRB796. Given available literature data on this compound as a well-characterized inhibitor of p38α with nM cellular activity this was highly concerning. To understand the underlying cause of this lack of response we examined the immunofluorescence signal from cells treated with BIRB796 (1 µM) or DMSO. We observed a distinct loss of signal in the treated cells compared to the control when the heat challenge was omitted, suggesting that BIRB796 enters the cell but disrupts antibody recognition of p38α (Supplementary Figure 2G).21 This finding is further corroborated by the observation of p38α stabilization in live HL-60 cells using CETSA with AlphaScreen® detection (Supplementary Figure 2H), i.e. in a setting where remaining levels of soluble protein is significantly diluted upon cell lysis thus allowing for compound dissociation. While the combined use of unheated and heated samples thus allows for mechanistic studies, i.e. the loss of antibody recognition clearly differs between ligands, we also acknowledge the need for additional affinity reagents or detection protocols. Pilot screen for binders of p38α In order to demonstrate the compatibility of the CETSA imaging platform described above with the demands of high throughput screening, we performed a pilot screen of five 384-well microtiter plates, four plates containing small molecules and one control plate with DMSO only. The small molecule compound set consisted of 1,120 small molecules originated from a kinase directed library with drug-like properties

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from the CBCS compound collection (www.cbcs.se) at the Science for Life Laboratory (www.scilifelab.se). To observe protein stabilization in thermal shift assays relatively high ligand concentrations are needed to exceed the dissociation constant, i.e. saturation prompts larger thermal shifts. Hence these small molecules were screened at 50 µM to facilitate hit identification. We anticipated that any acute toxicity due to small molecule treatment would easily be identified by cell counting at the imaging stage. Given the significance of consistent heating for the assay, columns 1 and 23 contained the negative control (DMSO) and columns 2 and 24 had the positive control (1 µM AMG 548), such that the signal could be monitored across the screening set, as well as across each individual plate. Overall, reasonable Z’-factors, calculated based on quantification of single cell fluorescence values, and a consistent assay window were observed across all five plates (Figure 2, Supplementary Figures 3A-D). After data analysis, a threshold of 30% stabilization was applied, resulting in 14 hits and a 1% hit rate. The elevated hit rate was not considered alarming given the application of a kinase-targeted library. In addition to the analysis of single cell fluorescent signal, number of cells per well was monitored and five wells with the number of cells significantly lower that average were identified (data not shown) but none corresponded to any hit compound. Three of these were from the control plate suggesting a technical problem during cell dispensing (Supplementary Figure 3B). We also examined the location of all hits and the signal across the screening plates to ensure the quality of hits to prioritize (Supplementary Figures 3E-F). After analysis of numerical data, images corresponding to the active compounds were reviewed manually to eliminate any further “false positives”. This revealed that one out of 14 wells was identified as hit due to fluorescence debris present in the well. Example images of a screening plate are shown in Supplementary Figure 4. Hit confirmation and validation To confirm stabilization of p38α we probed the hit compounds in ITDRFCETSA experiments in live A431 cells including additional cytoplasmic staining to monitor possible changes in cell morphology caused by compounds. The majority (12/14) of the compounds confirmed the screening results and reproduced a weak stabilization of p38α, while two small molecules failed to recapitulate the stabilization observed in the screen in this experiment (Figure 3A, Supplementary Figures 5, 6). One of them was a compound identified as “false positive” due to the fluorescent debris present in

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the well as discussed before. Promisingly, compound CBK037438 proved to stabilize p38α with an ITDRFCETSA midpoint in the low µM range, without inducing any changes in cell morphology. Since high content imaging allows for resolution of individual cells we also sampled the intensity distribution over all identified cells per well at each concentration of the ITDRFCETSA and representative illustrations for two compounds are shown (Figure 4A, Supplementary Figure 7). Here the concentration-response of p38α stabilization appeared simultaneously at a per-well and the single-cell level. Importantly for both nuclei and cell mask intensity, no concentration response was observed. In an orthogonal assay we assessed the impact of the hit compounds on recombinant p38α kinase activity (Figure 3A, Supplementary Figures 5, 6). Overall, we observed a good correlation between the pITDRFCETSA values and the pIC50s from the activity assay (Figure 3C). One compound stabilized p38α, but showed no inhibition in the in vitro assay. Structural analysis of the hit compounds A closer examination of the hit compounds revealed a common core motif (Figure 3B, Supplementary Table 2). To the best of our knowledge, these structures have not been reported before. These compounds have been included in over 20 in house screens with various different readout measurements. This is the first screen any of these compounds has been classified as a hit (Supplementary Figure 8A). The series is however structurally related to the known p38α MAP kinase inhibitors, such as Losmapimod (GW856553) and AMG 548 as depicted in Figure 3B. The 2carboxamide-5-phenylsubstituted pyridine backbone is found in Losmapimod and the vic-pyridine and phenyl substitution pattern is present in AMG 548. Interestingly in the literature, 2-aminopyridine analogs of the hit series are reported as adenosine receptor antagonists28 and pyrazolopyridine derivatives as p38α inhibitors (Supplementary Figure 8B).29 In addition, regioisomeric carboxamide analogs are known and described as urotensin II receptor antagonists30 and A2B adenosine receptor antagonist.31 Follow-up studies and reproducibility In order for this method to be widely applicable it is important to determine if in situ target engagement can be observed in other cell line models. We briefly looked for stabilization of p38α in U2OS cells using the same protocol by probing a single

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concentration of the known inhibitors and one of the hit compounds. Though a smaller assay window was observed in this cell line, stabilization of p38α can be successfully measured (Figure 4B). The smaller assay window may be a function of the lower expression level of p38α in U2OS cells (Supplementary Figure 8C)32. To increase the applicability of this method, we further investigated the assay procedure to identify conditions where stabilization of p38α by BIRB796 could be observed. After exploring a number of modifications, we found that antigen retrieval after fixation facilitated the detection of the desired target-engagement event (Figure 4C). The apparent EC50s from the two protocols were in good agreement for the remaining inhibitors tested (Supplementary Figure 8D). This finding could help minimize the number of false negatives in screening campaigns and add an additional method to study the individual binding mode of each inhibitor. Given the novelty of the imaging CETSA approach we next decided to assess the reproducibility of our approach in additional labs. Assay reproduction is crucial for optimal guidance of compound activities, but can be challenging to achieve even across labs within the same organization33. For this reason the assay approach was repeated using a slightly modified protocol within the AstraZeneca R&D organization, with excellent quantitative agreement between both rank order and absolute ITDRFCETSA values (Supplementary Figures 9A-C). Methods to quantitate target engagement on unmodified drugs and protein targets in living systems are expected to benefit the drug discovery process by helping ensure translation across physiologically relevant models. Direct correlation of drug-target engagement with measurements of disease alleviation aid in both target validation and small-molecule triaging. Especially relevant for heterogeneous samples and in vivo experiments are methods that allow for multiplexed readouts with spatial resolution at the single-cell level. Here, we describe an in situ CETSA imaging platform that enables identification, and ranking of p38α binders. As compared to previously reported CETSA protocols, this assay relies on considerably fewer cells per well (2,000 v. 600,000), enabling measurements in precious samples. The assay is carried out in a single plate from cell seeding to detection, thereby minimizing the need for transfers in high throughput screening campaigns and reducing costs. Importantly it also removes the requirement for identification of antibody pairs that simultaneously recognize the native protein, likely extending the amenability to additional target proteins. In this

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protocol adherent cells are investigated in their native state, while remaining attached to the plate, thus removing washing or trypsinization steps that may alter compound availability and binding. Finally, imaging preserves spatial resolution of the individual cells allowing for target engagement measurements in mixed populations of cells or cell states. Unlike previous assay formats, here lack of signal due to cytotoxicity is simultaneously reported on by nuclear staining in each well. In this preliminary application we validate the assay concept and demonstrate the applicability to small-molecule screening. We identify a previously unreported series of small molecules that demonstrate binding to p38α and inhibit the enzyme in an in vitro activity assay. However, several key questions will need to be explored in further applications to truly assess the applicability of the approach. First, in the imaging format detection of ligand-target engagement is only possible when the antibody selectively reports on the native protein against a background of denatured and aggregated proteins. The ease of identifying suitable affinity reagents with good selectivity for all melting proteins is unknown. Second, we have used a water bath to administer the heat challenge to the microtiter plate but a dedicated device for this application with more flexibility and better heat transfer would certainly advance this method. Finally, p38α is relatively abundant in the cell line we investigated. We do not know at this point how the abundance of the target of interest will affect the ability to reliably measure a signal, although we expect this to correlate strongly to antibody affinity and selectivity, nor how variation in abundance across cell lines will affect assay optimizations. Further studies are necessary to investigate these aspects of in situ CETSA. Although not explored in this preliminary application, we believe that it will be possible to multiplex protein readouts by imaging. This would allow study of multiple targets simultaneously, or selective drug engagement studies in cocultures for example, not to mention study of drug and target localization.

Methods Image-based CETSA Protocol A general outline of the final assay protocol for in situ CETSA is available in Supplementary Table 1. The cell culture medium was aspirated from the cell culture flasks, the cell monolayers were washed twice with PBS and the cells were then detached by trypsinization, collected by centrifugation at 194 xg for 5 minutes, resuspended in medium and counted in a Burker chamber. 40 µl of cell suspension was

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dispensed into the wells of black 384-well clear-bottom imaging plates using a Multidrop (Thermo Fisher Scientific) to give a final cell density of 2000 cells/well. To minimize plate edge effects, the cells were allowed to settle at the bottom of the assay plates for 20 minutes at room temperature before placing them in a plastic container with damp cloths to ensure a humid atmosphere. The box with the assay plates was incubated for 2-3 days at 37°C and 5% CO2 in a humidified incubator. On the day of the experiment, the medium was first aspirated using a plate washer (Tecan) followed by the addition of 30 µl of compounds diluted in cell culture medium using a Bravo liquid handling station (Agilent) (see compound handling). Compoundtreated assay plates were sealed with a breathable plate seal followed by incubation at 37°C and 5% CO2 in a humidified incubator for 30 minutes. Next the plates were re-sealed with a tight adhesive aluminum foil to ensure that no water could leak into the wells during the subsequent heating in a water bath (Julabo TW12). During this step, it is critical to avoid air bubbles trapped under the assay plate. To prevent this 6 holes were drilled in the frame of the assay plates prior to seeding of the cells (supplementary Figure 2A). To avoid plastic particles entering the wells during this step, the plates were sealed with an adhesive aluminum foil prior to drilling. Furthermore, plates were carefully placed with the bottom of the plate angled towards the water surface to force any remaining air out from under the plates. The assay plates were heated while floating in the water bath for 3 minutes followed by an immediate transfer to another water bath with room tempered water for 5 minutes. To verify that the desired temperature was reached in the wells of the assay plates, an unsealed control plate containing the same volume of medium as the assay plate were placed alongside the plates in the water bath. The temperature in the control plate was constantly monitored using a Thermocouple Traceable lab thermometer (VWR) (supplementary Figure 2B). In our set up the temperature in the water bath was set 3°C above the desired temperature in order to obtain this temperature inside the wells of the assay plate. The heating challenge was followed by a fixation step. This was performed by dispensing 10 µl 16% (w/v) PFA directly to the assay plates using a Multidrop followed by 20 minutes incubation at room temperature. The PFA was then aspirated and the cells were subsequently washed with 300 µl PBS (overflow protocol) using a plate washer. Permeabilization was performed by the addition of 20 µl 0.1% (v/v) NP-40 followed by incubation at room temperature for 10 minutes and the subsequent washing procedure as described above (overflow protocol). Blocking was

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done by the addition of 15 µl 1% (w/v) BSA in PBS using a Multidrop followed by incubation at room temperature for 1 hour at room temperature or overnight at 4°C. The blocking solution was aspirated using a plate washer followed by manual addition of 10 µl rabbit anti-p38 antibody R1, diluted 1:500 in 1% (w/v) BSA in PBS. After incubation with primary antibody overnight at 4°C (or 1 hour at room temperature) the cells were washed with 300 µl PBS (overflow protocol) followed by manual addition of 10 µl secondary antibody, diluted 1:1000 in 1 % (w/v) BSA in PBS. After incubation for 1 hour at room temperature the cells were stained by the addition of 10 µl Hoechst diluted to 0.05 mg/mL in PBS and additional 10 minutes of incubation. For the hit confirmation ITDRFCETSA experiment 15 µl of HCS cell mask diluted to 200 ng/mL in PBS was added followed by 30 minutes incubation at room temperature. Next the cells were washed with 300 µl PBS (overflow protocol) followed by the addition of 60 µl PBS. The plates were sealed with an adhesive aluminum foil and images were acquired using an ImageXPress (Molecular Devices). Image acquisition and analysis Images were captured on ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices) using 2 (small screen) or 3 (ITDRFCETSA dose-response experiments) fluorescent channels: DAPI (387/447), GFP (472/520) and TexasRed (562/624). Typically, 4 images were taken per well using 10X Plan Fluor 0.3 NA objective. Automated laser autofocus was used and binning 2 was applied during acquisition. Images were stored as 16 bit, gray scale tiff files along with metadata in Oracle database for further analysis. Image analysis was performed using MetaXpress Custom Module. Cell nuclei were identified using DAPI images using the Find nuclei algorithm. Nuclear segmentation mask was then expanded out 3 pixels to identify cell boundaries. Average intensity of GFP channel was measured for each individual cell and then average per image and per well values were calculated. In addition, cell number was monitored in order to assess compound toxicity (See supplementary Figure 10 for representative images). For ITDRFCETSA dose response experiments additional images using TexasRed filter were acquired and analysis was performed using modified Custom Module. Cell boundaries were identified using the Cell Scoring algorithm with DAPI (nucleus) and TexasRed (cytoplasm). Average intensity for all acquired wavelengths and cell area for each individual cell were extracted for further data analysis (see supplementary Figure 11 for representative images).

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After image analysis per well data was analyzed in Excel templates for both the screening and ITDRFCETSA experiments. Plotting of the data was done using Graphpad prism or R (ggplot2 package). Supporting Information Available For reagent specifications, compound handling, cell culture, modified CETSA protocol and activity assay see accompanying Supporting Information. This material is available free of charge via the internet at http://pubs.acs.org.

Acknowledgments The authors kindly acknowledge Karolinska Institutet, SciLifeLab and the Swedish Research Council (Vetenskapsrådet) for funding.

Author Contributions H.Ax., H.Al., B.S-L., and T.L. conceived the study. H.Ax., H.Al. and B.S-L. performed the assay optimization and CETSA experiments at KI. S.L., U.K. and P.H. performed the CETSA experiments at AstraZeneca. H.Ax. ran the activity assay. M.O. designed the image analysis. M.V. performed the searches on the chemical structures and analysis of previous screens. B.S.-L. wrote the first draft of the manuscript. H.Ax., H.Al., B.S-L., M.O., M.V. and T.L contributed to the final manuscript preparation.

Competing Financial Interests The authors declare no competing financial interests.

References

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pharmacokinetic and pharmacological principles toward improving Phase II survival, Drug Discovery Today 17, 419-424. [5] Schürmann, M., Janning, P., Ziegler, S., and Waldmann, H. (2016) Small-Molecule Target Engagement in Cells, Cell Chem. Biol. 23, 435-441. [6] Simon, G. M., Niphakis, M. J., and Cravatt, B. F. (2013) Determining target engagement in living systems, Nat. Chem. Biol. 9, 200-205. [7] Robers, M. B., Dart, M. L., Woodroofe, C. C., Zimprich, C. A., Kirkland, T. A., Machleidt, T., Kupcho, K. R., Levin, S., Hartnett, J. R., Zimmerman, K., Niles, A. L., Ohana, R. F., Daniels, D. L., Slater, M., Wood, M. G., Cong, M., Cheng, Y.-Q., and Wood, K. V. (2015) Target engagement and drug residence time can be observed in living cells with BRET, 6, 10091. [8] Auld, D. S., Davis, C. A., Jimenez, M., Knight, S., and Orme, J. P. (2015) Examining LigandBased Stabilization of Proteins in Cells with MEK1 Kinase Inhibitors, Assay Drug Dev. Technol. 13, 266-276. [9] Dubach, J. M., Kim, E., Yang, K., Cuccarese, M., Giedt, R. J., Meimetis, L. G., Vinegoni, C., and Weissleder, R. (2017) Quantitating drug-target engagement in single cells in vitro and in vivo, Nat. Chem. Biol. 13, 168-173. [10] Vinegoni, C., Fumene Feruglio, P., Brand, C., Lee, S., Nibbs, A. E., Stapleton, S., Shah, S., Gryczynski, I., Reiner, T., Mazitschek, R., and Weissleder, R. (2017) Measurement of drugtarget engagement in live cells by two-photon fluorescence anisotropy imaging, Nat. Protoc. 12, 1472-1497. [11] Rutkowska, A., Thomson, D. W., Vappiani, J., Werner, T., Mueller, K. M., Dittus, L., Krause, J., Muelbaier, M., Bergamini, G., and Bantscheff, M. (2016) A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level, ACS Chem. Biol. 11, 2541-2550. [12] Martinez Molina, D., Jafari, R., Ignatushchenko, M., Seki, T., Larsson, E. A., Dan, C., Sreekumar, L., Cao, Y., and Nordlund, P. (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay, Science 341, 84-87. [13] Holdgate, G. A., and Ward, W. H. J. (2005) Measurements of binding thermodynamics in drug discovery, Drug Discovery Today 10, 1543-1550. [14] Martinez Molina, D., and Nordlund, P. (2016) The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In Situ Drug Target Engagement and Mechanistic Biomarker Studies, Annu. Rev. Pharmacol. Toxicol. 56, 141-161. [15] Savitski, M. M., Reinhard, F. B. M., Franken, H., Werner, T., Savitski, M. F., Eberhard, D., Molina, D. M., Jafari, R., Dovega, R. B., Klaeger, S., Kuster, B., Nordlund, P., Bantscheff, M., and Drewes, G. (2014) Tracking cancer drugs in living cells by thermal profiling of the proteome, Science 346. [16] Franken, H., Mathieson, T., Childs, D., Sweetman, G. M. A., Werner, T., Togel, I., Doce, C., Gade, S., Bantscheff, M., Drewes, G., Reinhard, F. B. M., Huber, W., and Savitski, M. M. (2015) Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry, Nat. Protoc. 10, 1567-1593. [17] Becher, I., Werner, T., Doce, C., Zaal, E. A., Togel, I., Khan, C. A., Rueger, A., Muelbaier, M., Salzer, E., Berkers, C. R., Fitzpatrick, P. F., Bantscheff, M., and Savitski, M. M. (2016) Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat, Nat. Chem. Biol. 12, 908-910. [18] Almqvist, H., Axelsson, H., Jafari, R., Dan, C., Mateus, A., Haraldsson, M., Larsson, A., Martinez Molina, D., Artursson, P., Lundback, T., and Nordlund, P. (2016) CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil, Nat. Commun. 7, 11040. [19] Axelsson, H., Almqvist, H., Seashore-Ludlow, B., and Lundback, T. (2016) Screening for Target Engagement using the Cellular Thermal Shift Assay - CETSA, In Assay Guidance

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Page 16 of 23

Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Legends Figure 1. Assay development in 384-well microtiter plate format using A-431 cells. (a) Antibody screening in cells treated with AMG 548 (1 µM,

) and DMSO (

)

the plate was heated to 52°C in a water bath followed by fixation of the cells, permeabilization and subsequent immunostaining. Error bars represent standard deviation of four replicates. (b) Antibody screening as described in a, but where the permeabilization of the cells was prior to fixation. (c) Representative images of positive, AMG 5481µM, and negative, DMSO, controls. Red- nuclear Hoechst staining, Green- p38α staining. (d) Titration of the primary and secondary antibodies on cells treated with 1µM AMG 548 (

,

,

) or DMSO (

,

,

). The plate was

heated to 52°C in a water bath. Error bars represent standard deviation of three replicates. (e) ITDRFCETSA curves for cells treated with known p38α inhibitors, PH797804 ( ),AMG 548 ( ),RWJ 67657 ( ), LY2228820 ( ), SB203850 ( ), BIRB796 ( ) and Skepinone-L ( ). The plate was heated to 52°C in a water bath. Error bars represent standard deviation of four replicates. (f) Correlation of the EC50s of p38α inhibitors as in part e with data from the same inhibitors in HL-60 cells detected using AlphaScreen® (compound names denoted in the graph).

Figure 2. Illustration of the results from the pilot screen of a kinase directed library in A431 cells. % stabilization of all wells tested grouped by plate with rows A, P and columns 1 and 24 removed. Wells treated with DMSO (0.5 %, ), library compound (50 µM, ) or positive control AMG 548 (1 µM, ). The blue dashed lines represents the hit threshold (30 %) applied. The plates were heated to 52°C in a water bath.

Figure 3. Hit confirmation and follow-up studies. (a) ITDRFCETSA curves for two representative compounds CBK037438 and CBK036770 ( ). The plate was heated to 52°C in a water bath. Inhibition curves from the p38α activity assay for the same compounds ( ). Error bars represent the standard deviation from three replicates for ITDRFCETSA and two replicates for the p38α activity assay. (b) Structure of the lead hit compound CBK037438 and its structural relationship to the known p38 inhibitors Losmapimod and AMG 548. The colors and green circle highlight common structural motifs found in this class of inhibitor. (c) Correlation between the pEC50 from the ITDRFCETSA and the pIC50 from the activity assay for the 14 hit compounds and the positive control AMG 548 (denoted in the figure).

17



Page 18 of 23

Figure 4. ITDRFCETSA curves for AMG 548. (a) Boxplot and histogram illustrating the distribution of average fluorescence intensity for p38α for each individual cell. The plate was heated to 52°C in a water bath. (b) Fluorescence intensity for the different treatments of U2OS cells for six compounds and DMSO at the indicated concentrations. For DMSO and AMG 548 the data represents the mean and standard deviation of 16 replicates. For the remaining inhibitors the mean and standard deviation of three replicates is plotted. (c) ITDRFCETSA curves for cells treated with BIRB796 either with ( ) or without ( ) antigen retrieval protocol.

Tables Table 1. Antibodies tested in the imaging assay.1 antibody

catalogue number

supplier

source

clonality

M1

33-1300

Invitrogen

mouse

monoclonal

M2

ab31828

Abcam

mouse

monoclonal

M3

ab89454

Abcam

mouse

monoclonal

M4

ab89688

mouse

monoclonal

M5

#9228

Abcam Cell Signalling

mouse

monoclonal

use in

epitope

WB, ELISA FC, WB, ELISA, ICC WB, ELISA WB WB, IF, FC

full-length recombinant C-terminus amino acids 200-360 full-length full-length

R1

ab17009

Abcam

rabbit

monoclonal

WB, ICC

internal sequence of p38 (amino acids 150-250)

R2

14064-1AP

Nordic Biosite

rabbit

polyclonal

WB

full-length

R3

sc-728

Santa Cruz

rabbit

polyclonal

R4

PA517713

Thermo Fisher

rabbit

polyclonal

WB, IHC, ELISA, IP WB, IHC, IP

1

N-terminus

C-terminus

WB= Western blot, ELISA = enzyme-linked immunosorbent assay, FC= flow cytometry, IF = immunofluorescence, ICC= immunocytochemistry, IHC=immunohistochemistry, IP= immunoprecipitation

18


Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


heat challenge

30 min pre-incubation

POSITIVE CONTROL



Figure 1

5000

b

DMSO

c

6000

DMSO

1 M AMG 548

3000 2000 1000 0

1 M AMG 548 4000

Positive control

4000

immunofluorescence

2000

3 R

2

1

R

R

4

5 M

M

3

2

anti-p38 antibodies

average intensity - background (no 1° antibody)

2500 1 M AMG 548 (1° @ 1:500)

2000

Negative control

anti-p38 antibodies

M

1 M

2

3 R

1

R

5

R

4

M

3

M

2

M

M

M

1

0

M

immunofluorescence

1 M AMG 548 (1° @ 1:1000) 1500

1 M AMG 548 (no 1°) DMSO (1° @ 1:500)

1000

DMSO (1° @ 1:1000)

500

DMSO (no 1°)

2° N

o

00 20 1:

00 10

1:

50

0

0

1:

f

160 140

AMG 548

% stabilization

120

RWJ 67657

100 80

LY2228820

60

SB203580

40

BIRB796

20

Skepinone-L

0 -20

-6

PH797804

-10

-8

-6

-4

-2

log(concentration) (M)

SB203580

HL-60 AlphaScreen®

1 2a 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 d 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 e 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 23

LY2228820 PH797804

-7

-8

RWJ 67657

AMG 548

-9 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 A431 imaging


Page 21 of Figure 2 23

% stabilization

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


180 160

0.5% DMSO

140

50 M library compound

120

1 M AMG 548

100 80 60 40 20 0 -20

0

500

1000

1500

2000



Figure 3

b

a

CBK037438

CBK036770 120 100 80

% stabilization

% stabilization

1 2 3 180 4 160 5 140 6 7 120 8 100 9 80 10 60 11 40 12 13 20 14 0 15 -20 16 -9 17120 18 100 19 20 80 21 60 22 23 40 24 20 25 26 0 27 -20 28 29 -40 30 31 c 32 33 10 34 35 8 36 37 38 6 39 40 4 41 42 2 43 44 45 0 0 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 23

60 40 20 0

-8

-7

-6

-5

-4

-20

-9 120

-8

-7

-6

-5

-4

CBK037438

Losmapimod

100 % inhibition

% inhibition

80 60 40 20 0

AMG 548

-20 -6

-5

-4

-3

-6

-40

log[cmpd] (M)

-5

-4

-3

log[cmpd] (M)

pEC50 activity assay

AMG 548

2

4

6

8

10

pEC50 ITDRFCETSA


Figure 4 Page 23 of 23 a -5.5

-6.0

-6.4

-6.9

-7.4

-7.9

-8.3

-8.8

-9.3

-9.7

DMSO

Integrated fluoresence intensity

log[AMG 548] -5.0 1 3000 2 3 4 5 6 7 2000 8 9 10 11 12 13 1000 14 15 16 17 18 19 0 20 21 0 100 22 b 23 1600 24 25 26 27 28 1400 29 30 31 32 1200 33 34 35 36 1000 37 38 39 40 800 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

100

0

100

0

100

0

100

0

100

0

100

0

100

count

c

BIRB796 BIRB796 antigen retrieval 160 140 120 100 80 60 40 20 0 -20

-10

-8

-6

log(concentration) (M)

A SB MG D 20 54 MS 8 PH 25 ( O 79 80 1 LY 78 (10 M) 22 04 0 RW 288 (10 M) J 20 0 C 67 (1 M BK 65 00 ) 03 7 67 (10 M) 70 0 (1 M 00 ) M )

% stabilization

100

immunofluorescence

0


-4

-2

0

100

0

100

0

100

In Situ Target Engagement Studies in Adherent Cells - ACS Chemical

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