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ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2021 Jul 12;12(8):1288–1294. doi: 10.1021/acsmedchemlett.1c00276

Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Jonathan D Mortison †,*, Ivan Cornella-Taracido †, Gireedhar Venkatchalam ‡, Anthony W Partridge ‡, Nirodhini Siriwardana †, Simon M Bushell †
PMCID: PMC8366000  PMID: 34413958

Abstract

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Determination of target engagement for candidate drug molecules in the native cellular environment is a significant challenge for drug discovery programs. The cellular thermal shift assay (CETSA) has emerged as a powerful tool for determining compound target engagement through measurement of changes to a protein's thermal stability upon ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA) that deploys a quantitative peptide tag for determination of compound target engagement in the native cellular environment using a high throughput, plate-based luminescence readout. We demonstrate that BiTSA can rapidly assess cellular target engagement of small molecule ligands against their cognate targets and highlight two applications of BiTSA for differentiating small molecules targeting mutant KRAS and TP53.

Keywords: Thermal shift assay, HiBiT, cellular target engagement, BiTSA, KRAS, TP53

Drug discovery campaigns often start with high throughput screens (HTS) aimed at the rapid identification of molecules that can be developed as drugs against a target protein of interest. These HTS are often conducted in less complex, ex cellulo systems, such as in vitro recombinant protein binding assays; however, the in vitro binding activity of a molecule may not translate to engagement of the desired protein target in the native cellular environment. Since rigorous validation of a preclinical molecule’s efficacy target can significantly improve its chances for clinical success,1−3 tools and methods for confirming “on target” activity of a candidate molecule are vital to the drug discovery process.

The cellular thermal shift assay (CETSA) is a powerful tool for determining target engagement for candidate drug molecules in the native cellular environment through monitoring changes in protein thermal stability upon ligand binding.4 Despite its general utility, CETSA can be limited by challenges in its operational workflow. For example, the workflow can require laborious separation of aggregated and soluble protein (Western blot CETSA) as well as require high quality antibodies for the specific detection of the protein under study (Western blot and AlphaLISA CETSA). To improve on these limitations, we applied a commercially available split luciferase system (NanoBiT technology, Promega Corp.) toward the development of a HiBiT thermal shift assay we call BiTSA, which is capable of rapid measurement of drug target engagement using a homogeneous, luminescence-based readout of HiBiT-tagged proteins in CRISPR-engineered cells (Figure 1a).

Figure 1.

Figure 1

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Establishing an in-cell HiBiT thermal shift assay (BiTSA). (a,b) Schemes showing compounds that bind a HiBiT-tagged target protein in cells may induce a change in that protein’s thermal stability, which can be assayed using a high-throughput luminescence readout. Compounds can induce positive (thermal stabilizing) or negative (thermal destabilizing) shifts to a protein’s melting/aggregation profile. (c) Proteins can be readily tagged with HiBiT at their endogenous genomic loci using CRISPR/Cas9. Control data is from a representative nontargeting sgRNA nucleofection in A549 cells. (d) HiBiT-tagged proteins have reliable melting/aggregation profiles in cells when using luminescence to track soluble protein. Melting curve data represent mean ± SD (n = 3 replicates) and are representative of at least two independent experiments. See the Supporting Information for cell line information and experimental details for each HiBiT tagging experiment.

The HiBiT-LgBiT system is a split version of NanoLuc luciferase5 consisting of a small, 11 amino acid peptide tag called HiBiT and its complementary partner LgBiT, which bind each other with high affinity (KD < 700 pM) and perform similarly to the parent NanoLuc with excellent sensitivity (subattomole detection limit) and linear dynamic range (>7 log10). Due to its small size (1.3 kDa), the HiBiT tag is minimally perturbing when appended onto a protein and can be incorporated readily into endogenous cellular proteins using CRISPR/Cas9 gene editing technology.6 In BiTSA, the HiBiT tag acts as a luminescent proxy for soluble versus aggregated protein, as the tag becomes inaccessible for complementation with its LgBiT partner upon protein denaturation and aggregation during thermal challenge (Figure 1b). Since proteins under study are tagged at their native genomic loci with HiBiT, BiTSA also allows for determination of target engagement under conditions that are reflective of native cellular proteostasis (i.e., under the control of endogenous promoters and regulatory elements), avoiding potential problems and artifacts that could arise with overexpression systems of tagged proteins (e.g., protein mislocalization or cell toxicity).7,8 While we were developing and validating the BiTSA system, others published methods for high throughput luminescent thermal shift assays (SplitLuc CETSA and NaLTSA) using plasmid-driven expression of HiBiT- or NanoLuc-tagged proteins,9,10 respectively. Thus, we sought to further advance the field to achieve similarly broad applicability but with the advantages of endogenous expression levels provided by our CRISPR-engineered BiTSA system.

To demonstrate the applicability of BiTSA, we tagged several cellular proteins with HiBiT and observed highly reproducible monitoring of thermal melting/aggregation for all proteins tested using luminescence. BiTSA was used to confirm small molecule target engagement with known small molecule inhibitors directed against kinase targets IRAK4 and p38 MAPK, as well as covalent inhibitors directed against G12C mutant KRAS. In those studies, shifts in the thermal protein aggregation curves induced by the targeted inhibitors could be readily tracked by luminescence. Additionally, we showed that covalent inhibitors of KRAS were specific for the G12C mutant form of KRAS, as they did not stabilize either HiBiT-tagged wild-type KRAS or HRAS against thermal denaturation. Finally, we deployed BiTSA to determine target engagement for two putative “pharmaco-chaperone” molecules, APR-246 and SCH529074, that are proposed to bind and reactivate mutated variants of the p53 tumor suppressor protein.11,12 However, in BiTSA, we were unable to confirm refolding and reactivation of R273H mutant p53 by either APR-246 or SCH529074.

In order to prototype and test the generality of a HiBiT thermal shift assay, we first performed CRISPR/Cas9 tagging with HiBiT on several protein targets across different protein classes, including p38 MAPK (MAPK14), IRAK4, AMPKβ1 (PRKAB1), KRAS, and p53 (TP53). For all HiBiT-tagged targets, a strong luminescent signal was achieved over background when LgBiT protein and NanoLuc substrate were added to lysates from the corresponding polyclonal cell pools (Figure 1c). Due to this robust HiBiT signal, we were able to test several proteins in BiTSA without the need to select clonal HiBiT-tagged cell lines. While polyclonal pools were sufficient for most of our BiTSA examples, we did choose to isolate clonal lines for KRAS and HRAS to enable mutant-specific (G12C vs WT) and isoform-specific (KRAS vs HRAS) BiTSA. The choice of HiBiT tagging at either the N- or C-termini of each of these proteins was based on previously successful efforts using CRISPR-Cas9 (personal communication with Promega; Supporting Information Materials and Methods) or, in the case of p53, previously known modification of the target protein with a FLAG peptide tag13 of similar size to the HiBiT tag.

All proteins tested showed highly reproducible cellular thermal melting profiles that could easily be tracked using luminescence. The tested protein targets showed a range of melting/aggregation temperatures (Tagg) from as low as ∼43 °C for TP53 to a high of ∼66 °C for KRAS (Figure 1d), which were consistent from run to run. In most cases, protein melting temperatures in live cell BiTSA were well in line with those previously reported (e.g., data from ProteomicsDB;14,15Table S1). One notable exception was KRAS, which showed a more than 10 °C higher Tagg in BiTSA relative to a previous report for KRAS CETSA.16 This prior KRAS work, however, was performed using lysate CETSA in a different cell line, and so this Tagg difference was not unexpected as protein melting can vary significantly across cell lines and assay formats17 (Figure S1). Additionally, since KRAS is anchored at the inner cell membrane where it forms localized nanoclusters,18 it is unsurprising to see differences in KRAS melting behavior between membrane disrupted lysate CETSA and live cell BiTSA.

Following establishment of melting curves for all the target proteins, we then looked for thermal stabilization of those proteins by known ligands, including compounds that have been previously shown to induce changes to protein melting upon binding as well as protein–ligand pairs that had not been previously characterized. We first studied in-cell target engagement with BiTSA using inhibitors of two kinases, MAPK14 and IRAK4, where thermal shift assays have previously established protein stabilization upon binding of orthosteric inhibitors.10,19 For MAPK14, binding of AMG-548 has been shown to induce a greater than 10 °C shift in the protein Tagg.10 In BiTSA we saw a similarly large, dose-dependent shift in the melting of MAPK14-HiBiT, with a ΔTagg = 12.4 °C at the highest dose (10 μM). Results were also highly consistent between BiTSA runs on different days and using different cell passages (Figure 2a). We also performed an isothermal dose–response (BiTSA-ITDR) experiment for AMG-548 at 52 °C, generating apparent binding curves for the molecule on MAPK14 that were in line with those previously reported in other thermal shift assay formats (EC50 = 35 nM using AlphaLISA19 and EC50 ≈ 10 nM using fluorescence imaging/AlphaLISA20 vs average EC50 = 2.06 nM in BiTSA) (Figure 2b). Similar to the BiTSA data, the BiTSA-ITDR results were highly consistent across different assay runs.

Figure 2.

Figure 2

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Testing BiTSA in live cells with known target/ligand pairs. (a) Two independent BiTSA runs demonstrating dose-dependent, in-cell target engagement of MAPK14 by AMG-548 in A549 cells. Replicate runs were conducted on different days with different cell passages. (b) BiTSA in-cell isothermal dose response (BiTSA-ITDR) at 52 °C showing AMG-548 target engagement in A549 cells (average EC50 = 2.06 nM). Replicate runs were conducted on different days with different cell passages. (c) Confirming in-cell target engagement of IRAK4 inhibitor, compound 5, using BiTSA. ΔTagg = 1.6 °C at 10 μM; p = 0.017 at 49.1 °C; Student’s t test (two tailed). (d) Negative in-cell BiTSA results for MK-8722 on its known target PRKAB1. BiTSA luminescence data are normalized to the DMSO-treated control samples at 37 °C, which were arbitrarily set to 100. BiTSA data represent mean ± SD (n = 2 replicates). BiTSA-ITDR luminescence data are normalized to the 10 μM AMG-548 samples, which were arbitrarily set to 100. BiTSA-ITDR data represent mean ± SD (n = 4 replicates). All data are representative of at least two independent experiments.

For IRAK4 BiTSA, we tested an amidopyrazole-based IRAK4 inhibitor (compound 5)21 that has previously shown excellent on-target potency and kinome selectivity. While the shifts in the melting temperature were not as pronounced as those with MAPK14 and AMG-548, compound 5 induced a small but significant shift (ΔTagg = 1.6 °C; p = 0.017) in IRAK4 melting at 10 μM (Figure 2c), which was in line with a previously reported thermal shift assay with other kinase inhibitors that bind IRAK4.10 Due to the low variability of luminescence measurements with HiBiT, this example with IRAK4 shows that even small shifts in thermal protein melting profiles can be reliably obtained with BiTSA.

While thermal shift assays can provide powerful confirmation of compound target engagement, they do have limitations in that not all binding events will lead to discernible changes in a protein’s melting and aggregation characteristics. As an example, a known agonist of AMP-activated protein kinase (AMPK), MK-8722, has been shown to directly bind the β-subunit of the heterotrimeric AMPK complex and activate its kinase activity.22 However, using BiTSA, we were unable to show target engagement of MK-8722 on the β1-subunit (PRKAB1), as the compound did not alter the protein’s melting profile, despite showing expected induction of AMPK and ACC phosphorylation under the tested cellular conditions (Figures 2d and S2). While it is challenging to parse the precise reasons for this result, many factors inherent to a protein target and its cellular context can affect the thermal melting profile of a protein in response to binding a small molecule. As such, cellular thermal shift assays like BiTSA should always be interpreted in conjunction with other lines of experimental evidence, as negative results do not preclude bona fide target engagement.

Since its discovery as an essential driver of tumor growth in many cancer types, the KRAS oncoprotein has been a high priority target for therapeutic drug development. Recently, clinical success of covalent inhibitors targeting the Switch II pocket of KRAS(G12C),23−25 a mutant subtype present in many solid tumors (e.g., lung adenocarcinoma and colorectal carcinoma), has been met with significant enthusiasm, as KRAS had previously been recalcitrant to drugging. Since there are many mutant subtypes of KRAS in cancer as well as related members in the RAS protein family, the development of KRAS inhibitors with selectivity over other RAS isoforms and mutants is critical for achieving the desired pharmacological profile as an anticancer drug. Toward that end, we demonstrate BiTSA as a tool for determining the selectivity of KRAS-targeting compounds and confirming the clinical-stage inhibitor sotorasib (AMG 510) as a selective KRAS(G12C) inhibitor over KRAS(WT) and the related RAS family member HRAS.

To determine the feasibility of developing a thermal shift assay for KRAS target engagement, we first demonstrated that the covalent inhibitor, ARS-1620, could significantly stabilize G12C mutant KRAS in a traditional Western blot CETSA experiment (Figure 3a). With those promising results in hand, we then engineered a HiBiT tag onto the N-terminus of KRAS in an NCI-H358 human lung adenocarcinoma cell line with a G12C mutation for deployment in BiTSA. Due to the heterozygous mutational status on KRAS in NCI-H358 cells and incomplete homology-directed repair efficiency during CRISPR/Cas9 gene editing, we were able to select individual clonal cell lines from our initial polyclonal cell pool where the HiBiT tag had been incorporated either solely on a KRAS(G12C) mutant allele or solely on a KRAS(WT) allele. Additionally, in the same cell background, we were able append a HiBiT tag on the related RAS protein family member, HRAS, allowing us to determine compound selectivity across RAS isoforms.

Figure 3.

Figure 3

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Testing covalent KRAS G12C inhibitors in BiTSA. (a) Traditional Western blot CETSA experiment demonstrating significant thermal stabilization of KRAS(G12C) by the covalent inhibitor ARS-1620. (b) KRAS BiTSA showing dose-dependent, in-cell thermal stabilization of KRAS(G12C) by the covalent inhibitor AMG 510. (c) BiTSA in-cell isothermal dose response showing comparative target engagement for AMG 510, ARS-1620, and ARS-1620 atropisomer on KRAS(G12C). (d) BiTSA in-cell isothermal dose response showing selectivity of AMG 510 for G12C mutant KRAS over wild-type KRAS and HRAS. BiTSA luminescence data are normalized to the DMSO-treated control samples at 37 °C, which were arbitrarily set to 100. BiTSA data represent mean ± SD (n = 2 replicates). BiTSA-ITDR luminescence data are normalized to 10 μM AMG 510 samples in KRAS(G12C), which were arbitrarily set to 100. Thermal melting in BiTSA-ITDR was performed at 75 °C, and data represent mean ± SD (n = 3 replicates). All data are representative of at least two independent experiments.

Since the melting/aggregation temperature for KRAS is greater than 65 °C where cellular membrane permeability has likely become compromised, inhibitors tested in BiTSA were washed out prior to thermal challenge to limit overestimation of target engagement due to compound leakage through the membrane. AMG 510 induced a significant dose-dependent stabilization of KRAS(G12C) in NCI-H358 cells relative to control treatment (Figure 3b), and at the 10 and 1 μM dose points more than 50% luminescence signal from HiBiT-KRAS remained even at the highest tested temperature (75 °C). Since AMG 510 covalently labels the mutant cysteine 12 in KRAS, this may in part explain the large thermal shift, as the modification could significantly impact the aggregation properties of unfolded KRAS.

Using BiTSA-ITDR, we were also able to compare target engagement of AMG 510 relative to another G12C inhibitor, ARS-1620 (Figure 3c). After 1 h of treatment, ARS-1620 demonstrated clear target engagement in the ITDR experiment at both 10 and 1 μM concentrations, resulting in a significant increase in HiBiT luminescence signal. By contrast, the inactive atropisomer of ARS-1620 did not induce any stabilization of KRAS(G12C), as expected due to its inability to covalently modify KRAS(G12C). At comparable concentrations to those tested with ARS-1620, AMG 510 also demonstrated very high levels of target engagement, with discernible KRAS stabilization above baseline starting even at the 10 nM compound dose. Notably, the BiTSA-ITDR curves for the covalent inhibitors were approximately 10-fold right-shifted relative to previously reported KRAS labeling experiments24 (e.g., EC50 ≈ 300 nM vs 30 nM for AMG 510), highlighting a liability in thermal shift assays where thermal stability curves may not represent absolute binding affinities for tested compounds. Nevertheless, in BiTSA-ITDR, AMG 510 and ARS-1620 maintain the same relative difference in their apparent binding affinities (approximately 20-fold) as those previously reported,24 demonstrating that BiTSA-ITDR can still serve as a powerful tool for cross comparison and rank ordering of compounds.

Since AMG 510 is reported to only label KRAS with a G12C mutation in the Switch II pocket, we tested the inhibitor’s selectivity in BiTSA against HiBiT-tagged wild-type KRAS and related RAS family member HRAS (which has an identical Switch II pocket with KRAS). In both HiBiT-tagged wild-type KRAS and HRAS, we did not observe thermal stabilization of either protein (Figure 3d), thus confirming the selectivity of AMG 510 for the only the G12C mutant KRAS and demonstrating the utility of BiTSA as a method for differentiating target engagement across highly related proteins.

TP53 is the most frequently mutated gene in human cancers, leading to inactivation of the critical tumor suppressor protein p53. Rescuing the function of mutant p53 has been the focus of many drug discovery efforts to slow tumor growth, and small molecules have been recently described that reactivate mutant p53 through direct binding to the p53 target protein. Two small molecules, SCH529074 and APR-246, have been described as putative “pharmaco-chaperones” for some p53 hotspot mutants (e.g., R173H and R273H) that restore the ability of p53 to bind target DNA promoter sites and drive gene transcription.11,12,26 While some evidence for p53 binding has been presented, unambiguous cellular engagement of p53 has not been demonstrated for these molecules. CETSA studies have previously shown that p53 undergoes substantial thermal stabilization upon binding its consensus DNA target sequences,27 and so we envisioned that BiTSA could serve as a robust method for further characterizing the mechanism of action for these proposed p53 rescue compounds. Should these compounds be able to rescue p53 function, we anticipated being able to measure p53 thermal stabilization upon re-engagement of its cellular DNA target sequences.

To investigate the putative p53 refolding activity for SCH529074 and APR-246, we developed an MDA-MB-468 cell line with an N-terminal HiBiT tag on p53, which maintained a similar thermal melting profile to that previously described.27 The MDA-MB-468 cell line contains an endogenous R273H mutation in p53 and is sensitive to both compounds as demonstrated by reduced cellular proliferation and activation of p53 target genes upon compound treatment. Additionally, there is in vitro biochemical evidence for both compounds directly engaging mutant forms of p53.11,12 We looked for thermal stabilization of mutant R273H p53 in BiTSA upon treatment of MDA-MB-468 cells with either SCH529074 or APR-246 but were unable to see evidence for p53 refolding as indicated by a lack of compound-induced changes to the thermal melting profile (Figure 4). Even at a high 100 μM dose of APR-246, no significant changes to p53 thermal melting behavior were observed. While negative results in the thermal shift assay do not explicitly exclude p53 target engagement by these compounds, they do raise questions around whether they are bona fide reactivators of p53. Recently, new data has also shown that expression of SLC7A11 rather than TP53 mutational status is a superior determinant of sensitivity to APR-246 treatment,28 and a comprehensive study of p53 reactivators (including SCH529074 and APR-246) failed to confirm their ability to stabilize p53 mutants,29 thus further confounding understanding of these compounds’ primary efficacy targets and mechanisms.

Figure 4.

Figure 4

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Assessing TP53 pharmaco-chaperones in BiTSA. (a) BiTSA data lacking clear in-cell target engagement and refolding of R273H mutant p53 by SCH529074 and potential compound assay interference. (b) BiTSA data lacking clear in-cell target engagement and refolding of R273H mutant p53 by APR-246. BiTSA data represent mean ± SD (n = 2 replicates). BiTSA luminescence data are normalized to the DMSO-treated control samples at 37 °C, which were arbitrarily set to 100. All data are representative of at least two independent experiments.

Finally, the p53 BiTSA data was also irregular at higher doses of SCH529074, showing a significant loss of luminescence signal above 5 μM of compound and showing no temperature dependence (Figure 4a). This unusual behavior could potentially be attributed to compound-induced protein aggregation or other modes of assay interference. As a result, our BiTSA data suggest that SCH529074 potentially mediates p53 destabilization rather than stabilization in cells at high doses and could be mediating its primary antiproliferative effects through an alternative mechanism to p53 refolding and reactivation.

Using Promega’s HiBiT-LgBiT split luciferase system, we have established a robust thermal shift assay, BiTSA, that enables quantitative tracking of protein thermal stability while maintaining endogenous cellular expression conditions. By comparison to the laborious protocol for traditional Western blot based CETSA, the simplified and rapid workflow in BiTSA makes it more amenable to high-throughput screening. Also, BiTSA does not require antibody-based protein detection methods, which can often suffer sensitivity and specificity limitations (e.g., narrow dynamic range or antibody cross-reactivity). Discriminating protein isoforms or mutants is a particularly salient use case for the HiBiT thermal shift assay, as demonstrated with the RAS protein family, since the tag can be engineered onto the desired genomic locus with CRISPR/Cas9 and thus expressed exclusively on the protein isoform or mutant under investigation

BiTSA, like other variants of the thermal shift assay, will not detect target engagement for every ligand/protein pair, as demonstrated in studies with the well characterized pair MK-8722/PRKAB1. However, in several test cases, we have demonstrated that it reliably captures protein thermal melting and recapitulates target engagement of known small molecules. BiTSA can reliably be deployed to compare and rank order target engagement across different molecules targeting the same binding pocket of a protein (KRAS(G12C) covalent inhibitors), or it can be utilized to further test confidence in a compound’s proposed mechanism of action (TP53 pharmaco-chaperones). Overall, BiTSA offers a method for researchers to interrogate their drug discovery hits and leads, providing a high-throughput readout to inform on cellular target engagement with endogenous protein expression levels. These attributes should help researchers advance molecules toward the clinic with greater speed and confidence.

Acknowledgments

We would like to thank Marie Schwinn and Kristin Riching of Promega for their help with NanoBiT technology and guidance on CRISPR/Cas9 engineering of HiBiT tags in cells. We would like to thank Benjamin Ruprecht and Jim Tata for providing feedback on the manuscript. We would also like to thank Tony Siu and Mark Demma for their consultation around TP53 pharmaco-chaperone molecules.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00276.

  • Materials and Methods including general procedures for HiBiT tagging, BiTSA, and BiTSA-ITDR (PDF)

  • Protein melting temperatures (XLSX)

  • Clonal amplicon sequencing data (XLSX)

Author Present Address

§ Cedilla Therapeutics, Cambridge, MA 02142, USA

Author Present Address

∥ Remix Therapeutics, Cambridge, MA 02149, USA

Author Contributions

J.D.M wrote the paper. J.D.M., I.C.T., G.V., and A.W.P. designed research. J.D.M, G.V., and N.S. performed research and analyzed data. I.C.T., S.M.B., and A.W.P supervised research.

The authors declare the following competing financial interest(s): Authors are current or former employees of Merck & Co., Inc. and MSD (Merck, Sharp and Dohme Corp.).

Supplementary Material

ml1c00276_si_001.pdf (686.3KB, pdf)
ml1c00276_si_002.xlsx (13.2KB, xlsx)
ml1c00276_si_003.xlsx (93.7KB, xlsx)

References

  1. Stefaniak J.; Huber K. V. M. Importance of Quantifying Drug-Target Engagement in Cells. ACS Med. Chem. Lett. 2020, 11 (4), 403–406. 10.1021/acsmedchemlett.9b00570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Morgan P.; Brown D. G.; Lennard S.; Anderton M. J.; Barrett J. C.; Eriksson U.; Fidock M.; Hamren B.; Johnson A.; March R. E.; Matcham J.; Mettetal J.; Nicholls D. J.; Platz S.; Rees S.; Snowden M. A.; Pangalos M. N. Impact of a five-dimensional framework on R&D productivity at AstraZeneca. Nat. Rev. Drug Discovery 2018, 17 (3), 167–181. 10.1038/nrd.2017.244. [DOI] [PubMed] [Google Scholar]
  3. Morgan P.; Van der Graaf P. H.; Arrowsmith J.; Feltner D. E.; Drummond K. S.; Wegner C. D.; Street S. D. A. Can the flow of medicines be improved? Fundamental pharmacokinetic and pharmacological principles toward improving Phase II survival. Drug Discovery Today 2012, 17 (9–10), 419–424. 10.1016/j.drudis.2011.12.020. [DOI] [PubMed] [Google Scholar]
  4. Molina D. M.; Jafari R.; Ignatushchenko M.; Seki T.; Larsson E. A.; Dan C.; Sreekumar L.; Cao Y. H.; Nordlund P. Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay. Science 2013, 341 (6141), 84–87. 10.1126/science.1233606. [DOI] [PubMed] [Google Scholar]
  5. Hall M. P.; Unch J.; Binkowski B. F.; Valley M. P.; Butler B. L.; Wood M. G.; Otto P.; Zimmerman K.; Vidugiris G.; Machleidt T.; Robers M. B.; Benink H. A.; Eggers C. T.; Slater M. R.; Meisenheimer P. L.; Klaubert D. H.; Fan F.; Encell L. P.; Wood K. V. Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate. ACS Chem. Biol. 2012, 7 (11), 1848–1857. 10.1021/cb3002478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Schwinn M. K.; Machleidt T.; Zimmerman K.; Eggers C. T.; Dixon A. S.; Hurst R.; Hall M. P.; Encell L. P.; Binkowski B. F.; Wood K. V. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide. ACS Chem. Biol. 2018, 13 (2), 467–474. 10.1021/acschembio.7b00549. [DOI] [PubMed] [Google Scholar]
  7. Ratz M.; Testa I.; Hell S. W.; Jakobs S. CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells. Sci. Rep. 2015, 5, 9592. 10.1038/srep09592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eguchi Y.; Makanae K.; Hasunuma T.; Ishibashi Y.; Kito K.; Moriya H. Estimating the protein burden limit of yeast cells by measuring the expression limits of glycolytic proteins. eLife 2018, 7, e34595. 10.7554/eLife.34595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Martinez N. J.; Asawa R. R.; Cyr M. G.; Zakharov A.; Urban D. J.; Roth J. S.; Wallgren E.; Klumpp-Thomas C.; Coussens N. P.; Rai G.; Yang S. M.; Hall M. D.; Marugan J. J.; Simeonov A.; Henderson M. J. A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase. Sci. Rep. 2018, 8, 9472–9487. 10.1038/s41598-018-27834-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dart M. L.; Machleidt T.; Jost E.; Schwinn M. K.; Robers M. B.; Shi C.; Kirkland T. A.; Killoran M. P.; Wilkinson J. M.; Hartnett J. R.; Zimmerman K.; Wood K. V. Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift. ACS Med. Chem. Lett. 2018, 9 (6), 546–551. 10.1021/acsmedchemlett.8b00081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Demma M.; Maxwell E.; Ramos R.; Liang L. Z.; Li C.; Hesk D.; Rossman R.; Mallams A.; Doll R.; Liu M.; Seidel-Dugan C.; Bishop W. R.; Dasmahapatra B. SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53. J. Biol. Chem. 2010, 285 (14), 10198–10212. 10.1074/jbc.M109.083469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Zhang Q.; Bykov V. J. N.; Wiman K. G.; Zawacka-Pankau J. APR-246 reactivates mutant p53 by targeting cysteines 124 and 277. Cell Death Dis. 2018, 9, 439. 10.1038/s41419-018-0463-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. He F.; Borcherds W.; Song T. J.; Wei X.; Das M.; Chen L. H.; Daughdrill G. W.; Chen J. D. Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (18), 8859–8868. 10.1073/pnas.1903077116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. https://www.proteomicsdb.org/.
  15. Samaras P.; Schmidt T.; Frejno M.; Gessulat S.; Reinecke M.; Jarzab A.; Zecha J.; Mergner J.; Giansanti P.; Ehrlich H. C.; Aiche S.; Rank J.; Kienegger H.; Krcmar H.; Kuster B.; Wilhelm M. ProteomicsDB: a multi-omics and multi-organism resource for life science research. Nucleic Acids Res. 2020, 48 (D1), D1153–D1163. 10.1093/nar/gkz974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guo X.; Zhao W.; Liu Z.; Wang J. Spiclomazine displays a preferential anti-tumor activity in mutant KRas-driven pancreatic cancer. Oncotarget 2018, 9 (6), 6938–6951. 10.18632/oncotarget.24025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Seashore-Ludlow B.; Axelsson H.; Lundback T. Perspective on CETSA Literature: Toward More Quantitative Data Interpretation. SLAS Discovery 2020, 25 (2), 118–126. 10.1177/2472555219884524. [DOI] [PubMed] [Google Scholar]
  18. Zhou Y.; Prakash P.; Liang H.; Cho K. J.; Gorfe A. A.; Hancock J. F. Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output. Cell 2017, 168 (1–2), 239. 10.1016/j.cell.2016.11.059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jafari R.; Almqvist H.; Axelsson H.; Ignatushchenko M.; Lundback T.; Nordlund P. Martinez Molina, D., The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 2014, 9 (9), 2100–2122. 10.1038/nprot.2014.138. [DOI] [PubMed] [Google Scholar]
  20. Axelsson H.; Almqvist H.; Otrocka M.; Vallin M.; Lundqvist S.; Hansson P.; Karlsson U.; Lundback T.; Seashore-Ludlow B. In Situ Target Engagement Studies in Adherent Cells. ACS Chem. Biol. 2018, 13 (4), 942–950. 10.1021/acschembio.7b01079. [DOI] [PubMed] [Google Scholar]
  21. McElroy W. T.; Tan Z.; Ho G.; Paliwal S.; Li G. Q.; Seganish W. M.; Tulshian D.; Tata J.; Fischmann T. O.; Sondey C.; Bian H.; Bober L.; Jackson J.; Garlisi C. G.; Devito K.; Fossetta J.; Lundell D.; Niu X. D. Potent and Selective Amidopyrazole Inhibitors of IRAK4 That Are Efficacious in a Rodent Model of Inflammation. ACS Med. Chem. Lett. 2015, 6 (6), 677–682. 10.1021/acsmedchemlett.5b00106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Myers R. W.; Guan H. P.; Ehrhart J.; Petrov A.; Prahalada S.; Tozzo E.; Yang X. D.; Kurtz M. M.; Trujillo M.; Trotter D. G.; Feng D. Q.; Xu S. Y.; Eiermann G.; Holahan M. A.; Rubins D.; Conarello S.; Niu X. D.; Souza S. C.; Miller C.; Liu J. Q.; Lu K.; Feng W.; Li Y.; Painter R. E.; Milligan J. A.; He H. B.; Liu F.; Ogawa A.; Wisniewski D.; Rohm R. J.; Wang L. Y.; Bunzel M.; Qian Y.; Zhu W.; Wang H. W.; Bennet B.; Scheuch L. L.; Fernandez G. E.; Li C.; Klimas M.; Zhou G. C.; van Heek M.; Biftu T.; Weber A.; Kelley D. E.; Thornberry N.; Erion M. D.; Kemp D. M.; Sebhat I. K. Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy. Science 2017, 357 (6350), 507–511. 10.1126/science.aah5582. [DOI] [PubMed] [Google Scholar]
  23. Janes M. R.; Zhang J. C.; Li L. S.; Hansen R.; Peters U.; Guo X.; Chen Y. C.; Babbar A.; Firdaus S. J.; Darjania L.; Feng J.; Chen J. H.; Li S. W.; Li S. S.; Long Y. O.; Thach C.; Liu Y.; Zarieh A.; Ely T.; Kucharski J. M.; Kessler L. V.; Wu T.; Yu K.; Wang Y.; Yao Y.; Deng X. H.; Zarrinkar P. P.; Brehmer D.; Dhanak D.; Lorenzi M. V.; Hu-Lowe D.; Patricelli M. P.; Ren P. D.; Liu Y. Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor. Cell 2018, 172 (3), 578–589. 10.1016/j.cell.2018.01.006. [DOI] [PubMed] [Google Scholar]
  24. Canon J.; Rex K.; Saiki A. Y.; Mohr C.; Cooke K.; Bagal D.; Gaida K.; Holt T.; Knutson C. G.; Koppada N.; Lanman B. A.; Werner J.; Rapaport A. S.; San Miguel T.; Ortiz R.; Osgood T.; Sun J. R.; Zhu X. C.; McCarter J. D.; Volak L. P.; Houk B. E.; Fakih M. G.; O’Neil B. H.; Price T. J.; Falchook G. S.; Desai J.; Kuo J.; Govindan R.; Hong D. S.; Ouyang W. J.; Henary H.; Arvedson T.; Cee V. J.; Lipford J. R. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 2019, 575 (7781), 217–223. 10.1038/s41586-019-1694-1. [DOI] [PubMed] [Google Scholar]
  25. Hallin J.; Engstrom L. D.; Hargis L.; Calinisan A.; Aranda R.; Briere D. M.; Sudhakar N.; Bowcut V.; Baer B. R.; Ballard J. A.; Burkard M. R.; Fell J. B.; Fischer J. P.; Vigers G. P.; Xue Y. H.; Gatto S.; Fernandez-Banet J.; Pavlicek A.; Velastagui K.; Chao R. C.; Barton J.; Pierobon M.; Baldelli E.; Patricoin E. F.; Cassidy D. P.; Marx M. A.; Rybkin I. I.; Johnson M. L.; Ou S. H. I.; Lito P.; Papadopoulos K. P.; Janne P. A.; Olson P.; Christensen J. G. The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discovery 2020, 10 (1), 54–71. 10.1158/2159-8290.CD-19-1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Fransson A.; Glaessgen D.; Alfredsson J.; Wiman K. G.; Bajalica-Lagercrantz S.; Mohell N. Strong synergy with APR-246 and DNA-damaging drugs in primary cancer cells from patients with TP53 mutant High-Grade Serous ovarian cancer. J. Ovarian Res. 2016, 9 (1), 27–36. 10.1186/s13048-016-0239-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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.; Drewes G. Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 2014, 346 (6205), 1255784. 10.1126/science.1255784. [DOI] [PubMed] [Google Scholar]
  28. Fujihara K. M.; Corrales-Benitez M.; Cabalag C. S.; Zhang B. Z.; Ko H. S.; Liu D. S.; Simpson K.; Haupt Y.; Haupt S.; Phillips W. A.; Clemons N. J.. SLC7A11 is a superior determinant of APR-246 (Eprenetapopt) response than TP53 mutation status. bioRxiv, November 29, 2020, ver. 1. 10.1101/2020.11.29.398875. [DOI] [PubMed] [Google Scholar]
  29. Chen S.; Wu J. L.; Liang Y.; Tang Y. G.; Song H. X.; Wu L. L.; Xing Y. F.; Yan N.; Li Y. T.; Wang Z. Y.; Xiao S. J.; Lu X.; Chen S. J.; Lu M. Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site. Cancer Cell 2021, 39 (2), 225–239. 10.1016/j.ccell.2020.11.013. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

ml1c00276_si_001.pdf (686.3KB, pdf)
ml1c00276_si_002.xlsx (13.2KB, xlsx)
ml1c00276_si_003.xlsx (93.7KB, xlsx)

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