1932

Abstract

A drug must engage its intended target to achieve its therapeutic effect. However, conclusively measuring target engagement (TE) in situ is challenging. This complicates preclinical development and is considered a key factor in the high rate of attrition in clinical trials. Here, we discuss a recently developed, label-free, biophysical assay, the cellular thermal shift assay (CETSA), which facilitates the direct assessment of TE in cells and tissues at various stages of drug development. CETSA also reveals biochemical events downstream of drug binding and therefore provides a promising means of establishing mechanistic biomarkers. The implementation of proteome-wide CETSA using quantitative mass spectrometry represents a novel strategy for defining off-target toxicity and polypharmacology and for identifying downstream mechanistic biomarkers. The first year of CETSA applications in the literature has focused on TE studies in cell culture systems and has confirmed the broad applicability of CETSA to many different target families. The next phase of CETSA applications will likely encompass comprehensive animal and patient studies, and CETSA will likely serve as a very valuable tool in many stages of preclinical and clinical drug development.

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2016-01-06
2024-06-17
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Literature Cited

  1. Kenakin T, Bylund DB, Toews ML, Mullane K, Winquist RJ, Williams M. 1.  2014. Replicated, replicable and relevant–target engagement and pharmacological experimentation in the 21st century. Biochem. Pharmacol. 87:64–77 [Google Scholar]
  2. Durham TB, Blanco MJ. 2.  2015. Target engagement in lead generation. Bioorg. Med. Chem. Lett. 25:998–1008 [Google Scholar]
  3. Simon GM, Niphakis MJ, Cravatt BF. 3.  2013. Determining target engagement in living systems. Nat. Chem. Biol. 9:200–5 [Google Scholar]
  4. Nelson B. 4.  2012. The lessons of failure: Clinical flops are helping researchers define the keys to successful cancer drug trials. Cancer Cytopathol. 120:359–60 [Google Scholar]
  5. Morgan P, Van Der Graaf PH, Arrowsmith J, Feltner DE, Drummond KS. 5.  et al. 2012. Can the flow of medicines be improved? Fundamental pharmacokinetic and pharmacological principles toward improving Phase II survival. Drug Discov. Today 17:419–24 [Google Scholar]
  6. Auld DS, Thorne N, Maguire WF, Inglese J. 6.  2009. Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. PNAS 106:3585–90 [Google Scholar]
  7. Schmidt C. 7.  2010. GSK/Sirtris compounds dogged by assay artifacts. Nat. Biotechnol. 28:185–86 [Google Scholar]
  8. Guha M. 8.  2011. PARP inhibitors stumble in breast cancer. Nat. Biotechnol. 29:373–74 [Google Scholar]
  9. Liu X, Shi Y, Maag DX, Palma JP, Patterson MJ. 9.  et al. 2012. Iniparib nonselectively modifies cysteine-containing proteins in tumor cells and is not a bona fide PARP inhibitor. Clin. Cancer Res. 18:510–23 [Google Scholar]
  10. Phillips KA, Van Bebber S, Issa AM. 10.  2006. Diagnostics and biomarker development: priming the pipeline. Nat. Rev. Drug Discov. 5:463–69 [Google Scholar]
  11. Zinn N, Hopf C, Drewes G, Bantscheff M. 11.  2012. Mass spectrometry approaches to monitor protein-drug interactions. Methods 57:430–40 [Google Scholar]
  12. Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA. 12.  et al. 2013. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341:84–87 [Google Scholar]
  13. Crothers DM. 13.  1971. Statistical thermodynamics of nucleic acid melting transitions with coupled binding equilibria. Biopolymers 10:2147–60 [Google Scholar]
  14. Schellman JA. 14.  1975. Macromolecular binding. Biopolymers 14:999–1018 [Google Scholar]
  15. Brandts JF, Lin LN. 15.  1990. Study of strong to ultratight protein interactions using differential scanning calorimetry. Biochemistry 29:6927–40 [Google Scholar]
  16. Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P. 16.  2006. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 357:289–98 [Google Scholar]
  17. Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ Jr. 17.  2006. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. PNAS 103:15835–40 [Google Scholar]
  18. Pantoliano MW, Bone RF, Rhind AW, Salemme FR. 18.  2000. Microplate thermal shift assay apparatus for ligand development and multi-variable protein chemistry optimization. US Patent No. 6036920 [Google Scholar]
  19. Kopec J, Schneider G. 19.  2011. Comparison of fluorescence and light scattering based methods to assess formation and stability of protein–protein complexes. J. Struct. Biol. 175:216–23 [Google Scholar]
  20. Dahlroth SL, Gurmu D, Schmitzberger F, Engman H, Haas J. 20.  et al. 2009. Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus. FEBS J. 276:6636–45 [Google Scholar]
  21. Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC. 21.  2008. Microscale fluorescent thermal stability assay for membrane proteins. Structure 16:351–59 [Google Scholar]
  22. Guettou F, Quistgaard EM, Raba M, Moberg P, Löw C, Nordlund P. 22.  2014. Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2. Nat. Struct. Mol. Biol. 21:728–31 [Google Scholar]
  23. Larsson EA, Jansson A, Ng FM, Then SW, Panicker R. 23.  et al. 2013. Fragment-based ligand design of novel potent inhibitors of tankyrases. J. Med. Chem. 56:4497–508 [Google Scholar]
  24. Fedorov O, Marsden B, Pogacic V, Rellos P, Müller S. 24.  et al. 2007. A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. PNAS 104:20523–28 [Google Scholar]
  25. Holdgate GA, Ward WH. 25.  2005. Measurements of binding thermodynamics in drug discovery. Drug Discov. Today 10:1543–50 [Google Scholar]
  26. Matulis D, Kranz JK, Salemme FR, Todd MJ. 26.  2005. Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44:5258–66 [Google Scholar]
  27. Asial I, Cheng YX, Engman H, Dollhopf M, Wu B. 27.  et al. 2013. Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell. Nat. Commun. 4:2901 [Google Scholar]
  28. Mourão MA, Hakim JB, Schnell S. 28.  2014. Connecting the dots: the effects of macromolecular crowding on cell physiology. Biophys. J. 107:2761–66 [Google Scholar]
  29. Muga A, Moro F. 29.  2008. Thermal adaptation of heat shock proteins. Curr. Protein Pept. Sci. 9:552–66 [Google Scholar]
  30. Weibrecht I, Leuchowius KJ, Clausson CM, Conze T, Jarvius M. 30.  et al. 2010. Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev. Proteom 7:401–9 [Google Scholar]
  31. Gillet JP, Gottesman MM. 31.  2010. Mechanisms of multidrug resistance in cancer. Methods Mol. Biol. 596:47–76 [Google Scholar]
  32. Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundbäck T. 32.  et al. 2014. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 9:2100–22 [Google Scholar]
  33. Eglen RM, Reisine T, Roby P, Rouleau N, Illy C. 33.  et al. 2008. The use of AlphaScreen technology in HTS: current status. Curr. Chem. Genomics 1:2–10 [Google Scholar]
  34. Nordlund P, Martinez Molina D, Lundbäck T. 34.  2015. Methods for determining ligand binding to a target protein using a thermal shift assay US Patent Appl. No. 20150133336 [Google Scholar]
  35. Nordlund P. 35.  2012. Methods for determining ligand binding to a target protein using a thermal shift assay Patent No. GB2490404 [Google Scholar]
  36. Phimister EG, Huang J. 36.  2013. Tracking drugs. N. Engl. J. Med. 369:1168–69 [Google Scholar]
  37. Savitski MM, Reinhard FB, Franken H, Werner T, Savitski MF. 37.  et al. 2014. Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346:1255784 [Google Scholar]
  38. Werner T, Becher I, Sweetman G, Doce C, Savitski MM, Bantscheff M. 38.  2012. High-resolution enabled TMT 8-plexing. Anal. Chem. 84:7188–94 [Google Scholar]
  39. Chahrour O, Cobice D, Malone J. 39.  2015. Stable isotope labelling methods in mass spectrometry-based quantitative proteomics. J. Pharm. Biomed. Anal. 113:2–20 [Google Scholar]
  40. Zhang L, Holmes IP, Hochgräfe F, Walker SR, Ali NA. 40.  et al. 2013. Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling. J. Proteome Res. 12:3104–16 [Google Scholar]
  41. Salisbury CM, Cravatt BF. 41.  2007. Activity-based probes for proteomic profiling of histone deacetylase complexes. PNAS 104:1171–76 [Google Scholar]
  42. Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN. 42.  et al. 2010. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467:596–99 [Google Scholar]
  43. Latif M, Saeed A, Kim SH. 43.  2013. Journey of the ALK-inhibitor CH5424802 to Phase II clinical trial. Arch. Pharm. Res. 36:1051–54 [Google Scholar]
  44. Wahlin S, Harper P. 44.  2010. Skin ferrochelatase and photosensitivity in mice and man. J. Investig. Dermatol. 130:631–33 [Google Scholar]
  45. Gelot P, Dutartre H, Khammari A, Boisrobert A, Schmitt C. 45.  et al. 2013. Vemurafenib: an unusual UVA-induced photosensitivity. Exp. Dermatol. 22:297–98 [Google Scholar]
  46. Dovega R. 46.  2014. Structural and functional studies of proteins in cell signaling and cancer PhD Thesis, Karolinska Inst., Stockholm [Google Scholar]
  47. Leroy B, Girard L, Hollestelle A, Minna JD, Gazdar AF, Soussi T. 47.  2014. Analysis of TP53 mutation status in human cancer cell lines: a reassessment. Hum. Mutat. 35:756–65 [Google Scholar]
  48. Walerych D, Napoli M, Collavin L, Del Sal G. 48.  2012. The rebel angel: mutant p53 as the driving oncogene in breast cancer. Carcinogenesis 33:2007–17 [Google Scholar]
  49. Brehme M, Hantschel O, Colinge J, Kaupe I, Planyavsky M. 49.  et al. 2009. Charting the molecular network of the drug target Bcr-Abl. PNAS 106:7414–19 [Google Scholar]
  50. Yin SQ, Wang JJ, Zhang CM, Liu ZP. 50.  2012. The development of MetAP-2 inhibitors in cancer treatment. Curr. Med. Chem. 19:1021–35 [Google Scholar]
  51. Ahuja V, Sharma S. 51.  2014. Drug safety testing paradigm, current progress and future challenges: an overview. J. Appl. Toxicol. 34:576–94 [Google Scholar]
  52. Huber KVM, Salah E, Radic B, Gridling M, Elkins JM. 52.  et al. 2014. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature 508:222–27 [Google Scholar]
  53. Gad H, Koolmeister T, Jemth A-S, Eshtad S, Jacques SA. 53.  et al. 2014. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature 508:215–21 [Google Scholar]
  54. Bai L, Chen J, McEachern D, Liu L, Zhou H. 54.  et al. 2014. BM-1197: a novel and specific Bcl-2/Bcl-xL inhibitor inducing complete and long-lasting tumor regression in vivo. PLOS ONE 9:e99404 [Google Scholar]
  55. Aguilar A, Zhou H, Chen J, Liu L, Bai L. 55.  et al. 2013. A potent and highly efficacious Bcl-2/Bcl-xL inhibitor. J. Med. Chem. 56:3048–67 [Google Scholar]
  56. Khoo KH, Verma CS, Lane DP. 56.  2014. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat. Rev. Drug Discov. 13:217–36 [Google Scholar]
  57. Tan BX, Brown CJ, Ferrer FJ, Yuen TY, Quah ST. 57.  et al. 2015. Assessing the efficacy of Mdm2/Mdm4-inhibiting stapled peptides using cellular thermal shift assays. Sci. Rep. 5:12116 [Google Scholar]
  58. Cromm PM, Spiegel J, Grossmann TN. 58.  2015. Hydrocarbon stapled peptides as modulators of biological function. ACS Chem. Biol. 10:1362–75 [Google Scholar]
  59. Qin JJ, Wang W, Voruganti S, Wang H, Zhang WD, Zhang R. 59.  2015. Identification of a new class of natural product MDM2 inhibitor: in vitro and in vivo anti-breast cancer activities and target validation. Oncotarget 6:2623–40 [Google Scholar]
  60. Miettinen TP, Björklund M. 60.  2014. NQO2 is a reactive oxygen species generating off-target for acetaminophen. Mol. Pharm. 11:4395–404 [Google Scholar]
  61. Wu JM, Hsieh TC. 61.  2011. Resveratrol: a cardioprotective substance. Ann. N.Y. Acad. Sci. 1215:16–21 [Google Scholar]
  62. Leung KK, Shilton BH. 62.  2015. Quinone reductase 2 is an adventitious target of protein kinase CK2 inhibitors TBBz (TBI) and DMAT. Biochemistry 54:47–59 [Google Scholar]
  63. Winger JA, Hantschel O, Superti-Furga G, Kuriyan J. 63.  2009. The structure of the leukemia drug imatinib bound to human quinone reductase 2 (NQO2). BMC Struct. Biol. 9:7 [Google Scholar]
  64. Chan-Penebre E, Kuplast KG, Majer CR, Boriack-Sjodin PA, Wigle TJ. 64.  et al. 2015. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models. Nat. Chem. Biol. 11:432–37 [Google Scholar]
  65. Bradley WD, Arora S, Busby J, Balasubramanian S, Gehling VS. 65.  et al. 2014. EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation. Chem. Biol. 21:1463–75 [Google Scholar]
  66. Dollery CT. 66.  2014. Lost in Translation (LiT): IUPHAR review 6. Br. J. Pharmacol. 171:2269–90 [Google Scholar]
  67. Swinney DC. 67.  2013. The contribution of mechanistic understanding to phenotypic screening for first-in-class medicines. J. Biomol. Screen 18:1186–92 [Google Scholar]
  68. Moffat JG, Rudolph J, Bailey D. 68.  2014. Phenotypic screening in cancer drug discovery—past, present and future. Nat. Rev. Drug Discov. 13:588–602 [Google Scholar]
  69. Martinez Molina D, Nordlund P. 69.  2014. The cellular thermal shift assay as a tool for setting individual treatment schemes. Cancer Res. 74:3479 (Abstr.) [Google Scholar]
  70. Sackton KL, Dimova N, Zeng X, Tian W, Zhang M. 70.  et al. 2014. Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C. Nature 514:646–49 [Google Scholar]
  71. Malik R, Khan AP, Asangani IA, Cieślik M, Prensner JR. 71.  et al. 2015. Targeting the MLL complex in castration-resistant prostate cancer. Nat. Med. 21:344–52 [Google Scholar]
  72. Sidrauski C, Tsai JC, Kampmann M, Hearn BR, Vedantham P. 72.  et al. 2015. Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife 4:e07314 [Google Scholar]
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