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Bright and stable luminescent probes for target engagement profiling in live cells

Nature Chemical Biology volume 17, pages 1168–1177 (2021)Cite this article

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Abstract

The pace of progress in biomedical research directly depends on techniques that enable the quantitative interrogation of interactions between proteins and other biopolymers, or with their small-molecule ligands. Time-resolved Förster resonance energy transfer (TR-FRET) assay platforms offer high sensitivity and specificity. However, the paucity of accessible and biocompatible luminescent lanthanide complexes, which are essential reagents for TR-FRET-based approaches, and their poor cellular permeability have limited broader adaptation of TR-FRET beyond homogeneous and extracellular assay applications. Here, we report the development of CoraFluors, a new class of macrotricyclic terbium complexes, which are synthetically readily accessible, stable in biological media and exhibit photophysical and physicochemical properties that are desirable for biological studies. We validate the performance of CoraFluors in cell-free systems, identify cell-permeable analogs and demonstrate their utility in the quantitative domain-selective characterization of Keap1 ligands, as well as in isoform-selective target engagement profiling of HDAC1 inhibitors in live cells.

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Fig. 1: Chemical structures and photophysical and physicochemical characterization of representative CoraFluors.
Fig. 2: Biochemical validation of CoraFluors with the HSFP6xHis test system.
Fig. 3: CoraFluors enable versatile and domain-specific interrogation of Keap1–Keap1 and Keap1–small molecule interactions.
Fig. 4: CoraFluor performance in TR-FRET ligand-displacement assays for HDAC1-HaloTag in crude lysate from transiently transfected HEK293T cells.
Fig. 5: CoraFluors enable TR-FRET based target engagement profiling in live cells.

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Data availability

The main data, including raw data, supporting the findings of this study are available within the Article and its Supplementary Information files. Extra data (NMR and LC/MS data files) are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Wegner, K. D., Jin, Z., Lindén, S., Jennings, T. L. & Hildebrandt, N. Quantum-dot-based Förster resonance energy transfer immunoassay for sensitive clinical diagnostics of low-volume serum samples. ACS Nano 7, 7411–7419 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Blay, V., Tolani, B., Ho, S. P. & Arkin, M. R. High-throughput screening: today’s biochemical and cell-based approaches. Drug Discov. Today 25, 1807–1821 (2020).

    Article  CAS  Google Scholar 

  3. Busby, S. A. et al. Advancements in assay technologies and strategies to enable drug discovery. ACS Chem. Biol. 15, 2636–2648 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Sanderson, M. J., Smith, I., Parker, I. & Bootman, M. D. Fluorescence microscopy. Cold Spring Harbor Protoc. https://doi.org/10.1101/pdb.top071795 (2014).

  5. Cho, U. et al. Ultrasensitive optical imaging with lanthanide lumiphores. Nat. Chem. Biol. 14, 15–21 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Cho, U. & Chen, J. K. Lanthanide-based optical probes of biological systems. Cell Chem. Biol. 27, 921–936 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Francés-Soriano, L. et al. In situ rolling circle amplification Förster resonance energy transfer (RCA-FRET) for washing-free real-time single-protein imaging. Anal. Chem. 93, 1842–1850 (2021).

    Article  PubMed  CAS  Google Scholar 

  8. Moore, E. G., Samuel, A. P. S. & Raymond, K. N. From antenna to assay: lessons learned in lanthanide luminescence. Acc. Chem. Res. 42, 542–552 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bünzli, J.-C. G. Lanthanide luminescence for biomedical analyses and imaging. Chem. Rev. 110, 2729–2755 (2010).

    Article  PubMed  CAS  Google Scholar 

  10. Ergin, E., Dogan, A., Parmaksiz, M., E Elcin, A. & M Elcin, Y. Time-resolved fluorescence resonance energy transfer [TR-FRET] assays for biochemicalprocesses. Curr. Pharm. Biotechnol. 17, 1222–1230 (2016).

  11. Zwier, J. M. & Hildebrandt, N. in Reviews in Fluorescence 2016 (ed. Geddes, C. D.) 17–43 (Springer, 2017).

  12. Wu, Y.-T. et al. Quantum dot-based FRET immunoassay for HER2 using ultrasmall affinity proteins. Small 14, 1802266 (2018).

    Article  CAS  Google Scholar 

  13. Cardoso Dos Santos, M. et al. Time-gated FRET nanoprobes for autofluorescence-free long-term in vivo imaging of developing zebrafish. Adv. Mater. 32, 2003912 (2020).

    Article  CAS  Google Scholar 

  14. Degorce, F. et al. HTRF: a technology tailored for drug discovery—a review of theoretical aspects and recent applications. Curr. Chem. Genomics 3, 22–32 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Algar, W. R., Hildebrandt, N., Vogel, S. S. & Medintz, I. L. FRET as a biomolecular research tool—understanding its potential while avoiding pitfalls. Nat. Methods 16, 815–829 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Coussens, N. P. et al. Assay Guidance Manual (Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2020).

  17. Hildebrandt, N., Wegner, K. D. & Algar, W. R. Luminescent terbium complexes: superior Förster resonance energy transfer donors for flexible and sensitive multiplexed biosensing. Coord. Chem. Rev. 273–274, 125–138 (2014).

    Article  CAS  Google Scholar 

  18. Sy, M., Nonat, A., Hildebrandt, N. & Charbonnière, L. J. Lanthanide-based luminescence biolabelling. Chem. Commun. 52, 5080–5095 (2016).

    Article  CAS  Google Scholar 

  19. Zwier, J. M. et al. A fluorescent ligand-binding alternative using tag-lite® technology. J. Biomol. Screening 15, 1248–1259 (2010).

    Article  CAS  Google Scholar 

  20. Xu, J. et al. Octadentate cages of Tb(III) 2-hydroxyisophthalamides: a new standard for luminescent lanthanide labels. J. Am. Chem. Soc. 133, 19900–19910 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Juillerat, A. et al. Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem. Biol. 10, 313–317 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Mathis, G. & Bazin, H. in Lanthanide Luminescence: Photophysical, Analytical and Biological Aspects (eds Hänninen, P. & Härmä, H.) 47–88 (Springer, 2011).

  24. Rodenko, O., Fodgaard, H., Tidemand-Lichtenberg, P., Petersen, P. M. & Pedersen, C. 340-nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays. Opt. Express 24, 22135–22143 (2016).

    Article  CAS  PubMed  Google Scholar 

  25. Johnson, T. W., Dress, K. R. & Edwards, M. Using the golden triangle to optimize clearance and oral absorption. Bioorg. Med. Chem. Lett. 19, 5560–5564 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Peterson, S. N. & Kwon, K. The HaloTag: improving soluble expression and applications in protein functional analysis. Curr. Chem. Genomics 6, 8–17 (2012).

    Article  CAS  Google Scholar 

  27. Cuadrado, A. et al. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat. Rev. Drug Discov. 18, 295–317 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Lu, M. et al. Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway. Eur. J. Med. Chem. 146, 251–259 (2018).

    Article  CAS  PubMed  Google Scholar 

  29. Tong, B. et al. Bardoxolone conjugation enables targeted protein degradation of BRD4. Sci. Rep. 10, 15543 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dayalan Naidu, S. et al. C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape. Sci. Rep. 8, 8037 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Poore, D. D. et al. Development of a high-throughput Cul3-Keap1 time-resolved fluorescence resonance energy transfer (TR-FRET) assay for identifying Nrf2 activators. SLAS Discov. 24, 175–189 (2019).

    CAS  PubMed  Google Scholar 

  32. Lee, S., Abed, D. A., Beamer, L. J. & Hu, L. Development of a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay for the inhibition of Keap1-Nrf2 protein-protein interaction. SLAS DISCOVERY: Advancing the Science of Drug Discovery 26, 100–112 (2021).

    CAS  Google Scholar 

  33. Cleasby, A. et al. Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO. PLoS ONE 9, e98896 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Tran, K. T. et al. A comparative assessment study of known small-molecule Keap1−Nrf2 protein–protein interaction inhibitors: chemical synthesis, binding properties and cellular activity. J. Med. Chem. 62, 8028–8052 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Hulme, E. C. & Trevethick, M. A. Ligand binding assays at equilibrium: validation and interpretation. Br. J. Pharmacol. 161, 1219–1237 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jarmoskaite, I., AlSadhan, I., Vaidyanathan, P. P. & Herschlag, D. How to measure and evaluate binding affinities. eLife 9, e57264 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wilson, B. D. & Soh, H. T. Re-evaluating the conventional wisdom about binding assays. Trends Biochem. Sci. 45, 639–649 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Badr, C. E. et al. Obtusaquinone: a cysteine-modifying compound that targets Keap1 for degradation. ACS Chem. Biol. 15, 1445–1454 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu, J. et al. Treatment of obesity with celastrol. Cell 161, 999–1011 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Davies, T. G. et al. Monoacidic inhibitors of the kelch-like ECH-associated protein 1: nuclear factor erythroid 2-related factor 2 (KEAP1:NRF2) protein–protein interaction with high cell potency identified by fragment-based discovery. J. Med. Chem. 59, 3991–4006 (2016).

    Article  CAS  PubMed  Google Scholar 

  41. Dubach, J. M. et al. In vivo imaging of specific drug-target binding at subcellular resolution. Nat. Commun. 5, 3946 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Robers, M. B. et al. Target engagement and drug residence time can be observed in living cells with BRET. Nat. Commun. 6, 10091 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Vinegoni, C., Feruglio, P. F., Gryczynski, I., Mazitschek, R. & Weissleder, R. Fluorescence anisotropy imaging in drug discovery. Adv. Drug Delivery Rev. 151–152, 262–288 (2019).

    Article  CAS  Google Scholar 

  44. Mohandessi, S., Rajendran, M., Magda, D. & Miller, L. W. Cell-penetrating peptides as delivery vehicles for a protein-targeted terbium complex. Chem. A Eur. J. 18, 10825–10829 (2012).

    Article  CAS  Google Scholar 

  45. Chen, T., Pham, H., Mohamadi, A. & Miller, L. W. Single-chain lanthanide luminescence biosensors for cell-based imaging and screening of protein–protein interactions. iScience 23, 101533 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Banks, C. A. S. et al. Differential HDAC1/2 network analysis reveals a role for prefoldin/CCT in HDAC1/2 complex assembly. Sci. Rep. 8, 13712 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Mazitschek, R., Patel, V., Wirth, D. F. & Clardy, J. Development of a fluorescence polarization based assay for histone deacetylase ligand discovery. Bioorg. Med. Chem. Lett. 18, 2809–2812 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bradner, J. E. et al. Chemical phylogenetics of histone deacetylases. Nat. Chem. Biol. 6, 238–243 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Marks, B. D., Fakhoury, S. A., Frazee, W. J., Eliason, H. C. & Riddle, S. M. A substrate-independent TR-FRET histone deacetylase inhibitor assay. J. Biomol. Screening 16, 1247–1253 (2011).

    Article  CAS  Google Scholar 

  50. Methot, J. L. et al. Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2). Bioorg. Med. Chem. Lett. 18, 973–978 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Yung-Chi, C. & Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).

    Article  Google Scholar 

  52. Fuller, N. O. et al. CoREST complex-selective histone deacetylase inhibitors show prosynaptic effects and an improved safety profile to enable treatment of synaptopathies. ACS Chem. Neurosci. 10, 1729–1743 (2019).

    Article  CAS  PubMed  Google Scholar 

  53. Dai, L. et al. Horizontal cell biology: monitoring global changes of protein interaction states with the proteome-wide cellular thermal shift assay (CETSA). Annu. Rev. Biochem. 88, 383–408 (2019).

    Article  CAS  PubMed  Google Scholar 

  54. Asawa, R. R. et al. A comparative study of target engagement assays for HDAC1 Inhibitor profiling. SLAS Discov. 25, 253–264 (2020).

    CAS  PubMed  Google Scholar 

  55. Seashore-Ludlow, B., Axelsson, H. & Lundbäck, T. Perspective on CETSA literature: toward more quantitative data interpretation. SLAS Discov. 25, 118–126 (2020).

    PubMed  Google Scholar 

  56. Vinegoni, C. et al. Measurement of drug–target engagement in live cells by two-photon fluorescence anisotropy imaging. Nat. Protoc. 12, 1472–1497 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. England, C. G., Ehlerding, E. B. & Cai, W. NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjug. Chem. 27, 1175–1187 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Selvin, P. R. Principles and biophysical applications of lanthanide-based probes. Annu. Rev. Biophys. Biomol. Struct. 31, 275–302 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Ash, C., Dubec, M., Donne, K. & Bashford, T. Effect of wavelength and beam width on penetration in light–tissue interaction using computational methods. Lasers Med. Sci. 32, 1909–1918 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Latva, M. et al. Correlation between the lowest triplet state energy level of the ligand and lanthanide(III) luminescence quantum yield. J. Lumin. 75, 149–169 (1997).

    Article  CAS  Google Scholar 

  61. Horrocks, W. D. & Sudnick, D. R. Lanthanide ion probes of structure in biology. Laser-induced luminescence decay constants provide a direct measure of the number of metal-coordinated water molecules. J. Am. Chem. Soc. 101, 334–340 (1979).

    Article  CAS  Google Scholar 

  62. Beeby, A. et al. Non-radiative deactivation of the excited states of europium, terbium and ytterbium complexes by proximate energy-matched OH, NH and CH oscillators: an improved luminescence method for establishing solution hydration states. J. Chem. Soc. Perkin Trans. 2, 493–504 (1999).

    Article  Google Scholar 

  63. Neklesa, T. K. et al. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by NSF 1830941, NIH R21AI13298 and NIH R01AI143723 (to R.M.). N.C.P. was supported by a National Science Foundation Graduate Research Fellowship (DGE1745303). A.S.K. was supported by Research Travel Grant (Karlsruhe House of Young Scientists, Karlsruhe Institute of Technology, Germany). K.S. was supported by a Goldwater Scholarship and by the Northeastern University Office of Undergraduate Research and Fellowships. We thank S. J. Haggarty for generosity in providing access to tissue culture space and instrumentation, and S. A. Reis and Z. Rosenthal for guidance and helpful discussion. HA-EGFP-HaloTag2 was a gift from C. Crews (Addgene plasmid 41742). We would like to thank J. Qi (Dana-Farber Cancer Institute) for providing JQ1.

Author information

Authors and Affiliations

  1. Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA

    N. Connor Payne, Alena S. Kalyakina, Kritika Singh, Mark A. Tye & Ralph Mazitschek

  2. Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA, USA

    N. Connor Payne

  3. Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany

    Alena S. Kalyakina

  4. Department of Bioengineering, Northeastern University, Boston, MA, USA

    Kritika Singh

  5. Harvard Graduate School of Arts and Sciences, Cambridge, MA, USA

    Mark A. Tye

  6. Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Ralph Mazitschek

  7. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Ralph Mazitschek

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Contributions

R.M. conceived the study. R.M., N.C.P., A.S.K., K.S. and M.A.T. designed the experiments and analyzed the data. R.M., N.C.P. and A.S.K. developed the synthetic methodology. N.C.P., A.S.K. and M.A.T. synthesized and characterized compounds. N.C.P. optimized and performed cell-free assays and cellular target engagement studies. K.S. optimized bacterial expression and purification of HSFP6xHis. R.M. supervised all aspects of the project. R.M. and N.C.P. wrote the manuscript with input from all authors. All authors read, revised and approved the manuscript.

Corresponding author

Correspondence to Ralph Mazitschek.

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Competing interests

R.M. is a scientific advisory board (SAB) member and equity holder of Regenacy Pharmaceuticals, ERX Pharmaceuticals and Frequency Therapeutics. R.M., N.C.P. and A.S.K. are inventors on patent applications related to this work.

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Extended data

Extended Data Fig. 1 Three-dimensional representation of CoraFluor complex.

Model of macrotricyclic terbium complex with tertiary amide linker attachment (upper left). The model was generated in Chem-3D (ChemDraw, PerkinElmer, Waltham, MA). The terbium center is shown as a green sphere.

Extended Data Fig. 2 Synthetic scheme to access CoraFluor ligands.

Reagents and conditions: (a) TsCl, K2CO3, H2O, rt, 48 h; (b) NaOH, H2O, 4 °C, 2 h (43% over 2 steps); (c) ethylenediamine, 10 mol% p-TsOH, MePh, 60 °C, 24 h (92%); (d) HBr, AcOH, 115 °C, 24 h (> 95%); (e) ethyl 6-bromohexanoate, K2CO3, ACN, 80 °C, 12 h then KOH, H2O, 95 °C, 2 h; (f) HBr, AcOH, 115 °C, 24 h then EtOH, HBr (cat.), 85 °C, 2 h (54% over 2 steps); (g) 46, 47, or 48, DIPEA, N,N-dimethylformamide, rt, 12 h (> 95%); (h) 40, PyBOP, DIPEA, N,N-dimethylformamide, rt, 1-3 h, 2-5 mM (40-70%); (i) HBr, AcOH, 100 °C, 30 min then NaOH, H2O, rt, 10 min then aqueous HBr (> 95%); (j) isobutyl chloroformate, DIPEA, DCM, rt, 10 min then tetrafluorophenol, DMAP (cat.), rt, 12 h (60-80%).

Extended Data Fig. 3 CoraFluor-2 exhibits improved excitability at 405 nm.

(a) Visual comparison of luminescence intensities of CoraFluors under constant illumination with a 365 nm LED (left image) or a 405 nm laser diode (right image) demonstrates enhanced luminescence intensity of Cora-2-Halo compared to Cora-1-Halo with 405 nm but not 365 nm excitation. Excitation light is passed through the adjacent samples from the left, eliminating potential light filtering effects from Cora-2-Halo, which exhibits a higher molar absorptivity at the tested wavelengths. (10 μM CoraFluor in 50 mM HEPES buffer, pH 7.4). (b) Comparative quantitative analysis of excitation wavelength-dependent, time-resolved luminescence intensity of CoraFluors demonstrates that Cora-2-Halo offers superior signal intensity following 405 nm excitation (200 nM CoraFluors in 50 mM HEPES buffer, pH 7.4, constant photomultiplier gain, acquisition delay = 100 μs, and integration time = 50 μs). Excitation wavelength (bandwidth = 5 nm) was varied in 5 nm increments, and the TR-fluorescence response of Cora-1-Halo and Cora-2-Halo was measured relative to background (buffer alone). Data were background-corrected, normalized and are presented as mean ± SD (n = 16 technical replicates). Data were acquired on a Tecan SPARK plate reader in a white 384-well plate (Corning 3572).

Source data

Extended Data Fig. 4 Select photophysical characterization data for CoraFluors and linker-less complexes.

(a) Quantum yield plots for select terbium complexes. Data are representative of two independent experiments. (b) Background-corrected decay curves and calculated luminescence lifetimes for linker-less (12-14) and select CoraFluor complexes. Luminescence intensity values were normalized, ln-transformed and linear regression analysis was performed in Prism 8. Data are presented as the averaged signal of n = 50 independent measurements.

Source data

Extended Data Fig. 5 Characterization of Keap1 fluorescent tracers and their use in single-ligand displacement TR-FRET assays.

(a-e) Saturation binding of (a) FITC-KL9 against Keap1 (6xHis/GST) construct (1 nM) with 0.5 nM Tb-Anti-6xHis, (b) Cora-1-KL9 against Keap1 (His/GST) construct (1 nM) with 0.5 nM AF488-Anti-6xHis, (c) FITC/Cora-1-KL9 mixture against Keap1 (tag-free) construct (1 nM), (d) CDDO-FITC against Keap1 (6xHis/GST) construct (1 nM) with 0.5 nM Tb-Anti-6xHis, and (e) CDDO-FITC against Keap1 (tag-free) construct (5 nM) with 5 nM Cora-1-KL9. The equilibration dissociation constants (Kd and Kd,app) were calculated in Prism 8 (GraphPad Software) using a one-site-binding (a-d) or four-parameter (e) nonlinear regression fit model. (f) Off-rate measurements (koff) for FITC-KL9 (black) and CDDO-FITC (grey) tracers. (g) Keap1-Keap1 dimer off-rate (koff,dimer) measurement, determined by rapid dilution of FITC-KL9/Cora-1-KL9-saturated homodimer into buffer containing isomolar FITC-KL9/Cora-1-KL9 concentrations. (h-i) Dose-response curves for Keap1 inhibitor test set as measured in TR-FRET assays with recombinant, full-length Keap1 with N-terminal 6xHis/GST tags and FITC-KL9 tracer (h) or CDDO-FITC tracer (i). Conditions: 1 nM Keap1 (6xHis/GST) construct, 0.5 nM Tb-Anti-6xHis, and either (h) 5 nM FITC-KL9 or (i) 40 nM CDDO-FITC, 4 h incubations. See Supplementary Table 1 for measured IC50 values. In these dose-response assays, due to the formation of higher-order oligomeric complexes, we did not attempt to determine true Kd values of inhibitors from the measured IC50 values. However, relative potencies between the inhibitors profiled remained constant. Data are presented as mean ± SD (n = 3 technical replicates) and are representative of at least two independent experiments.

Source data

Extended Data Fig. 6 Cell permeability profiling of select CoraFluors with EGFP-HaloTag expression construct.

(a) Labeling of intracellular EGFP-HaloTag construct in HEK293T cells by Cora-2-Halo, but not Cora-1-Halo, in a dose-dependent manner. Cells were treated with the indicated concentrations of HaloTag-ligands (or DMSO control) in phenol red-free Opti-MEM for 4 h at 37 °C before being washed, lysed in the presence of 10 μM TMR-Halo, and assessed for competition of TMR-Halo labeling via SDS-PAGE. Blot is representative of two independent experiments. (b) Detection of specific TR-FRET signal between EGFP-HaloTag and Cora-2-Halo in live cells after treatment with 50 μM Cora-2-Halo for 4 h at 37 °C. Data are presented as mean ± SD (n = 16 technical replicates) and are representative of two independent experiments.

Source data

Extended Data Fig. 7 Mammalian expression and lysate-based quantification of HDAC1-HaloTag construct.

(a) Expression, Cora-1-Halo labeling, and TR-fluorescence-based quantification of HDAC1-HaloTag construct in HEK293T expression lysate. After incubation with 10 μM Cora-1-Halo, the lysate is gel filtrated to remove excess HaloTag ligand. Because labeling is stoichiometric (1:1 Cora-1-Halo:HDAC1-HaloTag), the concentration of Cora-1-Halo labeled HDAC1-HaloTag in the lysate can accurately be determined via a reference calibration curve (see Methods). In our experience, the yield of HDAC1-HaloTag from a single 15 cm dish of transfected HEK293T cells (~25 million cells) was between 25-50 μg, giving protein concentrations between 200-500 nM and, therefore, samples were diluted ~1:5 to remain within the standard curve (green square). Data are presented as mean ± SD of (n = 3 technical replicates) (b) Quantification of HDAC1-HaloTag (Cora-1-Halo labeled) in HEK293T cell overexpression lysate with AF488-HaloTrap. The labeled lysate was diluted 1:12 (275 μg/mL total protein) and incubated with varying concentrations of HaloTrap-AF488 (0-150 nM, 16-point). The concentration of Cora-1-Halo labeled HDAC1-HaloTag in the diluted lysate was determined by nonlinear regression analysis following a quadratic equilibrium-binding equation (see Methods). Data are presented as mean ± SD (n = 2 technical replicates). Data in a-b are representative of at least two independent experiments.

Source data

Extended Data Fig. 8 Biochemical validation of HDAC fluorescent tracers and inhibitors with purified, recombinant protein.

(a) Saturation binding curves for fluorescent HDAC tracers (SAHA-NCT, M344-FITC) using recombinant HDAC1. Conditions: 5 nM HDAC1 (6xHis/FLAG; 50051; BPS Biosciences Inc), 2.5 nM Tb-Anti-6xHis IgG, 2 h incubation. (b) Dose-response curves for HDAC inhibitor test set as measured in TR-FRET assay with recombinant HDAC1. Conditions: 5 nM HDAC1 (6xHis/FLAG; 50051; BPS Biosciences Inc), 2.5 nM Tb-Anti-6xHis IgG, 20 nM SAHA-NCT or 70 nM M344-FITC, 3 h incubation. (c) HDAC activity dose-response curves for HDAC inhibitor test set, as well as fluorescent HDAC tracers (SAHA-NCT, M344-FITC) used in this study toward recombinant HDAC1. Conditions: 5 nM HDAC1 (6xHis/FLAG; 50051; BPS Biosciences Inc), 18 μM MAZ1600 substrate (3x KM), 3 h incubation. See Supplementary Table 2 for measured IC50 and determined Kd values. Data are presented as mean ± SD (n = 3 technical replicates) and are representative of at least two independent experiments.

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Extended Data Fig. 9 Profiling cellular response of HDAC inhibitors with 0.25 μM SAHA-NCT.

Cellular dose-response curves for HDAC inhibitor test set as measured in TR-FRET assay with Cora-2-Halo labeled HEK293T cells expressing HDAC1-HaloTag, with 0.25 μM SAHA-NCT tracer present. Conditions: 25,000 cells/well (384-well plate; Corning 3574), 4 h incubation at 37 °C and 5% CO2. See Supplementary Table 2 for measured EC50 and apparent Ki (Ki,app) values. Data are presented as mean ± SD (n = 6 technical replicates) and are representative of two independent experiments.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–11 and notes 1 and 2.

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Source data for HSFP6xHis expression and purification.

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Payne, N.C., Kalyakina, A.S., Singh, K. et al. Bright and stable luminescent probes for target engagement profiling in live cells. Nat Chem Biol 17, 1168–1177 (2021). https://doi.org/10.1038/s41589-021-00877-5

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