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OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues

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Abstract

Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.

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Fig. 1: Design strategies to develop water-soluble and cell-membrane-permeant fluorophores for quantitative, live-cell and tissue imaging.
Fig. 2: ORFluors are a class of zwitterionic, water-soluble, cell-membrane-permeant fluorophores suitable for live-cell imaging.
Fig. 3: Erlotinib iPAI probe design.
Fig. 4: Superior live-cell imaging of drug distribution with ORFluor-labelled iPAI probes.
Fig. 5: Quantitative imaging of erlotinib iPAI probes in live cells.
Fig. 6: iPAI erlotinib imaging enables quantification of DTA in tissues.

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Data availability

All data associated with this study are presented in the article, extended data, source data, supplementary files or Supplementary Information. The data generated during the study are also available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

Custom­-written MATLAB code used to calculate drug target availability is available at https://doi.org/10.5281/zenodo.4004647.

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Acknowledgements

This work was supported by an ASPIRE Award from the Mark Foundation for Cancer Research (S.L.G.) as well as the National Cancer Institute (R21CA257942; S.L.G.). We thank M. Vescio (OHSU) and S. Kumarapeli (OHSU) for experimental assistance, and the OHSU Advanced Light Microscopy Core for imaging assistance.

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Authors and Affiliations

  1. Biomedical Engineering Department, Oregon Health & Science University, Portland, OR, USA

    Lei G. Wang, Antonio R. Montaño, Jason R. Combs, Nathan P. McMahon, Allison Solanki, Dani A. Szafran & Summer L. Gibbs

  2. Cancer Early Detection Advanced Research Center (CEDAR), Oregon Health & Science University, Portland, OR, USA

    Michelle M. Gomes & Kai Tao

  3. Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA

    Michelle M. Gomes, Kai Tao, William H. Bisson & Summer L. Gibbs

  4. Thayer School of Engineering, Dartmouth College, Hanover, NH, USA

    Kimberley S. Samkoe

  5. Department of Surgery, Dartmouth Health, Lebanon, NH, USA

    Kimberley S. Samkoe

  6. Department of Biomedical Engineering, Illinois Institute of Technology, Chicago, IL, USA

    Kenneth M. Tichauer

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  1. Lei G. Wang
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Contributions

L.G.W. and S.L.G. designed the study. L.G.W., A.R.M. and J.R.C. performed the probe synthesis. L.G.W. and A.R.M. completed all spectral measurements. L.G.W. performed confocal microscopy. W.H.B. completed the molecular docking studies. M.M.G. and K.T. completed the BLI studies. N.P.M. and A.S. conducted intracellular paired agent imaging studies. D.A.S. conducted in vivo biodistribution studies. K.S.S., K.M.T. and S.L.G. completed intracellular paired-agent imaging data analysis and interpretation. L.G.W. and S.L.G. wrote and edited the paper. All authors reviewed and approved the paper. S.L.G. supervised the project.

Corresponding author

Correspondence to Summer L. Gibbs.

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Competing interests

L.G.W., A.R.M. and S.L.G. are inventors on patent application PCT/US21/53806, ‘Zwitterionic cell-permeant and water-soluble rhodamine dyes for quantitative imaging applications’, submitted to the World Intellectual Property Organization and held by Oregon Health and Science University. This covers the composition and methods of use of the ORFluor compounds discussed herein. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Fluorescence live-cell images of SiR, SiR-Me and OF650.

Representative images of U2OS cells stained with a fluorescent on/off switchable rhodamine fluorophore SiR, b fluorescent always-on fluorophore SiR-Me, and c ORFluor OF650 at 30 nM for 30 min. Fluorescence images are shown normalized to one another for the three rhodamine dyes. Positive intracellular localization was observed in cells that were stained with SiR-Me, while negligible fluorescence signal was observed in the cells stained with either SiR or OF650. d Co-localization imaging showed SiR-Me preferentially localized to the mitochondria with high affinity and specificity, confirmed by co-staining with commercial mitochondria probe, MitoTracker Green (green). All cells were co-stained with Hoechst (blue) and examined from a minimum of three independent staining experiments. Scale bar, 100 μm.

Extended Data Fig. 2 Synthesis of ORFluor OF650 and its conjugatable versions.

ORFluor OF650 was prepared in six steps with an averaged chemical yield of 68%. A Vilsmeier–Haack reaction of dimethylbromoaniline precursor 23 gave benzaldehyde intermediate 24 in 88% yield. This was then followed by the addition of pre-lithiated 2-bromo-toluene (25), giving diaryl methanol intermediate 26 in decent yield (81%). The allyl-protected N-methyl bromoaniline 27 was activated by Lewis acid, ZnCl2, to participate in a nucleophilic substitution reaction with secondary alcohol 26, providing heterodimer 28 in reasonable yield (56%). Subsequent lithiation of heterodimeric bromide 28 using tert-BuLi and addition of dichlorodimethylsilane yielded the leuco-basic rhodamine 29 (60%). Deprotection (that is, allyl group removal) of 29, catalyzed by a Pd(PPh3)4 and dimethylbarbituric acid (DMBA) system, provided leuco-base rhodamine 30 in 68% yield. Introduction of a sulfonate group to the silicon-substituted rhodamine was achieved through alkylation of intermediate 30 using excess propane-1,3-sultone, followed by direct oxidation with p-chloranil, affording the final ORFluor OF650 (2) in 55% yield. To demonstrate that our synthetic strategy can be generalized to other asymmetrical silicon-substituted rhodamines and provide access to bioconjugatable derivatives, we modified the synthetic sequence to prepare carboxylic acid OF650COOH and N-hydroxysuccinimide (NHS) ester OF650NHS versions (Fig. 1d). Intermediate 31, bearing a functional handle, was metalated before reaction with compound 24, giving the diaryl methanolic intermediate 32 with a protected carboxyl group in 74% yield. The reaction protocols used to synthesize OF650 – until removal of the allylic protecting group – were again utilized to afford compound 35. Initial attempts at alkylation using propane-1,3-sultone followed directly by hydrolysis gave very poor yields. However, when an acid-catalyzed hydrolysis was performed first, releasing the carboxyl group, the free carboxylic acid intermediate (36) was given in 46% yield and subsequent alkylation and oxidation products were furnished by the aforementioned protocols. The free acid OF650COOH (6), treated with TSTU, was transformed to NHS ester OF650NHS (37) in 69% yield.

Extended Data Fig. 3 Extension of zwitterionic structural design strategy to the TMR analog.

ORFluor OF550 was prepared in three steps with an averaged chemical yield of 48%. Typically, an asymmetric rhodamine dye is prepared through condensation between a hydroxy benzophenone and an aniline derivative together in a one-to-one ratio at high temperature, under acidic conditions. In our case, we sought to efficiently prepare asymmetrical zwitterionic fluorophore derivatives by introducing required structural variables (that is, the phenyl ring motifs) at a later stage in the synthetic sequence. An Ullmann cross-coupling reaction of the commercially available dimethylaminophenol 38 and aryl bromide 39, catalyzed by CuI and 2-picolinic acid under basic conditions, afforded the diaryl ether intermediate 40. Alkylation of compound 40 with an excess amount of propane-1,3-sultone gave the sulfonated intermediate 41 (70%). Because the diaryl ether derivative 41 is prone to electronic activation, an acid catalyzed reaction should readily provide the oxidized fluorophore product via the Friedel–Crafts acylation–cyclization reaction. To ease the product recovery and purification process, a catalytic system consisting of ZnCl2 in ethanol was introduced to the condensation step. However, cyclization of compound 41 with a benzaldehyde 42 did not proceed until the temperature rose above 100 °C. A subsequent DDQ-mediated oxidation reaction was performed to push the reaction to completion in 33% yield over two steps. A bioconjugatable version OF550NHS was prepared using the aforementioned key intermediate 41. Using this same protocol and the orthogonal protecting group-bearing intermediate 43 allowed for the introduction of a functional handle (for example, protected carboxyl group). Deprotection under acidic conditions, followed by DDQ-mediated oxidation provided the OF550COOH (8) as a single regioisomer. The free acid OF550COOH (8) was then transformed into NHS ester OF550NHS (44) via treatment with TSTU in 48% yield.

Extended Data Fig. 4 Fast Airyscan confocal fluorescence imaging with SNAP-tag and HaloTag ligands.

Intracellular localization of OF650 (red) and OF550 (green) labeled SNAP-tag and HaloTag substrates in doxycycline inducible U2OS TetR-Mito-SNAP-tag expressing cells and TetR-ER-HaloTag expressing cells. All cells were co-stained with Hoechst (blue) prior to fixation, and examined from a minimum of three independent staining experiments. Scale bar = 10 μm. Single focal plane.

Extended Data Fig. 5 BLI binding determination of erlotinib and SiR and TMR labeled iPAI probes.

Representative BLI binding sensorgrams of erlotinib, SiR and TMR labeled targeted and untargeted probes, as well as untagged fluorophores SiR and TMR, binding to biotinylated recombinant EGFR-TKD immobilized on Super Streptavidin (SSA) biosensors. BLI sensorgrams show the association of each probe (0–90 s) at the indicated concentrations ranging from 37–3000 nM, followed by the subsequent dissociation in binding buffer without analytes (90–180 s). Kinetic data (Colored curve: observed experimental data) were fit globally using a simple Langmuir 1:1 binding model (Black dashed curve: fitted experimental data) in ForteBio Data Analysis HT 10.0 evaluation software to obtain the association rate constant ka, dissociation rate constant kd and the equilibrium dissociation constant KD.

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Supplementary Information

Supplementary Figs. 1–27 and full synthetic details of all compounds, their structural and optical characterization, as well as imaging experiments.

Reporting Summary

Real-time three-colour fast Airyscan confocal microscopy of U2OS TetR- ER-Halo-Mito-SNAP expressing cells labelled with OF650HALO, OF550SNAP and Hoechst 33342.

Real-time two-colour fast Airyscan confocal microscopy of U2OS TetR- ER-HaloTag expressing cells labelled with OF650HALO and Hoechst 33342.

Real-time two-colour fast Airyscan confocal microscopy of U2OS TetR- ER-HaloTag expressing cells labelled with OF550HALO and Hoechst 33342.

Real-time two-colour fast Airyscan confocal microscopy of U2OS TetR- Mito-SNAP-tag expressing cells labelled with OF650SNAP and Hoechst 33342.

Real-time two-colour fast Airyscan confocal microscopy of U2OS TetR- Mito-SNAP-tag expressing cells labelled with OF550SNAP and Hoechst 33342.

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Source Data Extended Data Fig. 5

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Wang, L.G., Montaño, A.R., Combs, J.R. et al. OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues. Nat. Chem. 15, 729–739 (2023). https://doi.org/10.1038/s41557-023-01173-6

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