Potent and selective photo-inactivation of proteins with peptoid-ruthenium conjugates

Journal name:
Nature Chemical Biology
Volume:
6,
Pages:
258–260
Year published:
DOI:
doi:10.1038/nchembio.333
Received
Accepted
Published online

Advances in high-throughput screening now enable the rapid discovery of bioactive small molecules, but these primary hits almost always exhibit modest potency. We report a strategy for the transformation of these hits into much more potent inhibitors without compound optimization. Appending a derivative of Ru(II)(tris-bipyridyl)2+, an efficient photosensitizer of singlet oxygen production, to synthetic protein-binding compounds results in highly potent and specific target protein inactivation upon irradiation with visible light.

At a glance

Figures

  1. Visible light–triggered inactivation of VEGFR2 by a ruthenium-peptoid conjugate.
    Figure 1: Visible light–triggered inactivation of VEGFR2 by a ruthenium-peptoid conjugate.

    (a) Chemical structure of RuGU40C. The modified Ru(II)(bpy)32+ complex and the GU40C peptoid are shown in red and blue, respectively. (b) Western blots showing the level of phospho-VEGFR2 (the active form of the receptor) and total VEGFR2 after receptor-expressing cells (PAE/KDR) were incubated under the conditions indicated. The duration of irradiation was 10 min. FGU40C, fluorescein-conjugated GU40C (see Supplementary Fig. 2). RuCON, a Ru(II)(bpy)32+-conjugated control peptoid that does not bind VEGFR2 (see Supplementary Fig. 2). (c) Dose dependence of the inhibition of autophosphorylation of VEGFR2 by RuGU40C with or without irradiation. (d) Effect of ruthenium-peptoid conjugates on the VEGF-induced formation of tubes by human umbilical vascular endothelial cells (HUVECs). HUVECs on Matrigel-coated plates were incubated under the conditions indicated and irradiated (10 min). 16 h after the addition of VEGF, degree of tube formation was evaluated by quantitative analysis (AngioQuant software; http://www.cs.tut.fi/sgn/csb/angioquant/) of images obtained using a light microscope (see Supplementary Fig. 4 for representative images). (e) Analysis of the specificity of RuGU40C-mediated inhibition of VEGFR2. The effect of the ruthenium-peptoid conjugate on hormone-mediated autophosphorylation (activation) of VEGFR2 and EGFR was examined by western blot in the presence and absence of irradiation (10 min) in cells that express both receptors (H441) and evaluated by quantitative analysis (ImageJ; http://rsbweb.nih.gov/ij/). Note that there is a basal level of phosph-VEGFR2 present even in the absence of VEGF treatment. Error bars are s.d.

  2. A hyperpotent CALI inhibitor of VEGFR2.
    Figure 2: A hyperpotent CALI inhibitor of VEGFR2.

    (a) Chemical structure of RuGU40C4. The modified Ru(II)(bpy)32+ complex and GU40C4 peptoid are shown in red and blue, respectively. (b) Dose-dependent inhibition of VEGF-induced autophosphorylation of VEGFR2 on PAE/KDR cells by RuGU40C4 with irradiation (10 min). Error bars are s.d.

  3. Visible light–triggered inactivation of the 26S proteasome by a ruthenium-peptoid conjugate.
    Figure 3: Visible light–triggered inactivation of the 26S proteasome by a ruthenium-peptoid conjugate.

    (a) Illustration of the 26S proteasome and gate opening of the 20S proteasome. (b) Chemical structure of RuRIP1. The modified Ru(II)(bpy)32+ complex and RIP1 peptoid are shown in red and blue, respectively. (c) Chymotrypsin-like peptidase activity of purified yeast 26S proteasome was measured in the presence of RuRIP1 with and without irradiation by monitoring the cleavage of the fluorogenic substrate Suc-LLVY-AMC. (d) The effect of RuRIP1 on the chymotrypsin-like activity of the 26S proteasome in HeLa cells with and without irradiation (30 min) was assessed by measuring luminescence generated by substrate (Suc-LLVY-aminoluciferin) cleavage. RFU, relative fluorescence units. Error bars are s.d.

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Author information

Affiliations

  1. Division of Translational Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    • Jiyong Lee,
    • D Gomika Udugamasooriya &
    • Hyun-Suk Lim
  2. Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.

    • Thomas Kodadek
  3. Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.

    • Thomas Kodadek
  4. Present addresses: Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA (J.L.), Advanced Imaging Center and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA (D.G.U.) and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA (H.-S.L.).

    • Jiyong Lee,
    • D Gomika Udugamasooriya &
    • Hyun-Suk Lim

Contributions

J.L. designed and performed experiments, analyzed data and wrote the manuscript. D.G.U. and H.-S.L. performed experiments. T.K. designed experiments, analyzed data and wrote the manuscript.

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The authors declare no competing financial interests.

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    Supplementary Methods and Supplementary Figures 1–15

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