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. 2012 Sep 24;13(14):2082-93.
doi: 10.1002/cbic.201200445. Epub 2012 Aug 20.

Rapid development of a potent photo-triggered inhibitor of the serine hydrolase RBBP9

Xiaodan Liu  1 , Melissa Dix, Anna E Speers, Daniel A Bachovchin, Andrea M Zuhl, Benjamin F Cravatt, Thomas J Kodadek
Affiliations

Rapid development of a potent photo-triggered inhibitor of the serine hydrolase RBBP9

Xiaodan Liu et al. Chembiochem. .

Abstract

The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors of these proteins. Traditional active-site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we designed a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated by visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases.

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Figures

Figure 1
Figure 1
Activity based labeling of RBBP9 with biotinylated fluorophosphonate probe. A) General mechanism for covalent modification of serine hydrolases active site by fluorophosphonate-biotin. B) Whole protein mass spectra for unmodified and FP-biotin modified RBBP9. C) Gel analysis for the recognition of FP-biotinylated RBBP9 by Streptavidin.
Figure 2
Figure 2
Mass spectra of on-bead tryptic digestion from Streptavidin-coated magnetic beads after incubation with RBBP9. A) Streptavidin-coated magnetic beads were incubated with unmodified RBBP9. B) Streptavidin-coated magnetic beads were incubated with FP-biotinylated RBBP9. C) Mass spectra of a standard in-solution RBBP9 digestion.
Figure 3
Figure 3
Structure of the hit compounds identified from the screening and the binding affinity evaluation by fluorescence polarization assay. A) Fluorescein-1. B) Fluorescein-6.
Figure 4
Figure 4
Comparison of inhibition potency of the chromophore-hit conjugates in the absence or presence of photo-activation. A) ABPP gel assay showing only light activated Ru(II)-6 blocks RBBP9 labeling by FP-Rh in a concentration dependent manner. B) ABPP gel assay showing only light activated Eosin-1 blocks RBBP9 labeling by FP-Rh in a concentration dependent manner.
Figure 5
Figure 5
Selective inhibition of RBBP9 in cellular proteome. Evaluation of A) Ru(II)-6, B) Eosin-1 by competitive ABPP in soluble proteome of HeLa cells (1 mg/mL). Recombinant human RBBP9 (400 nM) was doped into this proteome for comparison.
Figure 6
Figure 6
Photo-inactivated RBBP9 by Ru(II)-6 does not react with ABPP probe. A) The photo-inactivated RBBP9 does not response to Coomassie blue staining. Silver staining and Streptavidin-HRP blotting suggest that photo-damaged RBBP9 does not react with FP-biotin. B) Mass Spectra of RBBP9 labeling with FP-Biotin after incubation with Ru(II)-6 in the absence of presence of light activation. C) No endogenous level of RBBP9 was detected by western-blotting with anti-RBBP9 antibody. For the recombinant RBBP9 and RBBP9 doped in cell proteome complex, a ladder of higher molecular weight bands observed with increased Ru(II)-6 upon light activation.
Scheme 1
Scheme 1
Screening Strategy of ABPP Probe facilitated Peptoid Library Screening against Serine Hydrolases. RBBP9 was allowed to react with FP-biotin to yield the active site biotinylated protein. The FP-biotinylated RBBP9 was incubated with OBOC peptoid library. The hit compounds that bound to RBBP9 can be pull out with Streptavidin-coated magnetic beads by Streptavidin-biotin interaction.
Scheme 2
Scheme 2
Synthesis of a 4-mer peptoid library with 20 different amines (diversity: 160,000) for screening.
Scheme 3
Scheme 3
Synthesis of photo-activated inhibitors. A) Synthesis of Ru(II)-6 via alkyne-azide click reaction. B) Synthesis of Eosin-1 through the reaction of maleimide and thiol.
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References

    1. Lander ES, Linton LM, et al. Nature. 2001;409:860–921. - PubMed
    2. Venter JC, Adams MD, et al. Science. 2001;291:1304–1351. - PubMed
    3. Long JZ, Cravatt BF. Chem. Rev. 2011;111:6022–6063. - PMC - PubMed
    1. Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ. Nature. 1991;354:82–84. - PubMed
    1. Clark MA. Curr. Opin. Chem. Biol. 2010;14:396–403. - PubMed
    1. Shields DJ, Niessen S, Murphy EA, Mielgo A, Desgrosellier JS, Lau SK, Barnes LA, Lesperance J, Bouvet M, Tarin D, Cravatt BF, Cheresh DA. Proc. Natl. Acad. Sci. USA. 2010;107:2189–2194. - PMC - PubMed
    1. Bachovchin DA, Brown SJ, Rosen H, Cravatt BF. Nat Biotechnol. 2009;27:387–394. - PMC - PubMed

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