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

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.

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

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

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

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

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

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

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

Synthesis of a 4-mer peptoid library with 20 different amines (diversity: 160,000) for screening.

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.