Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9)

Abstract

We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme's active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.

Figure 1

Structures of HTS lead RBBP9 inhibitors. Normalized percent inhibition of RBBP9 (compounds at 7.94 µM) from fluopol-ABPP PubChem Bioassay AID 1537 is shown in parenthesis.

Figure 2

Selectivity profiling of oxime ester RBBP9 inhibitors. (A) Evaluation of RBBP9 inhibitors by competitive ABPP with the FP-Rh probe in the RBBP9-transfected HEK 293T soluble proteome at 20 µM (left panel) and at 200 µM (right panel). Fluorescent gels are shown in gray scale. (B) Evaluation of the selectivity of oxime ester 3 at indicated concentrations and (C) oxime ester 18 at 200 µM by competitive ABPP in the mouse brain membrane proteome doped with recombinant RBBP9.

Figure 3

Oxime ester compounds covalently inhibit RBBP9. (A) Recombinant RBBP9 was incubated with DMSO or inhibitor (100 µM) and each reaction was split into two fractions. One fraction was reacted directly with FP-Rh (left panels), and the other was filtered and then reacted with FP-Rh (right panels) to assess the reversibility of inhibition. (B) RBBP9 was incubated with DMSO or compound 2, digested with trypsin, and analyzed by LC-MS/MS. The spectral counts of peptides with a +138.00 Da modification on Ser75 are shown.

Figure 4

(A) Selectivity profiling of RBBP9 hit compounds (20 µM) for thiol-crossreactivity using a cysteine-reactive phenylsulfonate-rhodamine (PS-Rh) probe (5 µM, 1 hour). (B) Evaluation of oxime ester 21 by competitive ABPP in the mouse brain membrane proteome doped with recombinant RBBP9.