Development of small molecule inhibitors and probes of human SUMO deconjugating proteases

Abstract

Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.

Figure 1

Activity of Initial Lead Compounds Against hSENP1 using the ProSUMO Processing Assay. A. Purified recombinant ΔNhSENP1 (100nM) was pre-treated with JCP665, JCP666, JCP667 or JCP668 (0–100μM) for 30 min at room temperature followed by addition of hSUMO1-pro substrate. Cleavage of ProhSUMO1 was assessed by SDS-PAGE and visualized by Gelcode Blue protein stain reagent. ProhSUMO1 alone was included as a control in the lanes labeled (C). B. Activity of the first generation analog of JCP666 lacking the aza-aspartic acid side chain on the azide nitrogen, VEA260 was measured using the same assay conditions outlined in A.

Figure 2

Generation of a Library of Peptide Aza-Epoxides Based on VEA260. A. Structures of the P2 and P3 libraries along with the structure of VEA389, the direct analog of VEA260 containing a terminal amide in place of the carboxylic acid. B. Scheme for synthesis of the P2 library. The epoxide electrophile building block (1) is shown.

Figure 3

Structures of Acyloxymethyl Ketone (AOMK) Inhibitor Libraries. A. Structures of compounds from scaffold A containing the original P2 element of VEA260 and variable P1 and P3 elements. Inhibitors were synthesized as described in the supplemental methods section. Scaffold A was used to assess aryl group size, P1 aspartic acid versus glycine and importance of capping group. B. Structures of scaffold B AOMKs based on the natural amino acid sequence found in SUMO and Ubiquitin.

Figure 4

Activity of Aza-Epoxide and AOMK Inhibitors Against Recombinant hSENP Proteases. Recombinant purified ΔNhSENPs1, 2, 5, 6 and 7 were incubated with 50μM inhibitors. After addition of either Ac-QTGG-AFC for ΔNhSENPs 1, 2 and 5 or Ac-LRGG-AFC for ΔNhSENPs 6 and 7, the AFC production at 405 nm/510 nm for 10–60 min was measured using a fluorescence plate reader. Activity relative to the DMSO treated control was determined and average values from triplicate runs are shown as a heat map where red indicates 100% inhibition and white indicates 0% inhibition. See also Figures S1 and S2.

Figure 5

Development of Activity-Based Probes for hSENPs Based on the Most Potent AOMK and Aza-Epoxide Inhibitors. A. Structures of the AOMK and aza-epoxide probe scaffolds along with the peptide vinyl sulfone and vinyl methyl ester probes. For each probe the R group was either a biotin tag or Cy5 fluorescent tag as indicated in the name of the compound. B. Heat maps showing the activity of all the biotin versions of the probes (50 μM) against the recombinant hSENPs. C. Labeling of recombinant hSENP1 and hSENP2. ΔNhSENPs 1 and 2 or the catalytically dead mutant ΔNhSENP2 C548S (0.5μM) were labeled with probe (1μM) after pre-treatment with either with vehicle (–) or with VEA260 (epoxide inhibitor, 50 μM), VEA499 (AOMK inhibitor, 50 μM) or N-ethylmaleimide (NEM, 20 mM) as indicated. Proteins were resolved by SDS-PAGE and labeling visualized by biotin affinity blotting using horseradish peroxidase conjugated streptavidin for the biotinylated probes. Cy5 probe labeling was resolved by SDS-PAGE and visualized using a flatbed scanner. D. Purified recombinant ΔNhSENPs 1 and 2 (0.5μM) were treated with probe (1μM) for increasing time (30 minutes to 7 hours). Samples were visualized as described above. See also Figure S4.

Figure 6

Labeling of recombinant hSENP1 in complex Proteomes. A. Recombinant ΔNhSENP1 was spiked into hypotonic HEK293 cell lysates as 0.1% of the total protein mixture (25ng ΔNhSENP1 in 25ug of lysate). The spiked lysate was treated with decreasing concentrations of probes (Bio-VEA355, Bio-VEA505, Cy5-VEA355 and Cy5-VEA505) for 1 hour at 37ºC. Proteins were resolved by SDS-PAGE and visualized as described above. B. Recombinant ΔNhSENP1 spiked into hypotonic HEK293 cell lysates was treated with decreasing concentrations of apparent active full length SUMO VS and VME probes. Proteins were resolved by SDS-PAGE and visualized by western blot using an anti-HA antibody.