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. 2017 Aug;13(8):850-857.
doi: 10.1038/nchembio.2386. Epub 2017 Jun 5.

"VSports手机版" Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase

Daniel C Scott  1   2 , Jared T Hammill  3 , Jaeki Min  3 , David Y Rhee  4 , Michele Connelly  3 , Vladislav O Sviderskiy  1 , Deepak Bhasin  3 , Yizhe Chen  3 , Su-Sien Ong  3 , Sergio C Chai  3 , Asli N Goktug  3 , Guochang Huang  5 , Julie K Monda  1 , Jonathan Low  3 , Ho Shin Kim  3 , Joao A Paulo  4 , Joe R Cannon  4 , Anang A Shelat  3 , Taosheng Chen  3 , Ian R Kelsall  6 , Arno F Alpi  7 , Vishwajeeth Pagala  8 , Xusheng Wang  8 , Junmin Peng  1   8 , Bhuvanesh Singh  5 , J Wade Harper  4 , Brenda A Schulman  1   2 , R Kip Guy  3
Affiliations

"VSports在线直播" Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase

"VSports手机版" Daniel C Scott et al. Nat Chem Biol. 2017 Aug.

VSports在线直播 - Abstract

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions VSports手机版. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases. .

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Conflict of interest statement

Competing Financial Interest

The authors have filed a provisional application with the US Patent and Trademark Office on this work.

Figures

Figure 1
Figure 1. Discovery of small molecule inhibitors targeting N-Acetyl-UBE2M interaction with DCN1
(a) Model of a neddylation complex, highlighting DCN1 (pink) interactions with acetylated N-terminus of UBE2M (cyan), including structures of CUL1 (green)-RBX1 (red) and SKP1 (pale cyan)-FBXL3 (magenta)-Substrate (CRY2, light blue). Although ≈40 Å from the CUL1 neddylation site, the DCN1-AcUBE2M interaction accelerates neddylation. (b) Pulse-chase assays monitoring effects of the indicated concentrations of NAcM-HIT on DCN1-dependent (top, timescale 0–1 min) or DCN1-independent (bottom, timescale 0–12 min) neddylation from AcUBE2M to CUL2CTD. The gel scans are representative of multiple biological replicates. (c) Structure of DCN1 (surface colored by electrostatic potential) bound to NAcM-HIT (spheres, orange) aligned to DCN1 (omitted for clarity)-AcUBE2M (cyan) demonstrating NAcM-HIT binds to DCN1’s N-AcetylMet binding pocket.
Figure 2
Figure 2. Structure based optimization and development of a toolkit of small molecule probes inhibiting DCN’s interaction with UBE2M
(a) Close-up of DCN1 UBE2M binding site with NAcM-HIT (orange) superimposed on N-terminally acetylated UBE2M (cyan, 3TDU.pdb). The sub-pockets targeted during structure-based compound optimization are highlighted. (b) Chemical structures, nomenclature, and class of chemical probes targeting DCN’s N-Acetyl-Met binding pocket. NAcM-COV was synthesized and tested as a racemic mixture. Synthetic procedures and characterization data are in Supplementary Note. (c) Inhibition of DCN1 binding to an N-terminally acetylated peptide from UBE2M (TR-FRET assay, left) or DCN1 activation of N-terminally acetylated UBE2M-dependent neddylation (pulse-chase enzyme assay, right). TR-FRET values represent averages of an experiment performed in triplicate and the pulse-chase values are averaged from three independent experiments. (d) Comparison of co-crystal structures of DCN1 (surface electrostatic) bound to AcUBE2M (cyan, 3TDU.pdb), NAcM-HIT (orange), NAcM-OPT (green), and NAcM-COV (light blue).
Figure 3
Figure 3. Optimized NAcM inhibitors display exquisite selectivity for N-Acetyl pockets of DCN1 and DCN2
(a) Structure of complex containing N-Acetyl-Met peptide (purple), N-terminal acetyltransferase (hNaa50/NatA (gray), and CoA (pink) (4×5K.pdb). (b) Effects of NAcM-NEG (red), NAcM-OPT (green), and NAcM-COV (light blue) on human (Hs), S. cerevisiae (Sc), or S. pombe (Sp) Nat enzymes. Kd values inferred from enzymatic assays (Supplementary Fig. 10) are plotted on similarity based dendrogram. (c) Structural superimposition of DCN family members: DCN1 (100% identity, 0 RMSD, pink)-AcUBE2M (cyan, 3TDU.pdb), DCN2 (82%, 0.46, magenta)-AcUBE2M (pale cyan, 4GAO.pdb), DCN3 (36%, 1.35, violet)-AcUBE2F (teal, 4GBA.pdb), and DCN4 (34%, 1.57, purple). (d) Effects of probes on DCN1 neddylation activity. Kd values inferred from pulse-chase assays (Supplementary Fig. 11) plotted as in (b). (e) Subtle variations in targeted binding pocket determine NAcM selectivity profiles. Coloring as in (c) with residues potentially contributing to selectivity surrounding NAcM-OPT shown in stick representation.
Figure 4
Figure 4. NAcM inhibitors are on target and disrupt AcUBE2M interaction network
(a) NAcM-OPT and NAcM-COV, but not NAcM-NEG, disrupt co-immunoprecipitation of DCN1 with AcUBE2M. Experiments were performed with 293T cells expressing UBE2M with a C-terminal FLAG-HA tag at near-endogenous levels. Immunoblot of input and elutions from anti-FLAG immunoprecipitations were probed with the indicated antibodies (full gels shown in Supplementary Fig. 14) (b) N-acetyl-UBE 2M interactions decreased by >1.5-fold with P <0.05 (calculated using the moderated t statistic (two-sided) with Benjamini–Hochberg false discovery rate adjustment; n = 3 independent experiments) by NAcM-OPT (DCN 1, red; CAND 1, orange) or by association with a particular CRL (CUL 1, pink; CUL 2, green; CUL 3, blue; CUL 4A, purple; CUL 5, cyan) (Supplementary Data Set 6). The fold decrease in binding is indicated for each protein.
Figure 5
Figure 5. NAcM inhibitors reduce CUL neddylation in cells and mimic effects of shRNA knockdown of DCN1
(a) HCC95 and CAL-33 have high levels of DCN1 as demonstrated by immunoblot of total cell extracts of indicated cell lines (full gels shown in Supplementary Fig. 15a). (b) Effects of NAcM-NEG, NAcM-OPT, NAcM-COV, and NAcM-COVCTRL on cullin neddylation in HCC95 cells treated with DMSO, MLN4924 (single dose, 1 μM), or indicated compounds (10 μM, dosed at 0 and 24 hours). Cells were harvested at 48 hours, processed for immunoblotting, and probed with the indicated antibodies (full gels shown in Supplementary Fig. 15b). (c) NAcM-OPT mimics shRNA knockdown of DCN1. Immunoblot of total cell extracts from HCC95, or a line stably expressing a DCN1 shRNA, treated and processed as in (b) (full gels shown in Supplementary Fig. 15c). (d) Probe effects vary amongst cell lines. Immunoblot of total cell extracts from indicated cell lines, treated and processed as in (b). For all blots, * indicates non-specific bands in CUL1, CUL4A, and CUL5 immunoblots (full gels shown in Supplementary Fig. 15d–h).
Figure 6
Figure 6. NAcM-OPT inhibits neddylation in and prevents anchorage-independent growth of a DCN1 amplified cell line without causing changes in protein homeostasis
(a) Neither NAcM-OPT treatment nor DCN1 shRNA stabilizes cullin-RING ligase substrates in HCC95 cells. Cells treated and processed as in Fig. 5c using antibodies for the indicated proteins (full gels shown in Supplementary Fig. 16). (b) NAcM-OPT does not globally change protein homeostasis in HCC95 cells. TMT-quantification of total proteome following treatment with DMSO, MLN4924 (left, 1 μM), or NAcM-OPT (right, 10 μM) for 24 hours. MLN4924 causes >2-fold stabilization of 136 proteins (p-value <0.05), whereas NAcM-OPT treatment fails to stabilize any protein (Supplementary Dataset 8). Substrates examined in (a) are highlighted in red. (c) NAcM-OPT and NAcM-COV treatment blocks growth of HCC95 cells in soft agar. Representative data showing anchorage independent growth of HCC95 cells treated with NAcM-OPT (10 μM), NAcM-COV (10 μM), and NAcM-NEG (10 μM). (d) Quantification of results from (c).
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