Designed semisynthetic protein inhibitors of Ub/Ubl E1 activating enzymes

Article abstract of DOI:10.1021/ja9088549

(Chemical Equation Presented) Semisynthetic, mechanism-based protein inhibitors of ubiquitin (Ub) and ubiquitin-like modifier (Ubl) activating enzymes (E1s) have been developed to target E1-catalyzed adenylation and thioesterification of the Ub/Ubl C-terminus during the processes of protein SUMOylation and ubiquitination. The inhibitors were generated by intein-mediated expressed protein ligation using a truncated Ub/Ubl protein (SUMO residues 1-94; Ub residues 1-71) with a C-terminal thioester and synthetic tripeptides having a C-terminal adenosine analogue and an N-terminal cysteine residue. SUMO-AMSN (4a) and Ub-AMSN (4b) contain a sulfamide group as a nonhydrolyzable mimic of the phosphate group in the cognate Ub/Ubl-AMP adenylate intermediate in the first half-reaction, and these constructs selectively inhibit SUMO E1 and Ub E1, respectively, in a dose-dependent manner. SUMO-AVSN (5a) and Ub-AVSN (5b) contain an electrophilic vinyl sulfonamide designed to trap the incoming E1 cysteine nucleophile (Uba2 Cys173 in SUMO E1; Uba1 Cys593 in Ub E1) in the second half-reaction, and these constructs selectively, covalently, and stably cross-link to SUMO E1 and Ub E1, respectively, in a cysteine nucleophile-dependent manner. These inhibitors are powerful tools to probe outstanding mechanistic questions in E1 function and can also be used to study the biological functions of E1 enzymes. Copyright

Full text of DOI:10.1021/ja9088549

Published on Web 01/25/2010
Designed Semisynthetic Protein Inhibitors of Ub/Ubl E1 Activating Enzymes
Xuequan Lu,† Shaun K. Olsen,‡ Allan D. Capili,‡ Justin S. Cisar,†,§ Christopher D. Lima,*,‡,§ and
Derek S. Tan*,†,§,⊥
Molecular Pharmacology and Chemistry Program, Structural Biology Program, Tri-Institutional Training Program
in Chemical Biology, and Tri-Institutional Research Program, Memorial Sloan-Kettering Cancer Center,
1275 York AVenue, Box 422, New York, New York 10065
Received October 16, 2009; E-mail: limac@mskcc.org; tand@mskcc.org
Ubiquitin (Ub) and related ubiquitin-like (Ubl) proteins such as
SUMO serve as reversible, post-translational modifications of
protein substrates, impacting diverse cellular processes.1,2 The Ub/
Ubl modifier is coupled by its C-terminal carboxylate to specific
lysine side chains on target proteins via an isopeptide bond. Initial
steps in this process are catalyzed by a Ub/Ubl activating enzyme
(E1), which first adenylates the Ub/Ubl C-terminus to form a Ub/
Ubl-AMP intermediate and then transfers the Ub/Ubl to a conserved
cysteine on the E1 (Figure 1). The Ub/Ubl is then transthioesterified
onto a cysteine side chain of a conjugating enzyme (E2) and finally
transferred to a lysine side chain of the target protein, often mediated
Figure 1. Ub/Ubl activating enzymes (E1) catalyze adenylation of a Ub/
Ubl at its C-terminus followed by thioesterification at a conserved cysteine
of the E1 (Uba2 Cys173 in SUMO E1; Uba1 Cys593 in Ub E1).2
by a ligase (E3). Although structures of several E1s have been
reported,3,4 outstanding questions remain about the mechanisms of
these reactions. First, E1s surprisingly crystallize with substrates
bound in the active site rather than the in situ-formed, presumably
more tightly binding acyl-AMP intermediate,5 in contrast to other
enyzmes that catalyze adenylation reactions.6 Second, the conserved
E1 cysteine that serves as the nucleophile in the thioesterification
half-reaction is remote, >30 Å away from the adenylation active
site. These observations suggest that additional conformational
changes are required in both half-reactions.3 To investigate these
questions, we sought to develop mechanism-based inhibitors of E1s
that could then be used in pivotal structural and biochemical
studies.7 Selective inhibitors would also be useful probes for
dissecting E1 functions. We and others have used 5′-sulfonylad-
enosine-based small molecules to inhibit various mechanistically
(but not structurally) related enzymes that catalyze adenylation
reactions.8 We envisioned that such inhibitor design strategies might
also be effective for E1s and report herein the development of
semisynthetic, C-terminally modified Ub/Ubl proteins as novel,
selective E1 inhibitors.
Our initial efforts focused on small-molecule inhibitors consisting
of the conserved C-terminal diglycine motif of Ub/Ubl modifiers
linked covalently to a 5′-O-sulfamoyladenosine (AMS) nonhydro-
lyzable analogue of AMP (Figure S1 in the Supporting Informa-
tion).9 However, these compounds proved to be extremely weak
E1 inhibitors.9 Thus, to develop more potent inhibitors, we
investigated 5′-sulfonyladenosine-based modifications to the C-
termini of full-length Ub/Ubl proteins.
synthesized tripeptides CGG-AMSN (1) and CGG-AVSN (2) and
ligated them to SUMO∆ (3a) and Ub∆ (3b) thioesters produced by
the intein fusion protein method.9,11 SUMO-AMSN (4a) and Ub-
AMSN (4b) contain a sulfamide as a nonhydrolyzable analogue of
the phosphate in the Ub/Ubl-AMP intermediate in order to probe
the first half-reaction. SUMO-AVSN (5a) and Ub-AVSN (5b)
contain a vinyl sulfonamide electrophile designed to trap the
incoming cysteine nucleophile in the second half-reaction.12
We then set out to test the abilities of these constructs to bind
and inhibit SUMO E1 (Sae1 ·Uba2) and Ub E1 (Uba1). Gel-
filtration experiments demonstrated that SUMO-AMSN (4a) binds
SUMO E1 (Figure S2).9 Moreover, 4a effectively inhibits Uba2-
S-SUMO thioester formation (Figure 3a) as well as subsequent E1-
dependent SUMO conjugation to a substrate protein, RanGAP
(Figure S3), both in a dose-dependent fashion that can be overcome
with excess SUMO. This inhibition presumably occurs at the level
of the first half-reaction. Ub-AMSN (4b) similarly inhibits Uba1-S-
Ub thioester formation in a dose-dependent manner (Figure 3b).
Importantly, these two inhibitors are highly selective for their cognate
E1s and do not inhibit the corresponding noncognate E1s (Figure 3c).
Examination of the Ub/Ubl ·E1 cocrystal structures revealed that
the conserved C-terminal diglycine motif is preceded by a non-
conserved hydrophilic residue with a solvent-exposed side chain
not bound to the E1 or Ub/Ubl.3 This suggested that this residue
could be replaced with a cysteine to enable native chemical ligation
of synthetic tripeptides having C-terminal 5′-sulfonyladenosine-
based modifications and an N-terminal cysteine to truncated Ub/
Ubl∆ proteins having a C-terminal thioester (Figure 2).10 Thus, we
† Molecular Pharmacology and Chemistry Program.
‡ Structural Biology Program.
§ Tri-Institutional Training Program in Chemical Biology.
⊥ Tri-Institutional Research Program.
Figure 2. Semisynthesis of mechanism-based SUMO E1 and Ub E1
inhibitors 4 and 5 (Ub/Ubl∆ ) SUMO1-94; Ub1-71).2
9
1748 J. AM. CHEM. SOC. 2010, 132, 1748–1749
10.1021/ja9088549  2010 American Chemical Society

C O M M U N I C A T I O N S
D.S.T. is an Alfred P. Sloan Research Fellow. Support from the
NIH (R01 AI068038 to D.S.T., R01 GM065872 to C.D.L., F32
GM075695 to A.D.C.), the NYSTAR Watson Investigator Program
(D.S.T.), the Rita Allen Foundation (C.D.L.), William H. Goodwin
and Alice Goodwin and the Commonwealth Foundation for Cancer
Research, and the MSKCC Experimental Therapeutics Center is
gratefully acknowledged.
Supporting Information Available: Complete ref 7, detailed
procedures, and analytical data for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
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