Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP

Article abstract of DOI:10.1021/jacs.8b13193

Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragmentbased covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.

Full text of DOI:10.1021/jacs.8b13193

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Article
Fragment-based covalent ligand screening enables rapid
discovery of inhibitors for the RBR E3 ubiquitin ligase HOIP
Henrik Johansson, Yi-Chun Isabella Tsai, Ken Fantom, Chun-Wa Chung, Sandra Kümper, Luigi
Martino, Daniel A Thomas, H Christian Eberl, Marcel Muelbaier, David House, and Katrin Rittinger
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13193 • Publication Date (Web): 18 Jan 2019
Downloaded from http://pubs.acs.org on January 18, 2019
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Fragment-based covalent ligand screening enables rapid discovery of
inhibitors for the RBR E3 ubiquitin ligase HOIP
Henrik Johansson,†,‡ Yi-Chun Isabella Tsai,‡ Ken Fantom,§ Chun-Wa Chung,†,§ Sandra Kümper, †,‡ Luigi Martino,‡ Daniel
A. Thomas, § H. Christian Eberl,‖ Marcel Muelbaier,‖ David House,†* Katrin Rittinger‡*
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† Crick-GSK Biomedical LinkLabs, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom.
‡ Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT,
United Kingdom.
§ R&D Platform Technology & Science, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom.
‖ Cellzome GmbH, a GSK company, Meyerhofstraße 1, Heidelberg 69117, Germany
ABSTRACT: Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior
and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles
in several cellular signalling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains
are formed by the linear ubiquitin chain assembly complex (LUBAC), a multi-protein E3 ligase that consists of 3 subunits,
HOIP, HOIL-1L and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic
subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic
fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis and
protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase.
Furthermore, using cell-based assays and chemoproteomics we demonstrate that these compounds effectively penetrate
mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective
probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead
compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable
inhibitors of thioester-forming E3 ubiquitin ligases.
Introduction
of methionine 1, called linear or M1-linked chains.5-6 Linear
polyubiquitin chains play crucial roles in the regulation of
multiple cellular functions including immune and
inflammatory signaling via the NF-κB pathway, cell death
and cancer.7-10 LUBAC consists of three core components,
the RBR-domain containing subunits HOIP and HOIL-1L
plus SHARPIN,11-13 with HOIP constituting the catalytic
machinery required for linear chain formation (Figure
1B).14-16 Recent crystal structures of truncated HOIP have
provided valuable insight into the mechanism of linear
polyubiquitin chain formation.15, 17 However, in order to
interrogate the physiological function of LUBAC in a cellular
context, chemical tools that selectively target the E3 ligase
activity of HOIP would be of great value. To this end, a
handful of HOIP modulators have been reported, including
small molecule inhibitors BAY 11-7082 (1, Figure 1C)18 and
gliotoxin (2),19 as well as stapled peptides that target
protein-protein interactions at the HOIP/HOIL-1L and
Ubiquitination represents one of the most diverse post-
translational modifications of proteins and constitutes an
essential route for the regulation of protein signalling and
degradation. The process is carried out through a complex
interplay of enzymes (E1 ubiquitin-activating enzymes, E2
ubiquitin-conjugating enzymes, E3 ubiquitin ligases and
deubiquitinating (DUB) enzymes), which provides an
intricate network of regulation and substrate specificity
(Figure 1A).1-2 E3 ubiquitin ligases are the key determinants
for substrate specificity in this cascade and as such
represent attractive yet notoriously challenging
pharmacological targets. There are over 600 known human
E3 ligases that fall into three main classes (RING, HECT and
RBR) based on their structure and mechanism of ubiquitin
transfer, which in the case of HECT and RBR E3 ligases
involves the formation of a covalent thioester intermediate
with ubiquitin.3-4 However, a deeper understanding of their
underlying biology has long been hampered by the lack of
selective pharmacological tools. The linear ubiquitin chain
assembly complex (LUBAC) is a multi-protein E3 ubiquitin
ligase of the RBR family that catalyzes the formation of
polyubiquitin chains linked between the C-terminal
carboxylate of ubiquitin and the N-terminal α-amino group
HOIL-1L/SHARPIN
interfaces.20-22
Although
these
modulators all have demonstrated effects on LUBAC
activity, the molecules are also associated with drawbacks
that may limit their utility as tools. Compound (1), which
was originally identified as an inhibitor of TNFα-induced
phosphorylation of IκBα and NF-κB signaling, has been
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shown to covalently inhibit HOIP activity yet exhibits broad
reactivity across multiple proteins.18
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Figure 1 – Targeting HOIP using fragment-based covalent ligand screening. A) Schematic of the ubiquitination cascade
highlighting the formation of linear (M1) polyubiquitin chains (Ubn) by LUBAC, a reaction that proceeds via a covalent
thioester intermediate between the HOIP subunit and ubiquitin. B) Schematic representation of the HOIP E3 ligase,
highlighting the RBR which contains the active-site cysteine residue C885 (yellow circle) and the linear chain-determining
domain, LDD (amino acids 697- 1072). C) Small molecule LUBAC inhibitors (1) BAY 11-7082 and (2) gliotoxin. D) Overview
of our approach to develop covalent probes targeting the active site cysteine residue of HOIP using fragment-based covalent
ligand screening by protein LC-MS.
Compound (2) is a well-characterized fungal metabolite and
was recently identified in a high-throughput screen as an
inhibitor of LUBAC.19 However, (2) interacts with multiple
specific targets in mammalian cells,23-24 and as a complex
natural product it does not easily lend itself to structure-
based optimization. Stapled peptides suffer from similar
disadvantages as staple type and position, and changes in
peptide sequence can have profound effects on cell
permeability.25-27 Furthermore, a recent report describing a
novel MALDI-TOF MS-based E2/E3 ligase screening assay
identified the chemotherapeutic bendamustine as an
inhibitor of HOIP RBR in vitro, albeit additional studies are
warranted to verify its utility as a tool in a cellular setting.28
Design and synthesis of a structurally diverse
electrophilic fragment library: We synthesized a small
library of fragments linked to α,β-unsaturated methyl ester
electrophiles by amide bond formation between carboxylic
acid fragments, and amines (3) and (4) (Figure 2,
Supplementary Schemes S1, S2). Compound collections
based on the α,β-unsaturated methyl ester scaffold (3) have
been shown to exhibit a narrower reactivity profile
compared to other commonly used electrophiles for
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covalent approaches, such as acrylamides.29,
This
advantageous reactivity profile promotes hit identification
governed by molecular recognition rather than variations in
the reactivity of the electrophile.29 We chose to synthesize a
small number of amides using the extended amine (4),
based on the hypothesis that a longer linker between the
fragment and electrophile may allow a greater sampling of
potential protein interactions in the vicinity of the reactive
cysteine residue. Fragments were selected from the GSK
compound collection and commercial sources, based on
physicochemical properties such as molecular weight,
Inspired by recent reports describing the targeting of
reactive cysteine residues in proteins using fragment-based
covalent ligand screening of α,β-unsaturated methyl
esters,29-30 we decided to take a rational approach to target
the active-site cysteine residue in HOIP as a means to
covalently block its catalytic activity (Figure 1D). Herein, we
describe the first inhibitors targeting the active-site
cysteine of an RBR E3 ligase discovered by fragment-based
covalent ligand screening and go on to show that such
fragments serve as a useful starting point for structure-
based development of selective, cell-permeable probes to
interrogate the biology of LUBAC and other thioester-
forming E3 ubiquitin ligases.
clogP,
hydrogen
bond
acceptors
and
donors
(Supplementary Figure S1, Supplementary Table S1), and
clustered to ensure scaffold and functional group diversity.
In total 106 amides were synthesized in parallel to give a
physicochemically favorable and topologically diverse
screening set of electrophilic fragments (Figure 2). A major
concern of working with electrophilic compounds is their
potential for poor stability. We monitored the DMSO stock
solutions of the synthesized compounds over time to
Results and Discussion
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conclude that the vast majority were stable for >6 months oxidation and glutathione trapping (Supplementary Figure
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in DMSO when stored at 4 °C (Supplementary Figure S1).
Furthermore, evaluation of the chemical stability of a
representative set of compounds revealed good stability
over a range of pHs and overall good resistance towards
S1).
Identification of HOIP-reactive compounds by LC-MS
screening: In total, 104 compounds were sufficiently stable
in DMSO, and were pooled into 22 groups of 4-5 compounds
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Figure 2 – Design and synthesis of an electrophilic fragment library. A) Parallel synthesis of the electrophilic fragment
library from commercial and in-house sourced fragment carboxylic acids and the trifluoro acetic acid (TFA) salts of amines
(3) and (4) by amide bond formation, using coupling reagents 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxide hexafluorophosphate (HATU), propylphosphonic anhydride (T3P) or 1,1’-carbonyldiimidazole (CDI).
B) The fragment library members clogP plotted against molecular weight illustrates the physicochemical diversity of the
library. C) The fragment library members normalized principal moment of inertia (PMI) were calculated in Molecular
Operating Environment (MOE)33 based on low energy conformations and plotted as ratios in a triangular graph to illustrate
the molecular shape diversity of the library. The vertices of this triangular plot (0,1), (0.5,0.5) and (1,1) represent a perfect
rod, disc, and sphere, respectively.
across the range of concentrations tested, which was
(each compound separated in molecular weight by at least
limited by compound aqueous solubility, and it was
5 mass units) and screened against the recombinant RBR
therefore not possible to derive individual kinact and Kd
domain of HOIP using protein LC-MS at a 10-fold
stoichiometric excess of compound over protein (20 µM
compound). Careful consideration of physicochemical
properties during library design, and the adoption of a low
compound screening concentration enabled a facile and
robust LC-MS screening protocol with minimal sample
handling and no need for protein purification prior to
analysis. The initial library screen was carried out using
values. Nonetheless, the gradient of the derivative plot gives
the ratio kinact/Kd (0.97±0.01 M-1s-1).34 We conclude that Kd is
>400 µM for compound (5) and correspondingly, kinact will
be >0.0004 s-1 (Figure 3C, D, Supplementary Figure S2).
Based on the lower limit of Kd we can infer a ligand
efficiency, LE <0.24 for compound (5). This relatively
modest efficiency is in line with that observed for
comparable ligands targeting the NEDD4-1 HECT E3
HOIP RBR R1032A mutant protein, which behaves like wild
ligase.30 The LE may be reflective of the topology and
type,15 while all further confirmatory experiments were
available interactions in the binding site and should be
carried out on the wild type HOIP RBR domain. Among the
contextualized as other HOIP inhibitors are reported.
handful of preliminary hits that were identified (for full
Treatment of the active-site mutant HOIP RBR C885S with
screening data see Supplementary Figure S3), compound
compound (5) at a 100-fold stoichiometric excess did not
(5) stood out, producing >85% labelling after 24 h
result in protein labelling, strongly suggesting that
incubation (Figure 3A-C). Incubation of HOIP RBR with
compound (5) labels at the active site cysteine residue
compound (5) led to mono-labelling of the protein in a time-
(Figure 3E). This finding was further corroborated by
and concentration-dependent manner as determined by LC-
chymotryptic digest of the HOIP RBR-(5) adduct and LC-
MS. The concentration-response was analyzed according to
MS/MS analysis which unequivocally showed labelling on
the catalytic residue C885 (Supplementary Figure S5).
Having successfully identified compound (5) as a covalent
binder of the active site of the HOIP RBR E3 ligase, we
sought to determine the specificity of this labelling. We
performed an LC-MS-based counter screen of compound
(5) against E1, the E2s UbcH5c and UbcH7, the RBR
a two-step model of reversible ligand binding followed by
irreversible covalent modification (P + I ⇄ PI → PI*).34
Individual time courses were normalized to percentage
occupancy and fitted to a single exponential equation to give
kobs (Figure 3C) which was plotted against the concentration
of compound (5)(Figure 3D). The observed relationship
between kobs and compound concentration was linear
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domains of the E3 ligases HOIL-1L, RNF144A, HHARI and at just 2-fold excess. Unsurprisingly, compound (1) caused
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TRIAD3A, the HECT E3 ligases NEDD4L and E6AP, which all
form covalent thioester intermediates with ubiquitin, and
the two DUBs known to cleave linear polyubiquitin chains
OTULIN and CYLD-1 (Figure 3F). Compound (5) was
screened at a 10-fold stoichiometric excess, and for
comparison BAY 11-7082 (1) was used as a positive control
rapid and extensive labelling for most proteins while
compound (5) displayed encouraging selectivity for HOIP,
resulting in <40% labelling after 24 h incubation for all
other proteins screened (Figure 3F, Supplementary Figure
S6), indicating that compound (5) is a useful starting point
for further ligand optimization.
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Figure 3 – Compound (5) labels HOIP RBR at the active site cysteine residue C885 in a time- and concentration-
dependent manner. A) Compound (5) (20 µM) labelled HOIP RBR R1032A (2 µM) over time in the library screen. B) The
chemical structure of compound (5). C) Labelling of wild type HOIP RBR (2 µM) was validated by screening different
concentrations of (5). Each time course was normalized to percentage occupancy, fitted to a single exponential equation and
plotted in GraFit (v.7) as an average of three independent experiments to determine the observed first-order rate constant
(kobs). D) kobs measurements were plotted against the concentration of (5) to give the ratio of kinact/Kd as the slope of the
curve.34 E) The active-site C885S mutant of HOIP RBR (2 µM) was not labelled by compound (5) at 200 µM after 4 h, suggesting
that (5) targets the catalytic cysteine. F) LC-MS based counter screen of compounds (1) (2-fold excess) and (5) (10-fold
excess) against a panel of RBR E3 ligases (HOIL-1L, HHARI, TRIAD3A, RNF144A RBR domains), HECT E3 ligases (NEDD4L,
E6AP), E2 enzymes (UbcH5c, UbcH7), E1 enzyme and DUBs (OTULIN, CYLD-1). Data shown as percentage labelling after 0.5
and 24 h incubation.
Figure 4 – Compound (5) inhibits the formation of polyubiquitin chains by preventing loading of ubiquitin onto the
HOIP RBR domain. A) In vitro polyubiquitination assay monitoring the formation of unanchored polyubiquitin chains. Pre-
labelling of the HOIP RBR domain with compound (5) (middle) led to complete inhibition of polyubiquitin chain formation,
whereas treatment with the saturated analogue (6) had no effect on catalytic activity. B) In vitro ubiquitin transfer assay
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monitoring the sequential loading of N-terminally AlexaFluor647-labelled ubiquitin (Ub-N-A647) onto E1, UbcH7, HOIP RBR
domain and a C-terminally His-tagged ubiquitin substrate (Ub-C-His6). Pre-labelling of the HOIP RBR domain with compound
(5) (right) effectively prevented the loading of ubiquitin onto the E3, thereby inhibiting the formation of di-ubiquitin. For full
gels see Supplementary Figures S9 and S11.
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UbcH5c), ubiquitin and ATP to initiate unanchored
polyubiquitin chain synthesis. Labelling of HOIP fully
abolished chain formation when compared to DMSO control
(Figure 4A,
The HOIP-reactive hit compound inhibits enzyme
function: The functional effects of HOIP labelling were
investigated in an in vitro polyubiquitination assay. The
HOIP RBR domain was preincubated with compound (5) to
ensure full labelling and mixed with E1, E2 (UbcH7 or
possible binding modes in the active site of the HOIP RBR
domain for this chemical series.
Supplementary Figure S9). In contrast, treatment of HOIP
with the saturated analogue (6) (Scheme 1A) which is not
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able to form a covalent adduct had no inhibitory effect on
Structural analysis of the HOIP-inhibitor complex: To
polyubiquitin chain formation, demonstrating that the
rationalize the observed reactivity of the analogs tested and
labelling occurs through the α,β-unsaturated ester
to provide a basis for structure-based ligand optimization,
electrophile. Furthermore, a fluorescence-based ubiquitin
we solved the crystal structure of the RING2-LDD domain of
transfer assay that monitors the sequential formation of E1,
HOIP in covalent complex with inhibitor (5), at 2.15 Å
E2 and E3-ubiquitin thioesters,14 showed that labelling of
resolution (Figure 5B, Supplementary Table S5). Compound
the HOIP RBR domain with compound (5) inhibits
(5) is bound within the active site of HOIP with clear
formation of the E3~Ub thioester intermediate, but not any
contiguous electron density between C885 and the beta
of the previous steps, effectively blocking only ubiquitin
carbon of ester (5).
transfer onto the protein substrate (Figure 4B).
Exploration of SAR through targeted compound
modifications: To investigate the structural determinants
for compound recognition and labelling of the HOIP RBR
domain we synthesized a set of closely related analogs,
including saturated compounds (6) and (17), linker-
extended analog (9) and fragment control (10) (Scheme 1A,
D). Moreover, the pyridone scaffold was systematically
altered to identify key protein-binding elements, including
ring expansion (11a and b), aromatic rings (11c), changes
to the hydrogen bond donor and acceptor abilities (11d-f),
and fragment growth in different vectors (11g-i; Scheme
1B). Compound (11i) was part of the original screening
library. Lastly, we established a synthetic route based on
sequential alkaline hydrolysis and alkylation to enable
structural variation of the ester (13a and b), and the
synthesis of a clickable trans-cyclooctene (TCO) probe (15)
by amide bond formation between activated ester (13c),
and amine (14; Scheme 1C). The analogs were screened by
protein LC-MS against the HOIP RBR domain at a 10-fold
stoichiometric excess, and the percentage labelling was
measured after 0.5, 4, and 24 h incubation (Figure 5A,
Supplementary Figure S7). The data confirmed that
saturation of the α,β-unsaturated ester (6 and 17)
abolished activity. Furthermore, it showed that extension of
the linker between fragment and electrophile (9), or
removal of the fragment portion of the molecule (10)
resulted in significantly lower activity, suggesting that the
fragment is essential for establishing key molecular
interactions with the protein. The loss of reactivity
observed when the hydrogen bonding ability of the
pyridone was modified (e.g. analogues 11d-f) highlighted
the importance of this group for binding and labelling.
Alternative substitution patterns on the pyridone were
more tolerated. Ring-expanded, and aromatic analogs (11a-
c) gave a comparable or increased rate of labelling, while
fragment growth from different vectors of the pyridone (e.g.
11g vs 11h and 11i), or growing the ester substituent (13a,
13b and 15) had varying effects on activity, suggesting
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Scheme 1 – Analog synthesis. A) Control compounds (6), probe (15). D) Saturated control (17) was synthesized from
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6
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8
(9), and (10) were synthesized by amide bond formation
using amines (8), (4), and (3) respectively. B) Analogs (11a-
i) were synthesized by amide bond formation between the
corresponding carboxylic acid and amine (3). Compound
(11i) was synthesized as part of the original screening
library. C) Ester analogs (13a-c) were synthesized in two
steps from ester (11a) by alkaline hydrolysis and alkylation
reactions using the appropriate alkyl halide (ethyl iodide,
propargyl bromide and 4-nitrophenyl iodoacetate
respectively), and (13c) was further converted into TCO
carboxylic acid (16) and amine (8).
The cyclopentyl pyridone sits on a shallow ledge perfectly
placed for its aromatic ring to stack above F888 and be
anchored on one side by hydrogen bonds formed between
the pyridone and the backbone nitrogen and oxygen of
H889. This arrangement helps to explain why an extension
of the linker between the pyridone and Michael acceptor in
compound (9) led to reduced activity. Residues Y878, H889
and
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Figure 5 – Molecular determinants for interactions between the HOIP RING2 domain and compound (5). A) LC-MS
screening data plotted as percentage labelling of the HOIP RBR domain (2 µM) by key analogues (6), (9)-(11), (13), (15) (20
µM), and (17) (200 µM) after 0.5, 4, and 24 h incubation. For full screening data see Supplementary Figures S7, S8. B) Ribbon
representation of the HOIP RING2-LDD domain structure in complex with compound (5) at 2.15 Å resolution (PDB: 6GZY).
Zn2+ ions are shown as grey spheres and compound (5) as ball and stick. The inset shows a zoom into the active site showing
hydrogen bonds (dashed lines) made by compound (5) with water molecules (red spheres) and HOIP (top), and the pocket
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around the active site which accommodates compound (5) (bottom). C) Sequence alignment of RING2 domains of those RBR
E3 ligases that were screened against compound (5). The catalytic cysteine residue is highlighted in red, non-catalytic cysteine
residues in purple, and non-conserved residues contributing to the compound-binding pocket in HOIP are highlighted with a
blue asterisk.
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T891 enclose the other side of the ring system to form a
defined, but probably somewhat malleable site, as
demonstrated by the ability to accommodate small changes
in ring sizes and substitutions in this region of the
compound (e.g. 11a-c and 11g). Additional water-mediated
and direct hydrogen bonds are made by all the other
heteroatoms of (5) (Figure 5B). The ester chain of (5) lies
within a pocket orthogonal to the ledge, with the carbonyl
oxygen held in position by the hydroxyl side chain of S899,
in a conformation that may facilitate the reaction of the
catalytic cysteine and the Michael acceptor. Sequence
alignment of the RING2 domains of the RBR E3 ligases HOIP,
HOIL-1L, HHARI,
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Figure 6 – Compound (11a) labels and inhibits HOIP in HEK293T and MCF7 cells. A) Total ion count traces of the
dodecapeptide GGCMHFHCTQCR containing C885 after FLAG-IP and tryptic digest. Incubating HEK293T cells with
compounds (11a) and (11i) at 30 µM for 8 h gave significant labelling of overexpressed 3×FLAG-tagged HOIP RBR by
compound (11a, middle) but not (11i, bottom). B) LC-MS/MS analysis of the GGCMHFHCTQCR peptides from DMSO control
(top) and (11a)-treated cells (bottom), showing labelling of (11a) at C885 (green star). The samples were globally labelled
with iodoacetamide (purple asterisk) prior to tryptic digest, and methionine oxidation (yellow asterisk) was taken into
account. C) On-bead polyubiquitination assay. In-cell labelling of overexpressed 3×FLAG-tagged HOIP RBR by compound
(11a) (30 µM) led to a significant decrease in free polyubiquitin chain formation compared to DMSO and (11i) (30 µM)
controls. Non- (-ve), mock or 3×FLAG HOIP RBR C885A transfected HEK293T cells did not show formation of polyubiquitin
chains in the polyubiquitin assay. The data is representative of two independent experiments. D) NF-κB Luciferase reporter
and CellTiter-Glo® Luminescent cell viability (Promega) assays. Compound (11a) but not saturated analog (17) inhibited
NF-κB activity in HEK293T cells overexpressing full-length HOIP, HOIL-1L and SHARPIN in a dose-dependent manner (top),
with no effect on cell viability (bottom). The graphs show average data of three independent experiments, each carried out in
triplicate. E) Activity-based protein profiling (ABPP) of HEK293T cells overexpressing 3×FLAG-tagged HOIP RBR (wt and
C885A mutant). Cells were pre-incubated with DMSO (control and HOIP C885A) or compounds (11a), (11i) or (17) (30 µM)
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for 7 h, then treated with TCO probe (15) (3 µM) for 2 h and lysed. The cell lysates were treated with Cy5-Tz (3 µM) for 1 h.
F) ABPP of HEK293T (overexpressed HOIP RBR) and MCF7 cells (endogenous LUBAC expression), by competition of (15) (3
µM) with (11a) (30 µM), using quantitative MS. Specific labelling (maroon boxes) of HOIP (RNF31, Supplementary datafile)
was observed in both cell types. Treatments were carried out in triplicate. For full blots see Supplementary Figures S13, S16.
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TRIAD3A and RNF144A (Figure 5C) reveal sequence
variation around the active-site cysteine, with Y878, F888
and S899 that contact the compound via their side chains
not being conserved. This variation may account for some
of the selectivity observed in labelling HOIP over other
RBRs. Taken together, these observations suggest that
specific molecular interactions between the fragment and
protein help to position the electrophile favorably and
promote sufficient residence time to support covalent bond
formation.
selectivity for most protein labelling applications in cell
lysates and in live cells compared to other types of click
chemistries.38-39 In brief, HEK293T cells overexpressing
3×FLAG-tagged HOIP RBR domain were pre-treated with
(11a) followed by (15) before cell lysis. The impact of
pretreatment time and concentration of (11a) and (15) was
evaluated by in-gel fluorescence using Cy5-Tz
(Supplementary Figure S15). Pretreating the HEK293T cells
with 30 µM (11a) for 7 h, then 3 µM (15) for 2 h, followed
by lysis and TCO-Tz click reaction of the lysates gave
significant reduction of the in-gel fluorescence of HOIP RBR
compared to (11i), (17), DMSO and transfection controls
(Figure 6E). Encouraged by these results, we then used
quantitative MS to identify specifically labelled proteins.
HEK293T cells overexpressing 3×FLAG-tagged HOIP RBR,
as well as MCF7 cells endogenously expressing LUBAC were
treated with DMSO or (11a), then (15) as described.
Proteins labelled by (15) were enriched using Tz-beads,38
treated with trypsin for on-bead digest, and the resulting
peptides were analyzed by quantitative MS. To ensure
specificity, the residual capturing of every protein was
calculated as log2-fold change competition versus DMSO
control (mean ratio), p-values were calculated using
student’s t-test, and the results plotted so that specifically
bound proteins with high p-value and strong fold-change
populate the upper left quadrant (maroon boxes, Figure
6F). In addition to HOIP, 11 other proteins in HEK293T cells,
and 8 other proteins in MCF7 cells were identified as being
specifically covalently labelled by (11a). Among these
proteins, IRAK4 is a kinase upstream of NF-κB and therefore
labelling by (11a) could impact physiological NF-κB
signalling. However, in an IRAK4 substrate phosphorylation
assay, compound (11a) showed no inhibition at
concentrations up to 200 μM (Supplementary Figure S17),
indicating that it is unlikely to affect NF-κB activation.
Considering that no other RBR or HECT E3 ligases were
identified as off-targets in the proteomics experiment, and
that (11a) is an unoptimized fragment hit, the observed
cellular selectivity profile is striking. Furthermore, specific
labelling of HOIP in MCF7 cells provides the first evidence
of targeting endogenous LUBAC in cells, showing that (11a)
already constitutes a valuable tool compound and an
excellent starting point for further optimization.
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Targeting of HOIP in mammalian cells: To provide
further mechanistic characterization of the inhibitors we
evaluated HOIP targeting in mammalian cells. For this
purpose, we overexpressed the 3×FLAG-tagged HOIP RBR
domain in HEK293T cells and used the slightly more potent
analog (11a; Figure 5A). The cells were transiently
transfected with 3×FLAG HOIP RBR for 48 h, then incubated
in media supplemented with inhibitor (11a) or inactive
analogue (11i) at 30 µM for 8 h before cell lysis. Following
immunoprecipitation (IP) of the 3×FLAG-HOIP RBR domain
with anti-FLAG magnetic beads, the protein was digested on
the beads with trypsin. Subsequent LC-MS/MS analysis
showed that the HOIP RBR domain was significantly
labelled by compound (11a) but not (11i) at C885,
demonstrating that these inhibitors can effectively bind and
label HOIP RBR in live cells (Figure 6A,
B and
Supplementary Figure S12). Furthermore,
immunoprecipitated 3×FLAG-tagged HOIP RBR (wild type
and catalytically inactive C885A mutant) from HEK293T
cells was used for an in vitro polyubiquitination assay with
E1, UbcH7 and ubiquitin, to demonstrate that in-cell
labelling of over-expressed HOIP RBR with compound
(11a) leads to significant loss of the catalytic activity of
HOIP, compared to (11i) and controls (Figure 6C). Given the
central role of LUBAC in canonical NF-κB signalling,8 we
then adopted an NF-κB Luciferase reporter assay to further
demonstrate functional effects of HOIP inhibition. Treating
HEK293T cells overexpressing full length HOIP, HOIL-1L
and SHARPIN with compound (11a) overnight led to
inhibition of NF-κB activation in a concentration-dependent
manner, with an estimated IC50-value of 37 ± 4 µM (Figure
6D, top, Supplementary Figure S14). Conversely, saturated
analog (17) had no effect on NF-κB activity, and neither
compound had any effect on cell viability (Figure 6D,
bottom, Supplementary Figure S14). Finally, we assessed
the selectivity of compound (11a) on a proteome-wide level
using activity-based protein profiling (ABPP). ABPP is a
powerful technique that utilizes covalent probes as handles
for protein visualization or enrichment to evaluate protein
engagement in cells,35 an approach that has recently
enabled proteome-wide in-cell screening of covalent
fragments.36-37 We synthesized probe (15), based on the
structure of (11a) and functionalized with a TCO moiety as
enrichment handle to enable inverse-electron demand
Diels-Alder (IED-DA) click chemistry with tetrazine (Tz);
IED-DA reactions typically give superior efficiency and
Conclusion
Despite the crucial biological roles of E3 ubiquitin ligases
the development of selective tools that target their activity
has been limited to date. Here, we describe how fragment-
based covalent ligand screening allowed the rapid
identification of inhibitors targeting the active site cysteine
of the catalytic HOIP subunit of LUBAC, a member of the
RBR family of E3s. A diverse library of fragments linked to
α,β-unsaturated ester electrophiles was synthesized and
rapidly screened by protein LC-MS to identify compound
(5) as a covalent binder of HOIP. The established screening
protocol required minimal sample handling and enabled
expedient screening of multiple proteins. Biochemical
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subset of the electrophilic fragment library. We thank S.
characterization of (5) and its analog (11a) demonstrated
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Lehmann for assay support and M. Boesche, T. Rudi, M. Kloes-
Hudak, and K. Kammerer for assistance with mass
spectrometry. Henning Walczak (HOIP, HOIL-1L and SHARPIN)
and Teresa Thurston (pRDII Luc) are thanked for their kind
donation of plasmids for the NF-κB assay. Thau F. Ho is thanked
for the expression and purification of IRAK4 protein. Sean Lynn
is thanked for assistance with NMR, and Marcela Castro for
assistance with manuscript figure design and preparation. We
thank Andy Powell and David Powell for helpful discussions.
This work was supported by the Francis Crick Institute which
receives its core funding from Cancer Research UK (FC010142
to K.R.), the UK Medical Research Council (FC010142 to K.R.)
and the Wellcome Trust (FC010142 to K.R.). We also thank GSK
for its commitment to support fundamental discovery research
through the establishment of the Crick-GSK LinkLabs
partnership.
that these compounds label HOIP with promising
proteome-wide selectivity and effectively inhibit linear
polyubiquitin chain formation in vitro and in a cellular
environment. The combination of analog synthesis and a
high-resolution crystal structure of the HOIP RING2-LDD
domain in covalent complex with (5) provides the first
insight into the molecular requirements for fragment
recognition by HOIP and will allow the rational design of
inhibitors with increased activity and specificity for this
RBR family member. Taken together, our results show the
first proof-of-concept for screening and rational design of a
covalent inhibitor library designed to target the active site
of an RBR E3 ligase, an approach which is generally
applicable to other thioester-forming E3 ligases and should
help in the search for novel inhibitors of this important class
of enzymes.
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REFERENCES
ASSOCIATED CONTENT
1. Komander, D.; Rape, M., The ubiquitin code. Annu. Rev.
Biochem. 2012, 81, 203-29.
Detailed synthetic procedures for all synthesized compounds,
1H NMR spectra and LC-MS chromatograms for all library
compounds, 1H, 13C NMR, LC-MS and HRMS data for all
synthesized hit analogs, physicochemical analyses of the
screening library, details of recombinant protein expression
and purification, experimental procedures for biochemical
assays, TOF LC-MS protocol and protein deconvolution data for
2. Pickart, C. M.; Eddins, M. J., Ubiquitin: structures,
functions, mechanisms. Biochim. Biophys. Acta 2004, 1695
(1-3), 55-72.
3. Cohen, P.; Tcherpakov, M., Will the Ubiquitin System
Furnish as Many Drug Targets as Protein Kinases? Cell
2010, 143 (5), 686-693.
LC-MS-based
compound
screening,
protein
X-ray
crystallography, ABPP and cell based experiment, as well as a
supplementary datafile for the quantitative MS-based ABPP
experiment. The supporting information is available free of
charge on the ACS Publications website.
4. Huang, X.; Dixit, V. M., Drugging the undruggables:
exploring the ubiquitin system for drug development. Cell
Res 2016, 26 (4), 484-98.
AUTHOR INFORMATION
5. Kirisako, T.; Kamei, K.; Murata, S.; Kato, M.; Fukumoto, H.;
Kanie, M.; Sano, S.; Tokunaga, F.; Tanaka, K.; Iwai, K., A
ubiquitin ligase complex assembles linear polyubiquitin
chains. EMBO J. 2006, 25 (20), 4877-87.
Corresponding Authors
*katrin.rittinger@crick.ac.uk
*david.x.house@gsk.com
6. Tokunaga, F.; Sakata, S.; Saeki, Y.; Satomi, Y.; Kirisako, T.;
Kamei, K.; Nakagawa, T.; Kato, M.; Murata, S.; Yamaoka, S.;
Yamamoto, M.; Akira, S.; Takao, T.; Tanaka, K.; Iwai, K.,
Involvement of linear polyubiquitylation of NEMO in NF-
kappaB activation. Nat Cell Biol 2009, 11 (2), 123-32.
ORCID
Henrik Johansson: 0000-0002-9990-6261
Yi-Chun Isabella Tsai: 0000-0003-4123-5052
Chun-Wa Chung: 0000-0002-2480-3110
Sandra Kümper 0000-0002-5478-0503
Luigi Martino: 0000-0001-5871-312
H. Christian Eberl: 0000-0002-4159-5958
Marcel Muelbaier: 0000-0002-5414-0258
David House: 0000-0002-9635-2451
Katrin Rittinger: 0000-0002-7698-4435
7. Rieser, E.; Cordier, S. M.; Walczak, H., Linear
ubiquitination: a newly discovered regulator of cell
signalling. Trends Biochem. Sci. 2013, 38 (2), 94-102.
8. Sasaki, K.; Iwai, K., Roles of linear ubiquitinylation, a
crucial regulator of NF-kappaB and cell death, in the
immune system. Immunol. Rev. 2015, 266 (1), 175-89.
ACKNOWLEDGMENTS
We thank Andy Purkiss for help with data processing and
crystallographic analysis, the Diamond Light Source, Oxford,
UK for synchrotron access, Abigail Davies and Ashleigh Howes
for the 3×FLAG HOIP RBR pcDNA3.1(+) construct, Georgina
Green for managing compound DMSO stock solutions and
carrying out the DMSO stability LC-MS assays, Ruben Gomez-
Sanchez and Natalie Warren-Godkin for carrying out the
chemical stability assays, and Shenaz Bunally for determining
kinetic solubility and ChromLogD7.4 for the representative
9. Rittinger, K.; Ikeda, F., Linear ubiquitin chains: enzymes,
mechanisms and biology. Open Biol. 2017, 7 (4).
10. Hrdinka, M.; Gyrd-Hansen, M., The Met1-Linked
Ubiquitin Machinery: Emerging Themes of (De)regulation.
Mol. Cell. 2017, 68 (2), 265-280.
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 20
11. Ikeda, F.; Deribe, Y. L.; Skanland, S. S.; Stieglitz, B.;
complex (LUBAC). Bioorg. Med. Chem. 2018, 26 (6), 1179-
1188.
1
2
3
4
5
6
7
8
Grabbe, C.; Franz-Wachtel, M.; van Wijk, S. J.; Goswami, P.;
Nagy, V.; Terzic, J.; Tokunaga, F.; Androulidaki, A.;
Nakagawa, T.; Pasparakis, M.; Iwai, K.; Sundberg, J. P.;
Schaefer, L.; Rittinger, K.; Macek, B.; Dikic, I., SHARPIN forms
a linear ubiquitin ligase complex regulating NF-kappaB
activity and apoptosis. Nature 2011, 471 (7340), 637-41.
21. Yang, Y.; Schmitz, R.; Mitala, J.; Whiting, A.; Xiao, W.;
Ceribelli, M.; Wright, G. W.; Zhao, H.; Yang, Y.; Xu, W.;
Rosenwald, A.; Ott, G.; Gascoyne, R. D.; Connors, J. M.;
Rimsza, L. M.; Campo, E.; Jaffe, E. S.; Delabie, J.; Smeland, E.
B.; Braziel, R. M.; Tubbs, R. R.; Cook, J. R.; Weisenburger, D.
D.; Chan, W. C.; Wiestner, A.; Kruhlak, M. J.; Iwai, K.; Bernal,
F.; Staudt, L. M., Essential role of the linear ubiquitin chain
assembly complex in lymphoma revealed by rare germline
polymorphisms. Cancer Discov. 2014, 4 (4), 480-93.
12. Gerlach, B.; Cordier, S. M.; Schmukle, A. C.; Emmerich, C.
H.; Rieser, E.; Haas, T. L.; Webb, A. I.; Rickard, J. A.; Anderton,
H.; Wong, W. W.; Nachbur, U.; Gangoda, L.; Warnken, U.;
Purcell, A. W.; Silke, J.; Walczak, H., Linear ubiquitination
prevents inflammation and regulates immune signalling.
Nature 2011, 471 (7340), 591-6.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22. Fujita, H.; Tokunaga, A.; Shimizu, S.; Whiting, A. L.;
Aguilar-Alonso, F.; Takagi, K.; Walinda, E.; Sasaki, Y.;
Shimokawa, T.; Mizushima, T.; Ohki, I.; Ariyoshi, M.; Tochio,
H.; Bernal, F.; Shirakawa, M.; Iwai, K., Cooperative Domain
Formation by Homologous Motifs in HOIL-1L and SHARPIN
Plays A Crucial Role in LUBAC Stabilization. Cell Rep. 2018,
23 (4), 1192-1204.
13. Tokunaga, F.; Nakagawa, T.; Nakahara, M.; Saeki, Y.;
Taniguchi, M.; Sakata, S.; Tanaka, K.; Nakano, H.; Iwai, K.,
SHARPIN is a component of the NF-kappaB-activating linear
ubiquitin chain assembly complex. Nature 2011, 471
(7340), 633-6.
14. Stieglitz, B.; Morris-Davies, A. C.; Koliopoulos, M. G.;
Christodoulou, E.; Rittinger, K., LUBAC synthesizes linear
ubiquitin chains via a thioester intermediate. EMBO Rep.
2012, 13 (9), 840-6.
23. Waring, P.; Beaver, J., Gliotoxin and related
epipolythiodioxopiperazines. Gen. Pharmacol. 1996, 27 (8),
1311-6.
24. Dolan, S. K.; O'Keeffe, G.; Jones, G. W.; Doyle, S.,
Resistance is not futile: gliotoxin biosynthesis, functionality
and utility. Trends Microbiol. 2015, 23 (7), 419-28.
15. Stieglitz, B.; Rana, R. R.; Koliopoulos, M. G.; Morris-
Davies, A. C.; Schaeffer, V.; Christodoulou, E.; Howell, S.;
Brown, N. R.; Dikic, I.; Rittinger, K., Structural basis for
ligase-specific conjugation of linear ubiquitin chains by
HOIP. Nature 2013, 503 (7476), 422-426.
25. Chu, Q. M., R. E.; Hilinski, G.J.; Kim, Y.-W.; Grossman, T.
N.; Yeh, J. T.-H.; Verdine, G.L., Towards understanding cell
penetration by stapled peptides MedChemComm 2015, 6,
111-119.
16. Smit, J. J.; Monteferrario, D.; Noordermeer, S. M.; van
Dijk, W. J.; van der Reijden, B. A.; Sixma, T. K., The E3 ligase
HOIP specifies linear ubiquitin chain assembly through its
RING-IBR-RING domain and the unique LDD extension.
EMBO J. 2012, 31 (19), 3833-44.
26. Walensky, L. D.; Bird, G. H., Hydrocarbon-stapled
peptides: principles, practice, and progress. J. Med. Chem.
2014, 57 (15), 6275-88.
27. Cromm, P. M.; Spiegel, J.; Grossmann, T. N., Hydrocarbon
stapled peptides as modulators of biological function. ACS
Chem. Biol. 2015, 10 (6), 1362-75.
17. Lechtenberg, B. C.; Rajput, A.; Sanishvili, R.;
Dobaczewska, M. K.; Ware, C. F.; Mace, P. D.; Riedl, S. J.,
Structure of a HOIP/E2~ubiquitin complex reveals RBR E3
ligase mechanism and regulation. Nature 2016, 529 (7587),
546-50.
28. De Cesare, V.; Johnson, C.; Barlow, V.; Hastie, J.; Knebel,
A.; Trost, M., The MALDI-TOF E2/E3 Ligase Assay as
Universal Tool for Drug Discovery in the Ubiquitin Pathway.
Cell Chem. Biol. 2018, 25 (9), 1117-1127 e4.
18. Strickson, S.; Campbell, D. G.; Emmerich, C. H.; Knebel, A.;
Plater, L.; Ritorto, M. S.; Shpiro, N.; Cohen, P., The anti-
inflammatory drug BAY 11-7082 suppresses the MyD88-
dependent signalling network by targeting the ubiquitin
system. Biochem. J. 2013, 451 (3), 427-37.
29. Kathman, S. G.; Xu, Z.; Statsyuk, A. V., A Fragment-Based
Method to Discover Irreversible Covalent Inhibitors of
Cysteine Proteases. J. Med. Chem. 2014, 57 (11), 4969-4974.
19. Sakamoto, H.; Egashira, S.; Saito, N.; Kirisako, T.; Miller,
S.; Sasaki, Y.; Matsumoto, T.; Shimonishi, M.; Komatsu, T.;
Terai, T.; Ueno, T.; Hanaoka, K.; Kojima, H.; Okabe, T.;
Wakatsuki, S.; Iwai, K.; Nagano, T., Gliotoxin suppresses NF-
kappaB activation by selectively inhibiting linear ubiquitin
chain assembly complex (LUBAC). ACS Chem. Biol. 2015, 10
(3), 675-81.
30. Kathman, S. G.; Span, I.; Smith, A. T.; Xu, Z.; Zhan, J.;
Rosenzweig, A. C.; Statsyuk, A. V., A Small Molecule That
Switches a Ubiquitin Ligase From a Processive to a
Distributive Enzymatic Mechanism. J. Am. Chem. Soc. 2015,
137 (39), 12442-5.
31. Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.;
Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery,
J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.;
Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M.
20. Aguilar-Alonso, F.; Whiting, A. L.; Kim, Y. J.; Bernal, F.,
Biophysical and biological evaluation of optimized stapled
peptide inhibitors of the linear ubiquitin chain assembly
10
ACS Paragon Plus Environment

Page 11 of 20
Journal of the American Chemical Society
M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram,
V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y., Chemical and
computational methods for the characterization of covalent
reactive groups for the prospective design of irreversible
inhibitors. J. Med. Chem. 2014, 57 (23), 10072-9.
1
2
3
4
5
6
7
8
32. Cee, V. J.; Volak, L. P.; Chen, Y.; Bartberger, M. D.; Tegley,
C.; Arvedson, T.; McCarter, J.; Tasker, A. S.; Fotsch, C.,
Systematic Study of the Glutathione (GSH) Reactivity of N-
Arylacrylamides: 1. Effects of Aryl Substitution. J. Med.
Chem. 2015, 58 (23), 9171-8.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
33. Molecular Operating Environment (MOE), C. C. G. U.,
1010 Sherbooke St. West, Suite #910, Montreal, QC, Cana-
da, H3A 2R7 2018.
34. Strelow, J. M., A Perspective on the Kinetics of Covalent
and Irreversible Inhibition. SLAS Discov. 2017, 22 (1), 3-20.
35. Heal, W. P.; Dang, T. H.; Tate, E. W., Activity-based
probes: discovering new biology and new drug targets.
Chem. Soc. Rev. 2011, 40 (1), 246-57.
36. Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning,
B. D.; Gonzalez-Paez, G. E.; Chatterjee, S.; Lanning, B. R.;
Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F.,
Proteome-wide covalent ligand discovery in native
biological systems. Nature 2016, 534 (7608), 570-4.
37. Parker, C. G.; Galmozzi, A.; Wang, Y.; Correia, B. E.; Sasaki,
K.; Joslyn, C. M.; Kim, A. S.; Cavallaro, C. L.; Lawrence, R. M.;
Johnson, S. R.; Narvaiza, I.; Saez, E.; Cravatt, B. F., Ligand and
Target Discovery by Fragment-Based Screening in Human
Cells. Cell 2017, 168 (3), 527-541 e29.
38. Rutkowska, A.; Thomson, D. W.; Vappiani, J.; Werner, T.;
Mueller, K. M.; Dittus, L.; Krause, J.; Muelbaier, M.;
Bergamini, G.; Bantscheff, M., A Modular Probe Strategy for
Drug Localization, Target Identification and Target
Occupancy Measurement on Single Cell Level. ACS Chem.
Biol. 2016, 11 (9), 2541-50.
39. Murrey, H. E.; Judkins, J. C.; Am Ende, C. W.; Ballard, T. E.;
Fang, Y.; Riccardi, K.; Di, L.; Guilmette, E. R.; Schwartz, J. W.;
Fox, J. M.; Johnson, D. S., Systematic Evaluation of
Bioorthogonal Reactions in Live Cells with Clickable
HaloTag Ligands: Implications for Intracellular Imaging. J.
Am. Chem. Soc. 2015, 137 (35), 11461-75.
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Figure 1 – Targeting HOIP using fragment-based covalent ligand screening. A) Schematic of the
ubiquitination cas-cade highlighting the formation of linear (M1) polyubiquitin chains (Ubn) by LUBAC, a
reaction that proceeds via a cova-lent thioester intermediate between the HOIP subunit and ubiquitin. B)
Schematic representation of the HOIP E3 ligase, highlighting the RBR which contains the active-site cysteine
residue C885 (yellow circle) and the linear chain-determining domain, LDD (amino acids 697- 1072). C)
Small molecule LUBAC inhibitors (1) BAY 11-7082 and (2) glio-toxin. D) Overview of our approach to
develop covalent probes targeting the active site cysteine residue of HOIP using fragment-based covalent
ligand screening by protein LC-MS.
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Figure 2 – Design and synthesis of an electrophilic fragment library. A) Parallel synthesis of the electrophilic
frag-ment library from commercial and in-house sourced fragment carboxylic acids and the trifluoro acetic
acid (TFA) salts of amines (3) and (4) by amide bond formation, using coupling reagents 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU),
propylphosphonic anhydride (T3P) or 1,1’-carbonyldiimidazole (CDI). B) The fragment library members
clogP plotted against molecular weight illustrates the phys-icochemical diversity of the library. C) The
fragment library members normalized principal moment of inertia (PMI) were calculated in Molecular
Operating Environment (MOE)33 based on low energy conformations and plotted as ratios in a triangular
graph to illustrate the molecular shape diversity of the library. The vertices of this triangular plot (0,1),
(0.5,0.5) and (1,1) represent a perfect rod, disc, and sphere, respectively.
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Figure 3 – Compound (5) labels HOIP RBR at the active site cysteine residue C885 in a time- and
concentration-dependent manner. A) Compound (5) (20 µM) labelled HOIP RBR R1032A (2 µM) over time in
the library screen. B) The chemical structure of compound (5). C) Labelling of wild type HOIP RBR (2 µM)
was validated by screening different concentrations of (5). Each time course was normalized to percentage
occupancy, fitted to a single exponential equation and plotted in GraFit (v.7) as an average of three
independent experiments to determine the observed first-order rate con-stant (kobs). D) kobs
measurements were plotted against the concentration of (5) to give the ratio of kinact/Kd as the slope of the
curve.34 E) The active-site C885S mutant of HOIP RBR (2 µM) was not labelled by compound (5) at 200 µM
after 4 h, suggesting that (5) targets the catalytic cysteine. F) LC-MS based counter screen of compounds
(1) (2-fold excess) and (5) (10-fold excess) against a panel of RBR E3 ligases (HOIL-1L, HHARI, TRIAD3A,
RNF144A RBR domains), HECT E3 ligases (NEDD4L, E6AP), E2 enzymes (UbcH5c, UbcH7), E1 enzyme and
DUBs (OTULIN, CYLD-1). Data shown as percentage labelling after 0.5 and 24 h incubation.
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Figure 4 – Compound (5) inhibits the formation of polyubiquitin chains by preventing loading of ubiquitin
onto the HOIP RBR domain. A) In vitro polyubiquitination assay monitoring the formation of unanchored
polyubiquitin chains. Pre-labelling of the HOIP RBR domain with compound (5) (middle) led to complete
inhibition of polyubiquitin chain formation, whereas treatment with the saturated analogue (6) had no effect
on catalytic activity. B) In vitro ubiqui-tin transfer assay monitoring the sequential loading of N-terminally
AlexaFluor647-labelled ubiquitin (Ub-N-A647) onto E1, UbcH7, HOIP RBR domain and a C-terminally His-
tagged ubiquitin substrate (Ub-C-His6). Pre-labelling of the HOIP RBR domain with compound (5) (right)
effectively prevented the loading of ubiquitin onto the E3, thereby inhibiting the formation of di-ubiquitin.
For full gels see Supplementary Figures S9 and S11.
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Figure 5 – Molecular determinants for interactions between the HOIP RING2 domain and compound (5). A)
LC-MS screening data plotted as percentage labelling of the HOIP RBR domain (2 µM) by key analogues (6),
(9)-(11), (13), (15) (20 µM), and (17) (200 µM) after 0.5, 4, and 24 h incubation. For full screening data
see Supplementary Figures S7, S8. B) Ribbon representation of the HOIP RING2-LDD domain structure in
complex with compound (5) at 2.15 Å resolu-tion (PDB: 6GZY). Zn2+ ions are shown as grey spheres and
compound (5) as ball and stick. The inset shows a zoom into the active site showing hydrogen bonds
(dashed lines) made by compound (5) with water molecules (red spheres) and HOIP (top), and the pocket
around the active site which accommodates compound (5) (bottom). C) Sequence alignment of RING2
domains of those RBR E3 ligases that were screened against compound (5). The catalytic cysteine residue is
high-lighted in red, non-catalytic cysteine residues in purple, and non-conserved residues contributing to the
compound-binding pocket in HOIP are highlighted with a blue asterisk.
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Figure 6 – Compound (11a) labels and inhibits HOIP in HEK293T and MCF7 cells. A) Total ion count traces of
the dodecapeptide GGCMHFHCTQCR containing C885 after FLAG-IP and tryptic digest. Incubating HEK293T
cells with com-pounds (11a) and (11i) at 30 µM for 8 h gave significant labelling of overexpressed 3×FLAG-
tagged HOIP RBR by com-pound (11a, middle) but not (11i, bottom). B) LC-MS/MS analysis of the
GGCMHFHCTQCR peptides from DMSO control (top) and (11a)-treated cells (bottom), showing labelling of
(11a) at C885 (green star). The samples were globally la-belled with iodoacetamide (purple asterisk) prior
to tryptic digest, and methionine oxidation (yellow asterisk) was taken into account. C) On-bead
polyubiquitination assay. In-cell labelling of overexpressed 3×FLAG-tagged HOIP RBR by com-pound (11a)
(30 µM) led to a significant decrease in free polyubiquitin chain formation compared to DMSO and (11i) (30
µM) controls. Non- (-ve), mock or 3×FLAG HOIP RBR C885A transfected HEK293T cells did not show
formation of polyubiquitin chains in the polyubiquitin assay. The data is representative of two independent
experiments. D) NF-κB Luciferase reporter and CellTiter-Glo® Luminescent cell viability (Promega) assays.
Compound (11a) but not saturated analog (17) inhibited NF-κB activity in HEK293T cells overexpressing full-
length HOIP, HOIL-1L and SHARPIN in a dose-dependent manner (top), with no effect on cell viability
(bottom). The graphs show average data of three independent experiments, each done in triplicate. E)
Activity-based protein profiling (ABPP) of HEK293T cells overexpressing 3×FLAG-tagged HOIP RBR (wt and
C885A mutant). Cells were pre-incubated with DMSO (control and HOIP C885A) or compounds (11a), (11i)
or (17) (30 µM) for 7 h, then treated with TCO probe (15) (3 µM) for 2 h and lysed. The cell lysates were
treated with Cy5-Tz (3 µM) for 1 h. F) ABPP of HEK293T (overexpressed HOIP RBR) and MCF7 cells (endog-
enous LUBAC expression), by competition of (15) (3 µM) with (11a) (30 µM), using quantitative MS. Specific
labelling (maroon boxes) of HOIP (RNF31, Supplementary datafile) was observed in both cell types.
Treatments were done in trip-licate. For full blots see Supplementary Figures S13, S16.
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Scheme 1 – Analog synthesis. A) Control compounds (6), (9), and (10) were synthesized by amide bond for-
mation using amines (8), (4), and (3) respectively. B) Analogs (11a-i) were synthesized by amide bond for-
mation between the corresponding carboxylic acid and amine (3). Compound (11i) was synthesized as part
of the original screening library. C) Ester analogs (13a-c) were synthesized in two steps from ester (11a) by
alka-line hydrolysis and alkylation reactions using the appro-priate alkyl halide (ethyl iodide, propargyl
bromide and 4-nitrophenyl iodoacetate respectively), and (13c) was further converted into TCO probe (15).
D) Saturated con-trol (17) was synthesized from carboxylic acid (16) and amine (8).
149x271mm (300 x 300 DPI)
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