Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid

Article abstract of DOI:10.1002/anie.201912392

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent K_D of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Full text of DOI:10.1002/anie.201912392

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Chemical Proteomics and Phenotypic Profiling Identifies the Aryl
Hydrocarbon Receptor as a Molecular Target of the Utrophin
Modulator Ezutromid
Authors: Isabel V. L. Wilkinson, Kelly J. Perkins, Hannah Dugdale, Lee
Moir, Aini Vuorinen, Maria Chatzopoulou, Sarah E. Squire,
Sebastian Monecke, Alexander Lomow, Marcus Geese, Philip
D. Charles, Peter Burch, Jonathan M. Tinsley, Graham M.
Wynne, Stephen G. Davies, Francis X. Wilson, Fraydoon
Rastinejad, Shabaz Mohammed, Kay E. Davies, and Angela
J. Russell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201912392
Angew. Chem. 10.1002/ange.201912392
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10.1002/anie.201912392
Angewandte Chemie International Edition
Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a
Molecular Target of the Utrophin Modulator Ezutromid
Isabel V. L. Wilkinson1, Kelly J. Perkins2, Hannah Dugdale2, Lee Moir2, Aini Vuorinen1, Maria
Chatzopoulou1, Sarah E. Squire2, Sebastian Monecke3, Alexander Lomow3, Marcus Geese3, Philip D.
Charles4,6, Peter Burch,5 Jonathan M. Tinsley,5 Graham M. Wynne1, Stephen G. Davies,1 Francis X.
Wilson5, Fraydoon Rastinejad6, Shabaz Mohammed1,4, Kay E. Davies2, Angela J. Russell1,7*
1Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road,
Oxford, OX1 3TA, UK; 2Department of Physiology, Anatomy and Genetics, University of Oxford, Sir
3
Henry Wellcome Building of Gene Function, South Parks Road, Oxford, OX1 3PT, UK; Evotec
International GmbH, Manfred Eigen Campus, Essener Bogen 7, 22419 Hamburg, Germany,
4Department of Biochemistry, University of Oxford, South Parks Rd, Oxford, OX1 3QU, UK; 5Summit
Therapeutics plc., 136a Eastern Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4SB, UK; 6Target
Discovery Institute, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, OX3 7FZ, UK;
7Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3PQ, UK.
*To whom correspondence should be addressed: angela.russell@chem.ox.ac.uk, Tel: +441865275643
Abstract
Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the
dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential
therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100
was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and
evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however
trial endpoints were not met after 48 weeks. The objective of this study was to understand the
mechanism of action of ezutromid which could explain the lack of sustained efficacy and help
development of new generations of utrophin modulators.
Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR)
is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an
apparent KD of 50 nM and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists
also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical
applications including oncology and rheumatoid arthritis, could also be exploited in future DMD
therapies.
Introduction
Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle wasting disease that affects
approximately 1 in 3500-5000 boys.[1,2] DMD is caused by loss-of-function mutations in the dystrophin
gene. Thousands of mutations exist, with an estimated one-third caused by spontaneous mutations.[3,4]
Loss of dystrophin results in progressive muscle degeneration, with patients wheelchair-dependent by
their early teens and average life expectancy reduced to the late 20s to 30s, due to heart and respiratory
failure.[5,6]
Developments in the clinical standard of care have led to improvements in quality of life and longevity[7–
9] but there is currently no available cure for DMD. Several therapeutic strategies have reached the clinic,
including stop codon readthrough and exon skipping which target particular mutations, and gene
therapy which faces multiple challenges, including viral production and immune response.[10,11]
Utrophin is an autosomal paralogue of dystrophin and highly similar in sequence, size and function.[12]
Increasing utrophin in the dystrophin-deficient mdx mouse model[13] prevents the dystrophic
phenotype,[14] not only offering a potential therapy to all DMD patients regardless of mutation type but
also avoiding an immune response.[11,15]
1
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10.1002/anie.201912392
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The first-in-class utrophin modulator ezutromid (formerly SMT C1100) 1 was discovered using a
phenotypic screen for utrophin gene upregulation[16,17] and, after successful animal[18] and human phase
1 trials[19,20], progressed to an open-label Phase 2 study in DMD patients (NCT02858362, Summit
Therapeutics PLC). In January 2018, interim 24-week data demonstrated reduced muscle fibre damage
and increased levels of utrophin, providing the first evidence of ezutromid target engagement and proof
of mechanism.[21] However, these effects were not seen after the full 48 weeks of the trial, and thus
development of ezutromid was discontinued. Since the mechanism of action of ezutromid is unknown,
it is difficult to rationalise the lack of sustained clinical efficacy.
Phenotypic drug discovery is seeing a resurgence in popularity, and while the molecular target is
unknown, when combined with target identification, it can lead to discovery of novel targets for a
disease.[22,23] Target identification can also allow a rational compound design strategy, provide
biomarkers for monitoring efficacy in clinical trials and provide insights into potentially deleterious on-
and off-target side effects.
Methods to elucidate target and mechanism of action of small molecules have been extensively
reviewed.[24–30] They are generally divided into (1) direct methods, which identify the protein interacting
partner(s) of a compound, and (2) indirect phenotypic profiling methods, which reveal a compound’s
mechanism by comparing its effect on cell phenotype and gene expression with vehicle or inactive
controls, and compounds with known mechanism. Both of these approaches were used to elucidate the
target and mechanism of action of ezutromid.
Results
Probe design and validation
Affinity-based protein profiling (AfBPP)[31–33] is a chemical proteomics strategy to identify a molecule’s
direct target(s) within a cellular context. AfBPP requires development of active probes bearing tags or
‘click’ handles for bioorthogonal tag ligation[34] for enrichment by affinity chromatography. Enriched
proteins can either be identified by western blotting or digested for LC-MS/MS analysis (Figure 1a).
AfBPP can be enhanced by inclusion of a photoaffinity label on the probe to create a protein-ligand
covalent bond under UV irradiation, allowing discovery of low affinity binding partners.[35]
AfBPP was employed as a strategy for ezutromid, beginning with synthesis of active and inactive probes
each containing a photoaffinity label and a click handle. A trifluoromethyl-phenyl diazirine (TPD) was
chosen to replace ezutromid’s 5′ ethylsulfonyl moiety due to the similarity in sterics and electronics of
the diazirine and the sulfonyl group. Alkynyl-phenyl substituents (meta compound 2 and para
compound 3, Figure 1b) were selected to replace the naphthyl substituent of ezutromid to provide a
copper-catalysed azide-alkyne cycloaddition (CuAAC) click handle with minimal impact on the overall
size of the compound. Altering the substitution pattern of ezutromid’s naphthyl substituent from 2’ to
1’ has been previously shown to ablate activity,[17] and was therefore incorporated into the design of the
inactive control probe (compound 4, Figure 1b).
Synthesis of the dual-tagged probes was undertaken in the same manner for each of the probes (Figure
1b), beginning with microwave-assisted benzoxazole cyclisation[17] from 3-amino-4-hydroxybenzoic
acid and the corresponding acid chloride. EDC-mediated couplings afforded Weinreb amides which
were used to generate trifluoromethyl ketones 5 and 6 with the Ruppert-Prakash reagent.[36] Synthesis
of TPDs from trifluoromethyl ketones is well-established,[37] starting from oxime formation and
activation (O-tosylation) followed by liquid ammonia induced diaziridine cyclisation and lastly
oxidation to afford the diazirine. Sonagashira couplings with TMS-acetylene followed by silyl
deprotection furnished the final probes 2 and 3 in overall yields of 5-13% (reported in the
Supplementary methods).
2
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10.1002/anie.201912392
Angewandte Chemie International Edition
Figure 1: Design and synthesis of dual-tagged ezutromid probes for use in affinity-based protein
profiling. a) Outline of the AfBPP strategy for target identification of small molecule probes in live cells.
Probe-treated cells are UV irradiated to trigger covalent binding of the probe to its protein target. A click
reaction is performed to ligate a fluorophore or enrichment tag, such as biotin, to the probe. Enriched proteins
are subsequently analysed using western blot or mass spectrometry. b) Chemical structures of ezutromid (1)
ezutromid-based active (2-3) and inactive (4) probes, containing a diazirine as a photoaffinity label (yellow
shading) and alkyne click handles (green shading). c) Synthesis of probes 2 and 3.
Validation of individual probe bioactivity was performed using initial in vitro assessment of activity in
a murine cellular utrophin gene firefly luciferase reporter assay[17]. Probes 2 and 3 showed increases in
luciferase activity in the micromolar range, but control probe 4 showed no increase in luciferase activity
as expected (Figure 2a). Next, fold change in utrophin protein levels post probe treatment compared to
the vehicle control was evaluated by western blot analysis. Encouragingly, probes 2 and 3 were found
to increase utrophin protein ~1.5 fold, similar to other reported utrophin modulators[38,39] and the
endogenous utrophin modulator heregulin[40,41]. Further, much of the activity of the parent compound
ezutromid was retained in both mouse and human DMD cells (Figure 2b-d). Probe 4 was confirmed to
be inactive in all assays.
The fusion of myoblasts into mature myotubes is required during muscle development and repair, and
is dysregulated in dystrophic muscles[42]. Ezutromid and heregulin increased fusion of treated human
DMD myocytes into mature myotubes in a concentration-dependent manner (Supplementary Figure
S1), indicating improved myogenesis[43]. This behaviour was replicated in the para probe 3, but not 2
or 4, indicating conservation of physiological activity (Supplementary Figure S1c). On the basis of
the combined activity data, 3 appears the most similar to ezutromid’s phenotype and was taken forward
to target identification studies.
3
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10.1002/anie.201912392
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Figure 2: Biological activity of AfBPP probes 2, 3 and 4.
a) Probes 2 and 3, but not 4 show an increase in activity in a mouse H2K mdx cellular utrophin gene
firefly luciferase reporter assay, although to a lesser extent than ezutromid, n = 4, representative graph
shown. b) Probes 2 and 3 increase utrophin protein, but inactive probe 4 does not. Representative
utrophin western blots, top: probe treatment of mouse H2K mdx myoblasts, bottom: probe treatment of
human DMD myoblasts. H, heregulin; M, protein standard (in kDa). Uncropped blots are presented in
Supplementary Figure S4. c-d) Probes 2 and 3 increase utrophin protein levels comparable to heregulin
and ezutromid after 24 h of compound treatment in mouse H2K mdx cells (c) and human DMD myoblasts
(d) determined by western blot. Bars represent means of utrophin levels normalised to total protein and
relative to DMSO, error bars are standard error of the mean, n = 3. * p < 0.05, ** p <0.005, ns = non-
significant.
In addition to a negative control probe, a competition control was desired to facilitate deconvolution of
therapeutically relevant targets from non-specific or irrelevant hits. This competitor compound is
ideally co-treated at an excess concentration to occupy target binding sites and block binding of the
photoaffinity probe. SMT022332 (probe 7, Figure 3d), an analogue of the second generation utrophin
modulator SMT022357 whose ability to upregulate utrophin levels and diminish dystrophic pathology
has been previously reported[44], was chosen as the competitor compound. SMT022332 (synthesis
reported in Supplementary Figure S2a and supplementary methods) demonstrates an ability
equivalent to ezutromid’s in utrophin mRNA and protein upregulation (Supplementary Figure S2b-
c). It also possesses significantly improved aqueous solubility, allowing dosage at the excess
concentrations appropriate for competition experiments.
4
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Target identification
A chemical proteomics strategy to identify interacting proteins was employed using the active dual-
tagged probe 3. This compound was used alongside two controls: (1) negative control probe 4 and (2)
SMT022332 7 dosed at >30× concentration to competitively block enrichment of 3-binding proteins.
Figure 3. Target identification by chemical proteomics.
a) In gel fluorescence of active probe 3, inactive probe 4 and active 3 with competitor 7, SMT022332 (d),
labelled H2K mdx proteome. M, protein standard (in kDa); red boxes, bands that appear stronger in active
probe treated samples than in the negative controls; blue stained gel represents total protein. b) Volcano
plot of proteins enriched by probe 3 in the presence and absence of competitor 7, top hits in red. c) Protein
enrichment by probe 3 compared to inactive probe 4 and competitor 7. Proteins strongly enriched by 3, but
not the controls, in red. d) Structure of competitor 7, SMT022332. e) Top hits from (c), full list of proteins
Initially, the protein-interaction profile of active probe 3 was assessed with gel-based labelling
experiments using live dystrophic mouse cells. Active probe 3 (3 μM), alongside DMSO vehicle,
negative control dual tagged probe 4 (3 μM) and competitor 7 (100 μM) were dosed for 2 h. After
irradiation (365 nm) and cell lysis, the probe-labelled lysate was subjected to a CuAAC-mediated click
reaction with TAMRA-azide, then SDS-PAGE. In gel fluorescence showed probe-dependent labelling
of proteins (Figure 3a), with little to no labelling in the vehicle (DMSO) control. Multiple proteins
were labelled in the three conditions, with only a subset of proteins outcompeted by SMT022332,
indicating a shortlist of specifically labelled targets amongst a majority of non-specific hits. The
labelling of proteins with probe 3 was also shown to be concentration-dependent (Supplementary
Figure S3).
Proteome-wide target identification proceeded with click-mediated ligation of probe-labelled proteins
to biotin, affinity enrichment with streptavidin beads and on-bead tryptic digest. Samples were analysed
by nano liquid chromatography–tandem mass spectrometry (nanoLC–MS/MS). Comparison of proteins
5
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10.1002/anie.201912392
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enriched by active probe 3 but displaced in the presence of competitor SMT022332 gave a list of high
occupancy binders (red proteins, Figure 3b). Combining differences in proteins identified in active
probe samples but not in the two controls delivered a shortlist of potential targets (Figure 3c-d, full list
of proteins identified available in Supplementary Information). To determine which potential targets
should be prioritised in follow-up studies, a phenotype profiling study of ezutromid was performed to
identify pathways which are differentially regulated post compound exposure.
Ezutromid was profiled in a DiscoverX BioMAP® Diversity PLUS study which examines changes in
the activity of 148 biomarkers across 12 different human primary cell-based co-culture systems upon
compound treatment[45] (Figure S4). Ezutromid was found to dose dependently decrease activity of
sIgG, and sIL-17F, indicating suppression of the immune response. Meanwhile, ezutromid caused
modest decreases in sPGE2, E-selectin and IL1α, which are hallmarks of an anti-inflammatory response.
Ezutromid’s profile most closely matched that of aryl hydrocarbon receptor (AhR) antagonist
CH223191[46] (Pearson correlation, r = 0.854) from a database of >4500 reference compounds.
AhR is a pleiotropic ligand-binding transcription factor which acts as an environmental sensor to control
complex transcriptional programmes in a ligand-, cell-type- and context-dependent manner.
Interestingly, AhR was found to be one of the most differentially enriched proteins in the AfBPP LC-
MS/MS experiment, comparing the active probe to both inactive controls (Figure 3c). Given the
phenotypic profiling results which suggest ezutromid behaves as an AhR antagonist and the
identification of AhR as a possible target in the chemical proteomics experiment, AhR was prioritised
for follow-up confirmatory and validation experiments.
Target validation
Binding of AhR to probe 3, but not negative control probe 4, was verified in human DMD myoblasts
by western blotting using an AhR-specific antibody (Figure 4a). The binding of both ezutromid and
SMT022332 to human AhR in cellulo was confirmed by competition of the probe 3 affinity capture.
Binding of ezutromid to a purified mouse AhR:ARNT complex (expression reported in the methods
section, supplementary information) was investigated by adapting a previously reported fluorescence
quenching assay[47,48]. This assay exploits ezutromid’s intrinsic fluorescence in solution which is
quenched upon protein binding and revealed a potent apparent KD of 50.1 ± 22.1 nM (Figure 4b).
a
AfBPP probe 3
-
+
-
+
-
+
-
+
-
negative control 4
+
-
ezutromid / µM
-
30
-
-
-
Figure 4: Target validation using
immunoblotting and fluorescence.
competitor 7 / µM
-
-
30
300
250
a) Immunoblot of AfBPP samples following
treatment of human DMD myoblast cells and
biotin enrichment. Selective enrichment of
AhR by active probe 3, but not inactive probe
130
AhR
95
M
b
4
is observed as well as enrichment
competition by ezutromid and SMT022332.
M, protein standard (in kDa); uncropped blot is
presented in Supplementary Figure 6. b)
Fluorescence of ezutromid (100 nM, 313 nm
excitation, 390 nm emission) measured after
incubation (16 h) with AhR:ARNT (5 – 10000
nM).
30000
KD = 50.1 ± 21.1 nM
20000
10000
100
101
102
103
104
Concentration / nM
6
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AhR binds structurally diverse small molecule agonists and antagonists, with agonists inducing nuclear
translocation of the receptor and increased transcription of AhR-responsive genes. To ascertain whether
ezutromid is an AhR agonist or antagonist, the effects of ezutromid on mRNA expression of AhR and
AhR-responsive genes Cyp1b1 and the AhR repressor (AhRR) were assessed in mdx mice and human
DMD myoblasts by RT-qPCR (Figure 5a). Ezutromid treatment more than halved AhRR expression
and led to an approximate 34% decrease in Cyp1b1 expression in both cell lines, indicating that
ezutromid behaves as an AhR antagonist. Decreased AhR repressor production saw a concomitant 2.0-
and 1.6- fold increase in AhR in mdx and human DMD myoblasts respectively.
Figure 5: Ezutromid behaves as an aryl hydrocarbon receptor antagonist.
a) Ezutromid treatment (+; 3 µM for 24 h; dark bars) increases full-length utrophin (Utrn) and AhR
mRNA expression in dystrophin-deficient mouse (mdx) and human DMD myoblasts
(DMD). Concomitantly, AhR-responsive genes AhRR and Cyp1b1 illustrate downregulation, indicating
antagonism of AhR signalling with compound treatment. Bars represent mean fold change relative to
DMSO (-; light bars) and normalised to S13 ± SD (n =3). Fold changes were all statistically significant
with p < 0.0001 (paired two-tailed Student t-test). b) Localisation of AhR induced by compound treatment
determined by immunofluroscence. AhR is mostly located in the cytoplasm of human DMD myoblasts;
ezutromid does not increase nuclear translocation, unlike agonist 2-(1′H-indole-3′-carbonyl)-thiazole-4-
carboxylic acid methyl ester (ITE), and co-treatment of ITE with ezutromid results in retention of AhR
in the cytoplasm.
7
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The ability of ezutromid to antagonise AhR activation was further investigated by immunofluorescence
of AhR localisation after compound treatment. Human DMD cells were treated with ezutromid (3 μM,
2 h) and imaging with an AhR-specific antibody revealed no induction of nuclear translocation, unlike
that observed with the potent endogenous AhR agonist ITE (2-(1′H-indole-3′-carbonyl)-thiazole-4-
carboxylic acid methyl ester, 1 μM, 2 h),[49,50] which was used as a positive control (Figure 5b). Co-
treatment of ezutromid with ITE led to retention of AhR in the cytoplasm (Figure 5b), manifesting the
ability of ezutromid to counteract a potent AhR agonist.
To determine whether AhR antagonism represents a viable mechanism to modulate utrophin in human
DMD cells, AhR antagonists GNF-351[51] and CH223191 were assessed for their effect on utrophin
protein levels by western blot (Figure 6). Gratifyingly, GNF-351 treatment (24 h) showed a significant
dose-dependent 2.7-fold increase in utrophin and a 3-fold increase in AhR protein (both p < 0.05). In
contrast, treatment with antagonist CH223191 appeared to increase utrophin, but gave no change in
AhR.
4.0
Utrophin
3.5
3.0
AhR
M
0
1 10 10
460
2.5
Utrophin
AhR
2.0
1.5
117
71
1.0
0.5
0.0
total
protein
DMSO
GNF351
1 µM
GNF351 CH223191
10 µM
10 µM
Figure 6: Differential effects of AhR antagonists on AhR and Utrophin protein levels.
The AhR antagonist GNF351 increases AhR and utrophin, while antagonist CH223191
increases utrophin without changing AhR, as determined by western blot analysis. Bars
represent means of utrophin (blue) and AhR (red) levels normalised to total protein and relative
to DMSO, error bars reflect standard error of the mean, n = 3. * denotes p < 0.05, M; protein
standard (in kDa).
Discussion
The utrophin modulator ezutromid was originally discovered using a phenotypic screen, and hence its
molecular targets were unknown. Phenotypic screening for drug discovery has many advantages and
disadvantages, but importantly, when it is combined with target identification, it can lead to the
discovery of novel therapeutic mechanisms. This is particularly significant for diseases where no good
targets are known. The identification and verification of AhR as a target for ezutromid represents the
first validation of a disease-modifying drug target in Duchenne muscular dystrophy. Our findings are
also consistent with other reported observations. For example, resveratrol, which while exerting
pleiotropic effects, has documented AhR antagonist activity[52] and has been shown to increase utrophin
expression and decrease inflammation in mdx mice.[53] Furthermore, AhR agonist PCB-126 has been
shown to decrease utrophin mRNA expression in rat liver progenitor cells.[54]
A mechanism by which AhR antagonism modulates utrophin has not yet been established. However,
prototypical AhR agonist TCDD has been shown to decrease activity and abundance of transcriptional
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coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α),[55] which stimulates
utrophin expression at neuromuscular junctions.[56] This dysregulation was removed by co-treatment
with an AhR antagonist, leading to stabilisation of active PGC1α. Treatment with AhR antagonists
alone could lead to an increase in PGC1α and consequently utrophin.
Ezutromid is extensively metabolised in humans and, moreover, repeated dosing of ezutromid led to a
reduction in exposure in both healthy volunteers and markedly in DMD patients in early phase clinical
trials.[19,57] This phenomenon may account for the observed positive clinical effect followed by a lack
of sustained efficacy in the Phase 2 clinical trial. An intriguing outstanding question is whether
ezutromid’s primary pharmacology also affects its overall exposure. In the short term, antagonism of
AhR, which regulates the xenobiotic response, is likely to suppress ezutromid metabolism. However,
we observe that ezutromid also increases AhR expression and decreases expression of the AhR
repressor, and therefore chronic administration studies may provide insights into whether this impacts
ezutromid metabolism in the longer term.
While numerous AhR agonists have been described, and are additionally found amongst existing
pharmaceuticals,[58] antagonists are fewer in number. Furthermore, AhR antagonists are typically free
of the dioxin-like carcinogenicity of some AhR agonists and are increasingly being investigated as
possible therapeutic agents in a diverse range of indications including multiple myeloma[59], treatment-
resistant triple-negative breast cancer[60], glioblastoma[61], head and neck squamous cell carcinoma[62]
,
atheroscelerosis[63], obesity[64] and rheumatoid arthritis[65]. Furthermore, AhR antagonism has a
promising application in stem cell expansion and development[66–68] and cancer immunotherapy[69]
.
While human and mouse AhR are known to have substantial differences in ligand specificity,[70] in this
work, ezutromid is shown to bind both species’ receptors. As a result, the identification of ezutromid
as a novel, nontoxic AhR antagonist represents an important addition to the literature by expanding the
toolbox available to interrogate AhR biology.
Conclusions
In this work, the protein interactome of ezutromid has been profiled and the aryl hydrocarbon receptor
has been identified and validated as a target through a combination of chemical proteomics and
phenotypic profiling. Ezutromid binds AhR potently and is revealed as a novel AhR antagonist,
inhibiting nuclear translocation and downregulating AhR responsive genes such as AhRR and Cyp1b1.
Signalling pathways connecting AhR to utrophin modulation now warrant further investigation given
that known AhR antagonists also upregulate utrophin protein expression, suggesting AhR is a valid
target for utrophin functional replacement therapies. This work could pave the way for the first target-
based drug discovery program in DMD as well as provide a biomarker for future clinical trials.
Furthermore, AhR modulation could be used in combination with other therapeutic approaches,
amplifying efficacy. Excitingly, the results of this work justifies research into a new approach to deliver
a utrophin modulator to benefit all DMD patients.
Acknowledgements
This work was supported by funding from the Engineering and Physical Sciences Research Council
(EPSRC) and the Medical Research Council (MRC) [grant numbers EP/L016044/1, I.V.L.W.,
1501AV003/CA2, S.E.S], Summit Therapeutics [I.V.L.W., H.D., L.M., A.V. & G.M.W.] and Muscular
Dystrophy UK [grant number RA4/3013/4, A.V., RA3/3073-3, K.P].
Utrophin qPCR data presented in Supplementary Figure 2b was collected by Tepnel Pharma Services,
Innovation House, Oaks Business Park, Crewe Road, Wythenshawe, Manchester, M23 9HZ. Optical
rotation of SMT022332 was determined at Chiralabs Ltd, Begbroke Centre for Innovation & Enterprise,
Begbroke Science Park, Begbroke Hill, Woodstock Road, Begbroke, Oxfordshire, OX5 1PF, UK.
Chiral separations were performed by Reach Separations Ltd. BioCity Nottingham, Pennyfoot Street,
Nottingham NG1 1GF, U.K.
9
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Table of Contents
Graphical abstract
Chemical proteomics and phenotypic profiling
illustrated the aryl hydrocarbon receptor (AhR) is a
target of ezutromid, the first-in-class utrophin
modulator for the treatment of Duchenne muscular
dystrophy. Ezutromid binds to AhR with an apparent
KD of 50 nM and functions as an AhR antagonist.
Other reported AhR antagonists also upregulate
utrophin, showing that this pathway, which is currently
being explored in other clinical applications including
oncology and rheumatoid arthritis, could also be
exploited in future DMD therapies.
Keywords: mechanism of action, medicinal chemistry, photoaffinity labelling, proteomics, target
identification
13
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