Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)**

Article abstract of DOI:10.1002/anie.202014457

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (K_i=38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

Full text of DOI:10.1002/anie.202014457

A Journal of the Gesellschaft Deutscher Chemiker
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International Edition Chemie
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Accepted Article
Title: Photochemical probe identification of a small-molecule inhibitor
binding site in Hedgehog acyltransferase (HHAT)
Authors: Thomas Lanyon-Hogg, Markus Ritzefeld, Leran Zhang,
Balazs Pogranyi, Milon Mondal, Lea Sefer, Callum D
Johnston, Claire E Coupland, Sebastian A Andrei, Joshua
Newington, Anthony I Magee, Christian Siebold, Edward
William Tate, Matthew J. Fuchter, and Jake L. Greenfield
This manuscript has been accepted after peer review and appears as an
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202014457
Link to VoR: https://doi.org/10.1002/anie.202014457

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COMMUNICATION
Photochemical probe identification of a small-molecule inhibitor
binding site in Hedgehog acyltransferase (HHAT)
[
a]
[b]
[b]
[b]
[b]
Thomas Lanyon-Hogg,* Markus Ritzefeld, Leran Zhang, Balazs Pogranyi, Milon Mondal,
[b]
[c]
[b]
[c]
[b]
Sebastian A. Andrei, Lea Sefer, Callum D. Johnston, Claire E. Coupland, Jake L. Greenfield,
[b]
[b]
[d]
[c]
Joshua Newington, Matthew J. Fuchter, Anthony I. Magee, Christian Siebold, and Edward W.
Tate*[b]
[
a]
Dr T. Lanyon-Hogg
Department of Pharmacology
University of Oxford
Oxford, OX1 3QT, UK
E-mail: thomas.lanyon-hogg@pharm.ox.ac.uk
[
b]
Dr. M. Ritzefeld, L. Zhang, B. Pogranyi, Dr. M. Mondal, Dr. S. A. Andrei, C. D. Johnston, Dr. J. L. Greenfield, J. Newington, Prof. M. J. Fuchter, Prof. E. W.
Tate
Department of Chemistry
Imperial College London
London, W12 0BZ, UK
E-mail: e.tate@imperial.ac.uk
Dr L. Sefer, C. E. Coupland, Prof. C. Siebold
Division of Structural Biology
Wellcome Centre for Human Genetics
University of Oxford
[
[
c]
d]
Oxford, OX3 7BN, UK
Prof. A. I. Magee
National Heart & Lung Institute
Imperial College London
London, SW7 2AZ, UK
Supporting information for this article is given via a link at the end of the document.
Abstract: The mammalian membrane-bound O-acyltransferase
Recent determination of the structure of a bacterial MBOAT, DltB,
provided the first insights into MBOAT architecture and
mechanism,[ revealing that DltB forms a transmembrane pore-
like structure with the active site in the centre of the pore, and
providing a rationale for how MBOATs can combine substrates
present on opposite sides of biological membranes.[5] De novo
computational predictions suggest GOAT may adopt a similar
structure,[6] and the first cryo-EM structures of a human MBOAT,
diacylglycerol O-acyltransferase 1 (DGAT1),[7] confirmed the
pore-like architecture of MBOATs and provided insights into the
catalytic mechanism. However, the mechanism of small molecule
MBOAT inhibition remains largely unknown.
(MBOAT) superfamily is involved in biological processes including
5]
growth, development and appetite sensing. MBOATs are attractive
drug targets in cancer and obesity; however, information on the
binding site and molecular mechanisms underlying small-molecule
inhibition is elusive. This study reports rational development of a
photochemical probe to interrogate a novel small-molecule inhibitor
binding site in the human MBOAT Hedgehog acyltransferase (HHAT).
Structure-activity relationship investigation identified single
enantiomer IMP-1575, the most potent HHAT inhibitor reported to-
date, and guided design of photocrosslinking probes that maintained
HHAT-inhibitory potency. Photocrosslinking and proteomic
sequencing of HHAT delivered identification of the first small-molecule
binding site in a mammalian MBOAT. Topology and homology data
suggested a potential mechanism for HHAT inhibition which was
confirmed via kinetic analysis. Our results provide an optimal HHAT
i
tool inhibitor IMP-1575 (K = 38 nM) and a strategy for mapping small
molecule interaction sites in MBOATs.
Members of the membrane-bound O-acyltransferase (MBOAT)
superfamily of proteins are involved in several critically important
biological pathways.[1] In humans, these include Wnt
PORCN),[
2]
Hedgehog
acyltransferase
(Porcupine;
acyltransferase (HHAT)[3] and ghrelin O-acyltransferase
GOAT)[4] which regulate Wnt and Hedgehog signaling, and
(
appetite sensing, respectively. These MBOATs are attractive
therapeutic targets in cancer and obesity,[1] and structural
information for mammalian MBOATs is highly sought after. The
membrane topology of various mammalian MBOATs has been
experimentally determined, supporting a conserved arrangement
of multiple transmembrane helices and catalytic residues.[1a]
Figure 1. Hedgehog acyltransferase (HHAT) function and topology. A) N-
acylation reaction of Sonic Hedgehog (SHH) with palmitoyl-CoA catalysed by
HHAT. B) Experimentally determined topology model for HHAT,[8] showing ten
transmembrane loops (1-10), two re-entrant loops (R1-2) and one
palmitoylation-tethered loop (PL). Yellow stars: sites of palmitoylation; green
stars: signature MBOAT residues involved in catalysis. C) Structure of RUSKI-
[9]
201, the only previously known highly selective HHAT inhibitor.
1
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Hedgehog signaling drives growth during development and
is reactivated in certain cancers, making HHAT an attractive
therapeutic target to block aberrant signaling.[1] HHAT S-acylates
Hedgehog signaling proteins, which then undergo S,N-acyl shift
to yield the final amide-linked product.[3] HHAT predominantly
attaches palmitic (C16:0) fatty acid, using palmitoyl-Coenzyme A
Table 1. Structure-activity relationship investigation of the amide substituent of
1 (RUSKI-201). Data represent mean and 95% confidence interval (CI, n = 3).
[3]
(
Pal-CoA) as the lipid donor substrate (Figure 1A). N-acylation
of Sonic Hedgehog protein (SHH) by HHAT and C-terminal auto-
O-cholesterylation[10] is required for signaling function. We
previously determined the membrane topology of HHAT, which
consists of ten transmembrane helices, two reentrant loops, and
one palmitoylation-tethered reentrant loop (Figure 1B).[8]
Remarkably these experimental analyses undertaken in live cells
placed the MBOAT signature residues His379 and Asp339 on
opposite sides of the membrane, raising questions regarding the
potential role of these residues in the catalytic mechanism. There
is one known series of small-molecule inhibitors of HHAT,[11] with
RUSKI-201 (1, Figure 1C) the only molecule in this class with
proven on-target cellular activity over
a
non-cytotoxic
[
9]
concentration range. 1 contains two undefined stereocenters,
with further scope for exploration of the structure-activity
relationship (SAR) to generate higher potency inhibitors. There is
also currently no information about this series’ binding mode at
HHAT, or indeed the binding site of any inhibitor of a mammalian
MBOAT. Photocrosslinking of ligands to proteins followed by
proteomic mass spectrometry-based sequencing is a powerful
method to identify binding sites.[12] However, mass spectrometry
studies remain technically challenging for integral membrane
proteins.[13] Here, we report a SAR investigation leading to
discovery of the most potent single-enantiomer HHAT inhibitor to
date, and rational design of a photochemical probe that was
applied to identify the small-molecule inhibitor binding site in
HHAT, providing the first insights into the mechanism of HHAT
inhibition.
The amide-linked side chain of 1 was selected for SAR
investigation. The 4,5,6,7-tetrahydrothieno[3,2-c]pyridine core (2)
was synthesized via Bischler–Napieralski cyclization,[14] or via a
shorter and higher yielding Pictet-Spengler cyclization (Scheme
S1). Acetylated derivative 3 was prepared to investigate the
importance of the aminoalkyl chain. Amide-linked sidechain
derivatives 4-6 were prepared via N-benzyl-protected
intermediates to enhance handling of the amines by decreasing
volatility (Scheme S2). 1 contains two undefined stereocenters, in
the central ring system and in the 2-methylbutylamino chain. To
investigate the stereochemical requirements for inhibition, (S)-2-
methylbutylamine was used to prepare 4 with defined sidechain
analysis (Table 1).
concentration (IC50
CI) = 1.4-2.8 µM).[15] Core 2 showed no inhibition, whereas
acetamide showed reduced activity (IC50 = 13 µM, 95%
CI = 7.4-24 µM), indicating the amide is required for activity and
that the aminoalkyl chain improves potency. The alkyl chain S-
enantiomer 4 exhibited ~2-fold reduced potency (IC50 = 3.7 µM,
1
exhibited
a half-maximal inhibitory
)
of 2.0 µM (95% confidence interval
(
3
95% CI = 1.7-8.2 µM) compared to 1, indicating stereochemistry
at this position marginally impacts activity. Moving the methyl
group by one position in 5 showed a minor decrease in potency
(
IC50 = 5.8 µM, 95% CI = 4.3-7.7 µM), whereas shortening the
alkyl chain to 6 modestly increased potency compared to 1
IC50 = 1.3 µM, 95% CI = 0.88-2.0 µM) whilst also removing one
stereochemistry.
3-Methylbutylamine
(5)
and
2-
(
methylpropylamine (6) derivatives were prepared to remove the
stereocenter. To investigate the importance of the sidechain
secondary amine, tertiary amine 7 and cyclic derivative 8 were
prepared from N-methyl isobutylamine and 3-methyl piperidine,
respectively. To further probe the role of the secondary amine,
urea derivative 9 was prepared from isopentylamine (Scheme S2).
We recently reported the acylation-coupled lipophilic
stereocenter. Tertiary amine derivatives 7 and 8, as well as urea
analogue 9, showed substantially reduced activity (Table 1).
The most potent compound, 6, contains an undefined
stereocenter in the dihydrothieno[3,2-c]pyridine core, and we
hypothesized that one enantiomer may optimally fit the HHAT
binding site. (+/-)-6 was therefore purified by chiral preparative
HPLC to obtain (+)-6 and (-)-6 in >99:1 enantiomeric ratio (Figure
S1). (-)-6 displayed no HHAT inhibition, whilst (+)-6 displayed two-
fold increased potency compared to (+/-)-6 (Table 2). The lead
inhibitor (+)-6, which we term IMP-1575, is the first sub-
micromolar small-molecule HHAT inhibitor reported to-date
induction of polarization (Acyl-cLIP) assay,
a facile and
universally applicable method to monitor enzymes processing
lipid post-translational modifications which uses the
hydrophobicity increase on lipidation to drive
a polarized
fluorescence readout.[15] Dose-response analysis of inhibition of
purified HHAT was conducted using real-time Acyl-cLIP
(
IC50 = 0.75 µM, 95% CI = 0.49-1.1 µM). IMP-1575 is an oil,
2
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Table 2. Development of single-enantiomer HHAT inhibitor (+)-6 (IMP-1575).
precluding determination of absolute stereochemistry by X-ray
methods, despite extensive efforts. Circular dichroism (CD)
spectra of IMP-1575 and (-)-6 were recorded (Figure S2), and
compared to those predicted by three different functional/basis-
set configurations (Figure S3).[16] Each of these predictions
indicate that (+)-6 (IMP-1575) is the (R)-enantiomer (Figure S4).
To further confirm the absolute stereochemistry of IMP-1575, two
enantioselective reduction methods predicted to yield (R)-2 were
performed (Scheme S3).[17] Asymmetric hydrogen transfer (AHT)
using RuCl(p-cymene)[(R,R)-TsDPEN] generated the highest
enantiomeric excess of (+)-2 in 92:8 er, and acylation of
enantiomerically-enriched 2 yielded an active HHAT inhibitor
(-)-6 does not inhibit HHAT, whereas (+)-6 (IMP-1575) is twice as potent as (+/-)-
6. Data represent mean and 95% CI (n = 3).
(
Figure S5). Taking these data together, the stereogenic center in
(
+)-6 (IMP-1575) was assigned as (R).
Figure 2. Photochemical probe identification of the small-molecule binding site in HHAT. A) Structure of photocrosslinking probes 10 and 11. B) Acyl-cLIP
assays showing probes are active HHAT inhibitors. Data represent mean and SEM (n = 3). C) UV-crosslinking (365 nm, 1 min) of 11 to purified HHAT and
competition with IMP-1575, analysed by CalFluor647 functionalisation followed by in-gel fluorescence and Coomassie staining. Image representative of two
independent experiments. D) Topology model of HHAT showing cytosolic (purple) and ER lumen (red) loops. Key residues are coloured as catalytic His379 and
Asp339 (green), probe-modified Pro212, Val213, His215, Glu399 and Val402 (cyan), palmitoylated cysteines (yellow), and residues that have had their topology
experimentally determined as cytosolic (blue) or luminal (orange). E) Homology model of HHAT, coloured as in (D), showing probe modified residues Pro212,
Val213, His215 in proximity to the central catalytic site on the cytosolic face of HHAT.
3
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fluorescence from dye that had not reacted with alkyne probe. 10
or 11 (10 µM) were incubated with purified HHAT with or without
UV irradiation (1 min). Functionalization with CalFluor-647 by
CuAAC followed by in-gel fluorescence analysis indicated
crosslinking of probe 11 to HHAT promoted by UV irradiation
(
(
Figure 2C), whereas probe 10 showed minimal crosslinking
Figure S6) under these conditions. Competing probe 11 binding
with 40-fold excess IMP-1575 reduced crosslinking to near
background levels (Figure 2C).
Having identified suitable conditions for photocrosslinking of
11 to HHAT, identification of the small-molecule binding site was
sought through crosslinking with protease digestion and tandem
LC-MS/MS analysis. To achieve maximum sequence coverage
purified HHAT (1 µg) was digested with trypsin, Glu-C, Proteinase
K, chymotrypsin, combinations of Lys-C and trypsin, or
ProteaseMAX and trypsin. Filter-aided sample preparation
(
(
FASP)[19] was used to remove n-dodecyl-β-D-maltopyranoside
DDM) detergent used to solubilized HHAT, and reduce and
alkylate peptides prior to analysis. Moderate-to-low sequence
coverage was achieved with all proteases (Table S1), with
chymotrypsin having greatest coverage (51%). Combined,
chymotrypsin, Proteinase K (41%), and trypsin (35%) gave 68%
overall sequence coverage (Figure S9). Purified HHAT and 11
(
25 µM) were irradiated for 3 min, followed by FASP processing
and digestion with trypsin, chymotrypsin, or Proteinase K. LC-
MS/MS identified two peptides with increased mass consistent
with crosslinking to 11, and MS/MS fragmentation indicated
modification of residues Pro212, Val213, His215, Glu399 and
Figure 3. IMP-1575 inhibition kinetics. A) Structure of IMP-1575. B) Effect of
IMP-1575 inhibition on palmitoyl-Coenzyme
A (Pal-CoA) kinetics. C)
Corresponding effect of Pal-CoA concentration on IMP-1575 inhibitory potency,
demonstrating IMP-1575 is a highly potent competative inhibitor with respect to
Val402 (Figure S10). Aliphatic diazirines have
a
general
Pal-CoA (K
i
= 38 nM, 95% CI = 29-46 nM).
[20]
preference for crosslinking to larger polar amino acids,
indicating the probe-modified residues detected in HHAT may be
directed by specific interactions with 11.
Collectively, SAR data demonstrated the amide carbonyl
was critical for HHAT binding, whereas the secondary amine
improved potency but was not essential and alteration of the alkyl
chain was well-tolerated. These new insights led to design of
photocrosslinking chemical probes 10 and 11 to investigate the
small-molecule binding site in HHAT (Figure 2A). In probe 10 the
secondary amine moiety was exchanged for a diazirine group,
and the terminal alkyl chain replaced with an alkyne; probe 11
contains an additional carbon between the carbonyl and diazirine.
The diazirine allows photoactivated chemical crosslinking to
nearby residues in HHAT, with the alkyne allowing biorthogonal
To investigate how the identified binding residues may be
involved in HHAT’s structure and function, residue positions were
compared to the experimentally determined membrane topology
[8]
of HHAT (Figure 2D). Probe-binding residues are located in re-
entrant loop 2 (Pro212, Val213 and His215), which is positioned
near the cytosolic face of the membrane, or immediately following
transmembrane helix 8 (Glu399 and Val402), which is also
positioned on the cytosolic membrane face. These residues are
distant in the HHAT primary sequence from the signature MBOAT
catalytic residues (Asp339 and His379), therefore a structural
model for HHAT was generated based on sequence homology to
functionalization
via
copper(I)-catalyzed
azide-alkyne
[5]
DltB (Figure 2E). Phyre2, SwissModel and Robetta (old and
cycloaddition (CuAAC, ‘click chemistry’) for subsequent analysis.
In silico SAR analysis using the inhibitory potency of compounds
new) servers were used to generate homology models of HHAT.
Models were initially screened based on agreement with previous
topology data.[8] Interestingly, all models shared the same fold
1-9 as a training set predicted both probes to be active HHAT
inhibitors (Figure S6). The sidechains were synthesized and
coupled to 2 (Scheme S4-5). Pleasingly, probes 10 and 11
retained inhibitory activity against HHAT (IC50 = 16 μM, 95%
CI = 12-20 µM and IC50 = 2.4 µM, 95% CI = 1.8-3.3 µM,
respectively, Figure 2B) demonstrating the key functional groups
were tolerated. Probe photoactivation by UV irradiation (365 nm)
was monitored by LC/MS, demonstrating an activation half-life of
between residues 94-206, with greater variability in the final two
predicted transmembrane helices at the N- and C-termini. A final
model was selected based on optimal hydrophobic packing of the
terminal membrane-spanning helices (Figure 2E). Predicted
transmembrane regions from topology studies were arranged
around a central pore (Figure 2E, shown in grey). Signature
MBOAT His379 was located at the center of the pore in close
proximity to the catalytically essential Asp339, suggesting a
central catalytic site. Inhibitor-binding residues Pro212, Val213,
and His215 were located adjacent to the proposed catalytic site,
whereas residues Glu399 and Val402 were not located in
proximity to the proposed catalytic site in this model (Figure 2E).
Collectively, this analysis suggested that there may be a
small-molecule inhibitor-binding site in HHAT located on the
3.3 min for probe 10 and 32 s for 11 (Figure S7). Protein
crosslinking was next investigated through CuAAC
functionalization with a fluorophore and in-gel fluorescence
readout. As an integral membrane protein, HHAT presents
substantial challenges in sample handling and cannot be
precipitated to remove excess fluorophore which otherwise binds
non-specifically (data not shown). Fluorogenic azide CalFluor-647
(
Figure S8)[18] was therefore used to reduce background
4
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Angewandte Chemie International Edition
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COMMUNICATION
cytosolic side of the ER membrane in proximity to the catalytic site. Career Fellowship to M.J.F) and the Leverhulme Trust (RPG-
As Pal-CoA is expected to approach HHAT from the cytosolic side
of the membrane, this suggested compounds from this series,
such as IMP-1575 (Figure 3A), may bind in competition with Pal-
CoA. IMP-1575 (50-0.023 μM) was therefore analyzed for effects
on HHAT kinetics at varying Pal-CoA concentrations (50-0.19 μM,
Figure 3B-C). Analysis using a mixed model equation for
inhibition[ generated α = 470 (95% CI = 320-620), indicative of
a competitive mode of inhibition for IMP-1575 with respect to Pal-
2
018-051, to J.L.G. and M.J. F.). T.L.-H. is a Career Development
Fellow funded by the Department of Pharmacology, University of
Oxford.
Keywords: Enzymes • Hedgehog acyltransferase • Hedgehog
signalling • Medicinal chemistry • Membrane-bound O-
acyltransferase • Photoaffinity labelling
21]
i
CoA (K = 38 nM, 95% CI = 29-46 nM). Taken together, these
data support identification of a small-molecule binding site in
HHAT that competes with Pal-CoA binding to disrupt enzymatic
function with high affinity.
[
1]
a) N. Masumoto, T. Lanyon-Hogg, U. R. Rodgers, A. D.
Konitsiotis, A. I. Magee, E. W. Tate, Biochem. Soc. Trans.
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We present here a new paradigm in rational development of
photocrosslinking chemical probes for MBOATs that can identify
small-molecule binding sites on these very challenging but
important targets. Probe 11 was designed based on new SAR
insights into known inhibitor 1, which retained required HHAT
inhibitory activity (Figure 2). Probe 11 was used here to identify a
small-molecule inhibitor binding site in an MBOAT for the first time.
Other mammalian MBOATs, such as PORCN and GOAT, have
known inhibitors and their investigation may be expedited by
technical advancements reported here, including use of
fluorogenic click regents for SDS-PAGE analysis, and FASP
processing in combination with multiple proteases to increase
sequence coverage. Photochemical tools based on substrates
may further allow mapping of key functional sites in HHAT and
other MBOATs. The limitations of biochemical topology analysis
and homology models for inhibitor design are significant,
therefore direct structural information for MBOATs remains an
important goal for future inhibitor development. We further
disclose IMP-1575, a single enantiomer small molecule, as the
most potent HHAT inhibitor reported to date that competes with
6
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concentrations below 10 μM, preventing accurate mechanistic
determination. Future studies of HHAT enzymology could take
advantage of IMP-1575, with the inactive enantiomer serving as
an ideal control. In this regard, the improved synthetic routes to
HHAT inhibitors (Schemes S1-3) presented here will significantly
accelerate future development of this series. In summary, we
present chemical tools and methodology to provide insight into
HHAT which may expedite future studies and drug discovery
efforts against this important target class.
[
[
13]
14]
[
[
[
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Acknowledgements
1
300; b) Z. Wu, M. Perez, M. Scalone, T. Ayad, V.
Ratovelomanana-Vidal, Angew. Chem. Int. Ed. Engl.
013, 52, 4925-4928.
This study was supported by Cancer Research UK grants
2
(
C6433/A16402 to A.I.M. and E.W.T, C29637/A20183 to E.W.T.,
[
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P. Shieh, V. T. Dien, B. J. Beahm, J. M. Castellano, T.
Wyss-Coray, C. R. Bertozzi, J. Am. Chem. Soc. 2015,
and C20724/A14414 and C20724/A26752 to C.S.), the European
Union Framework Program 7 (Marie Curie Intra European
Fellowship to M.R. and E.W.T., and to M.M. and E.W.T.), the
European Research Council under the European Union’s Horizon
137, 7145-7151.
[
[
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