Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

Article abstract of DOI:10.1016/j.bmcl.2020.127293

LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1 through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically precluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-modified compound, revealing a promising opportunity to target novel regions of the ligand binding pocket to alter LRH-1 target gene expression.

Full text of DOI:10.1016/j.bmcl.2020.127293

Bioorganic&MedicinalChemistryLetters30(2020)127293
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a new class of liver receptor homolog-1 (LRH-1) agonists by
photoredox conjugate addition
Jeffery L. Cornelisona,d, Michael L. Catob,d, Alyssa M. Johnsona, Emma H. D'Agostinob,
⁎
Diana Melchersc, Anamika B. Patelb, Suzanne G. Maysb,e, René Houtmanc, Eric A. Ortlundb,
,
⁎
Nathan T. Juia,
a Department of Chemistry, Emory University, Atlanta, GA 30322, United States
b Department of Biochemistry, Emory University, Atlanta, GA 30322, United States
c Precision Medicine Lab, Pivot Park, Antoni van Leeuwenhoek Building, Office1131/lab1333, Kloosterstraat 9, 5349 AB Oss, the Netherlands
A R T I C L E I N F O
A B S T R A C T
Keywords:
LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for
the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about
ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1
through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed
for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically pre-
cluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead
substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for
effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-
modified compound, revealing a promising opportunity to target novel regions of the ligand binding pocket to
alter LRH-1 target gene expression.
Nuclear receptor
LRH-1
Photoredox
Metabolic disease
Agonist
Liver receptor homolog-1 (LRH-1) is a member of the nuclear re-
ceptor (NR) family of ligand-regulated transcription factors that sense
lipophilic signaling molecules and produce biological responses. LRH-1
regulates a variety of cellular and organismal processes, including re-
verse cholesterol transport,1 steroidogenesis,2 endoplasmic reticulum
stress resolution,3 intestinal cell renewal,4 embryonic development,5
and bile acid biosynthesis.6–8 Its roles in lipid and glucose homeostasis
have drawn attention to LRH-1 as a potential target for treating type II
diabetes and non-alcoholic fatty liver disease (NAFLD),6 while its role
in intestinal cell renewal has made it a promising target for the treat-
ment of inflammatory bowel disease.9 Therefore, compounds that
modulate LRH-1 activity could be valuable for the treatment of multiple
diseases.
phospholipids, effective synthetic probes are required for character-
ization of LRH-1 biology. Towards this end, several laboratories have
made significant advances in developing potent LRH-1 modulators.13–15
Despite these advances, rational design has been difficult, in part be-
cause of the large, highly hydrophobic LRH-1 ligand binding pocket.
Due to this lipophilicity and a scarcity of sites for anchoring polar in-
teractions, even highly similar compounds can bind unpredictably,15,16
further complicating systematic agonist development.
Recently, our lab has identified key anchoring interactions in the
binding pocket that established the mechanism of binding for the pri-
vileged [3.3.0] bicyclic hexahydropentalene (6HP) substructure (shown
in Fig. 1, top), which was first identified by Whitby.17,18 Employing this
information has led to the design of more potent LRH-1 agonists
Dietary phospholipids are the putative endogenous ligands for LRH-
1,10 and a number of studies demonstrated that phosphatidylcholines
such as diundecanoylphosphatidylcholine (DUPC) and dilauroylpho-
sphatidylcholine (DLPC) preferentially activate LRH-1.6,11,12 However,
because of the low potency and poor physicochemical properties of
through optimization of R1 (“6N”|EC50 = 15
8 nM; Fig. 2, left),19 as
well as more strongly activating agonists, through optimization of R2
(“6HP-CA”|2.3
0.2-fold activation over vehicle; Fig. 2, right).20 To
further interrogate the structural requirements for LRH-1 activation by
6HP agonists, we sought to vary the bridgehead substituent (R3) (Fig. 1,
⁎ Corresponding authors.
E-mail addresses: eortlund@emory.edu (E.A. Ortlund), njui@emory.edu (N.T. Jui).
d JLC and MLC contributed equally.
e Present Address: Centre for Genomic Regulation, Calle del Dr. Aiguader, 88, 08003 Barcelona, Spain.
https://doi.org/10.1016/j.bmcl.2020.127293
Received 16 March 2020; Received in revised form 26 May 2020; Accepted 27 May 2020
Availableonline30May2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

J.L. Cornelison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127293
we first sought to investigate the necessity of substitution at this posi-
tion. Accordingly, our synthetic plan (shown in Scheme 1) involved
reduction of the enone function, followed by elaboration of the re-
sulting material to the corresponding endo-sulfamide or terminal acid
analogs, such that direct comparison with either of the parent com-
pounds would reveal the importance of R3. While a range of reducing
conditions were able to engage 1, we found that the cleanest profile was
observed in the presence of palladium on carbon and sodium borohy-
dride, followed by in situ triflation to afford 2 as a single regioisomer
(69% yield over two steps). This product could be utilized in a Negishi
coupling under Knochel conditions,24 where the SPhos-supported pal-
ladium catalyst afforded methyl decanoate derivative 3. Routine silyl
ether cleavage and saponification gave rise to 5, the direct analog of
6HP-CA lacking the bridgehead styrene.
To access the simplified endo-sulfamide analog, vinyl triflate 2 was
reacted with hexylzinc iodide under the same coupling conditions,
which after acidic alcohol deprotection, afforded 6 in moderate yield
(43%). Ley oxidation gave the corresponding ketone, which underwent
highly diastereoselective reductive amination with ammonia to afford
the endo amine 7. Sulfamide installation was accomplished using
chlorosulfonylisocyanate and tert-butanol, followed by acidic decom-
position of the resulting N-Boc sulfamide to afford analog 8.
Fig. 1. Design of Hexahydropentalene LRH-1 agonists.
We tested the biological activity of this simplified 6HP series using a
fluorescence polarization (FP) competition ligand binding assay re-
cently developed in our lab25 and a luciferase reporter assay to measure
LRH-1 transcriptional activity. We assessed the binding affinity (Ki), in-
cell potency (EC50), and efficacy (fold activation) of compounds 5 and
8, the direct analogs of 6HP-CA and 6N respectively, with the bridge-
head group entirely removed. The compound containing a sulfamide
anchoring group (8) demonstrated low nanomolar binding affinity
(Ki = 56 nM), while the compound with a charged tail (5) demon-
strated mid nanomolar affinity (Ki = 280 nM) (Fig. 3A). Strikingly,
removal of the bridgehead moiety entirely abolished the activity of both
compounds in luciferase reporter assays (Fig. 3B), suggesting that some
degree of steric occupancy is critical for the compounds’ ability to ac-
tivate LRH-1. Understanding that there is a requirement for some de-
gree of steric bulk at the bridgehead position, we searched for reaction
conditions that could engage alkene 1 via conjugate addition. Because
6HP derivatives bearing heteroatom substituents at this position are
known to be highly acid-sensitive,15,26 we targeted a protocol to forge
carbon–carbon bonds. However, in line with the dearth of reactions that
accept enones of this type, a broad survey of nucleophilic partners (e.g.
malonates, enolates, Gillman reagents, and other organometallics)
completely failed to provide the corresponding conjugate adducts.
We next turned our attention to radical coupling partners. Because
the Giese reaction operates through an early transition state, these
processes are less sensitive to steric hindrance.27–29 Drawing from our
own experience in radical conjugate addition, we found that aminoalkyl
Fig. 2. LRH-1 agonists previously reported by our lab with key polar groups
highlighted.
bottom). Because Whitby’s 3-component cyclization results in either
heteroatom- or vinyl-substitution at this position,21,22 we considered
the alternative synthetic approach that is outlined in Fig. 1, where the
installation of R3 would be accomplished through functionalization of
tetrasubstituted olefin 1. Although alkenes of this type are notoriously
unreactive,23 this plan was appealing because it would allow for
modular variation (or deletion) of R3, and regioselective enol-triflate
formation would allow for installation of different alkyl tails (R2).
To evaluate the feasibility of this proposal, we prepared olefin 1
through Pauson-Khand cyclization of an appropriately-substituted 1,6-
enyne (see SI for details). Because the parent styryl R3 group does not
appear to make any critical contacts in the ligand binding site of LRH-1,
Scheme 1. Synthesis of Unsubstituted Bridgehead Compounds. Reagents and conditions: (a) Pd/C, NaBH4, AcOH, PhMe, 23 °C; 1 h; (b) NaH, PhNTf2, 0–23 °C; 69%
yield over two steps; (c) SPhos Pd G3, SPhos, IZn(CH2)9CO2Me·LiCl, THF, 50 °C; 16 h; (d) SPhos Pd G3, SPhos, IZn(CH2)5CH3·LiCl, THF, 50 °C; 16 h; (e) conc. aq. HCl,
MeOH, 23 °C; 1 h, 41–43% yield over two steps; (f) LiOH, H2O, THF, 50 °C; 16 h, 97% yield; (g) TPAP, NMO, MeCN, 23 °C; 1 h, 81% yield; (h) i. Ti(OiPr)4, NH3,
t
MeOH, 23 °C; 5 h; ii. NaBH4, MeOH, 23 °C; 5 h, 59% yield; (i) chlorosulfonylisocyanate, BuOH, TEA, DCM, 0–23 °C; 1.5 h, 31% yield; (j) conc. aq. HCl, dioxane,
0–40 °C; 14 h, 77% yield.
2

J.L. Cornelison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127293
Fig. 3. Evaluation of Unsubstituted Bridgehead Compounds A) Fluorescence polarization (FP) evaluation of compounds 5 and 8. Data shown as mean
two independent experiments. Ki values are given with 95% confidence intervals in brackets. B) Luciferase reporter data for 5 and 8 shown as mean
three biological replicates.
SEM from
SEM from
triethylamine was united with 1 to give rise to the corresponding ad-
duct in 60% yield, as determined by NMR. Interestingly, these radical
species appear to be uniquely effective here, as other radical sources
(e.g. alkyl or aryl halides, carboxylates, NHPI esters) did not afford the
desired products. This distinctive reactivity is potentially owing to the
special electronic properties of the α-heteroatom alkyl radical com-
pared to aryl or unactivated alkyl radicals. Because a range of alkyla-
mines have been demonstrated as competent coupling partners for
Michael acceptors within this manifold, we presumed that this finding
would grant access to a library of substituted 6HP structures.
To predict the ability of 6HP derivatives with aminoalkyl sub-
stituents at the bridgehead (R3) position to promote binding to LRH-1,
we conducted an in silico screen of several amine conjugate addition
derivatives. Using the Glide software developed by Schrodinger,32
elaborated ligands derived from the Pauson-Khand product 1 (analo-
gous to an early 6HP LRH-1 agonist, RJW100)18 were docked and
scored. This series was conveniently selected for docking studies be-
cause we had a high-definition X-ray co-crystal structure of LRH-1
bound to the 6HP agonist RJW100 and a fragment of coregulator pro-
tein TIF-2 (PDB 5L11) to use as a reference. The scoring protocol pro-
vides XP GScores, which approximate the ΔG of binding (in kcal/mol)
for each compound and these scores were used to rank the potential for
Fig. 4. Strategy for R3 variation: Aminoalkyl radical conjugate addition via
photoredox catalysis.
radicals (readily accessed through a single electron oxidation/depro-
tonation sequence) readily engage olefin 1 (Fig. 4).30,31 More specifi-
cally, in the presence of an iridium photoredox catalyst and blue light,
Fig. 5. Top: The 6HP core used for docking studies and a representative sample of the screened bridgehead groups. Each group is given with the XP GScore assigned
by Glide which approximates binding energy in kcal/mol. Bottom: Overlay of RJW100 (gray; PDB 5L11) and predicted minimized energy pose of 9 (green) in the
LRH-1 ligand binding pocket. Key interactions with Thr352 through water and pi-stacking with His390, highlighted in blue sticks, are retained in both poses and
highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3

J.L. Cornelison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127293
Scheme 2. Synthesis of N,N-Dimethylaniline Bridgehead Compounds. Reagents and conditions: (a) N,N-dimethylaniline, Ir[dF(CF3)ppy]2dtbpy·PF6, blue LED, 23 °C;
16 h; (b) NaH, PhNTf2, 0–23 °C; 73% yield over two steps; (c) SPhos Pd G3, SPhos, IZn(CH2)9CO2Me·LiCl, THF, 50 °C; 16 h; (d) SPhos Pd G3 or Pd(OAc)2, SPhos, IZn
(CH2)5CH3·LiCl, THF, 50 °C; 16 h; (e) conc. aq. HCl, MeOH, 23 °C; 1 h, 49–54% yield over two steps; (f) LiOH, H2O, THF, 50 °C; 16 h, quant; (g) TPAP, NMO, MeCN,
23 °C; 1 h, 84% yield; (h) i. Ti(OiPr)4, NH3, MeOH, 23 °C; 5 h; ii. NaBH4, MeOH, 23 °C; 5 h, 34% yield; (i) chlorosulfonylisocyanate, tBuOH, TEA, DCM, 0–23 °C; 1.5 h;
(j) conc. aq. HCl, dioxane, 0–40 °C; 14 h, 26% yield over two steps.
each docked compound to bind to LRH-1 in a productive manner. A
diverse set of cyclic and acyclic, aliphatic and aromatic, and basic and
non-basic amines were docked and scored, and a selection of the results
are shown in Fig. 5. Hydrophobic groups were preferred over more
hydrophilic ones, with charged groups (such as protonated amines)
showing a significant drop in predicted binding affinity. Compounds
derived from N,N-dialkylanilines scored the best, with the ligand deri-
vatized from N,N-dimethylaniline scoring similarly to the parent mo-
lecule, RJW100. Overlaying the predicted binding pose of the N,N-di-
methylaniline-derived ligand with that of the known pose of RJW100
showed nearly perfect overlap throughout the structure, as shown in
the bottom of Fig. 5. This includes the exocyclic phenyl rings, despite
the difference in linker length between the phenyl ring and bridgehead
position. These data indicated that amine conjugate addition could be
utilized in the design of a new class of 6HP LRH-1 agonists. To evaluate
this idea, we conducted radical conjugate addition of dimethylaniline to
olefin 1 under the previously outlined photon-driven reaction condi-
tions. Again, in situ vinyl triflate formation gave rise to 10, containing
the completed 6HP core. As illustrated in Scheme 2, synthetic ela-
boration of this intermediate to the corresponding endo-sulfamide (15)
and terminal carboxylate (13) analogs proceeded according to the
previously developed protocols. Upon evaluation of these compounds
using FP competition (for binding) and luciferase reporter assays (for
LRH-1 transcriptional activity), we found that the bioactivity of this
series essentially parallels those of the analogous bridgehead styrenes
(Fig. 6).19,20 Specifically, endo-sulfamide 15 demonstrated greater in-
cell potency than the terminal carboxylate analog 13, which pre-
sumably results from direct interactions with the polar network deep
within the binding pocket (centered around the Thr352 hydroxyl which
mediates indirect contact with RJW100 and directly engages the
sulfamide on 6N).16,19 Further, the terminal carboxylic acid 13 showed
augmented efficacy relative to 15, which we propose arises from the
ligand contacting phospholipid-binding residues at the mouth of the
pocket.20 Importantly, Emax values for 13 and 15 were nearly identical
to those of bridgehead styrene analogs,19,20 demonstrating that the
aniline substitution preserved compound efficacy. Notably, while Ki
values for sulfamide-containing analogs 8 and 15 were comparable, 13
exhibited an affinity > 10-fold higher than that of 5 (Fig. 3A; Fig. 6A),
suggesting that inclusion of a hydrophobic bridgehead group is also
critical for the binding of phospholipid-mimicking ligands.
Encouraged by the results from FP competition and luciferase re-
porter assays, we assessed whether the aniline substituent promotes an
active conformation at the activation function surface (AFS), which
preferentially binds coregulator proteins that drive NR target gene ex-
pression.33 By determining how compounds drive recruitment of cor-
egulators, we can thoroughly examine whether the aniline bridgehead
group effects ligand-driven activation of LRH-1. Therefore, we used the
Microarray Assay for Real-time Coregulator-Nuclear Receptor Interac-
tion (MARCoNI), which quantifies binding of 154 peptides corre-
sponding to NR interaction motifs from 64 coregulators with a micro-
array platform.34 We compared the coregulator binding profile of 6N-
bound and 15-bound LRH-1 ligand-binding domain (LBD), relative to
apo-LRH-1. 6N demonstrated notable trends that involved decreased
binding to corepressors, such as nuclear receptor co-repressor
1
(NCOR1) and NCOR2, and an increased binding to coactivators, such as
p160/steroid receptor coactivator family member nuclear receptor
coactivator 1 (NCOA1) and mediator of RNA polymerase II transcrip-
tion subunit 1 (MED1). Importantly, these trends were mirrored in 15-
bound LRH-1 (Fig. 7, Fig. S1), demonstrating that the aniline sub-
stituent promotes a similar compound-mediated conformation of the
Fig. 6. Evaluation of N,N-Dimethylaniline Bridgehead Compounds A) FP evaluation of compounds 13 and 15. Data shown as mean
SEM from two independent
experiments. B) Luciferase reporter data for 13 and 15 shown as mean
SEM from three biological replicates. Ki and EC50 values are given with 95% confidence
intervals in brackets.
4

J.L. Cornelison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127293
Fig. 7. Microarray Assay for Real-time Coregulator-Nuclear Receptor Interaction (MARCoNI) comparing coregulator binding between 6N- and 15-bound LRH-1 LBD.
Log-fold change (LFC) of peptides corresponding to the binding interface of coregulators is indicated. * p < 0.05; ** p < 0.01 – Student’s t-test, FDR.
AFS as the styrene bridgehead of 6N. This shows that compounds with
the aniline bridgehead group effectively drive LRH-1 activity in a si-
milar fashion as previous agonists.
To determine the binding pose of the aniline-containing agonists,
we generated a co-crystal structure of 15 and the LRH-1 LBD (Fig. 8;
Table S1; PDB 6VIF). The sulfamide moiety, internal styrene, and bi-
cyclic core of the ligand assumed the same conformation seen for other
6HP agonists (Fig. 8, bottom left).16,19 Surprisingly, the exocyclic ani-
line moiety was rotated in the opposite direction from the styrene in
previous crystal structures (Fig. 8, bottom left).16,19 This is contrary to
the prediction made by Glide, which placed the aniline phenyl group
superimposed with the styrene phenyl group (Fig. 5). The observed
positioning supports the hypothesis that there are no specific interac-
tions made by the exocyclic bridgehead group and that the compound
efficacy granted by its inclusion are largely a result of space-filling
hydrophobic interactions. This unexpected aniline binding pose also
reveals unforeseen potential for this compound series. The aniline
phenyl group is oriented towards hydrophilic residues, providing novel
future targets in the highly lipophilic LRH-1 binding pocket (Fig. 8,
bottom right). Interestingly, these residues have been associated with
allosteric paths critical for communication between LRH-1 ligands and
the AFS,12 suggesting that modifications targeting these residues may
be an interesting route for agonist development. The LRH-1 activation-
function helix (AF-H) is also within ~6 Å, providing the opportunity to
directly modulate the dynamics of the AFS to induce unique gene ex-
pression profiles that may not be possible through indirect allosteric
modulation.
In conclusion, we have developed an alternative synthesis of the
standard 6HP scaffold used in modern LRH-1 agonists. This new
synthesis allows for the modular modification of the bridgehead group
to investigate the role of the α-styrene in 6HP LRH-1 agonists. Previous
alterations to the bridgehead group, including heteroatom substitutions
Fig. 8. Co-crystal structure of 15 and the LRH-1 LBD reveals unexpected
binding pose for the bridgehead aniline group (PDB 6VIF). Top: LRH-1 (gray)
and 15 (C = green, O = red, N = blue, S = yellow) in the binding pocket with
the bridgehead aniline group oriented toward AF-H. Coregulator peptide frag-
ment TIF-2 is shown in dark gray. Middle: Ligand FO-FC omit map showing
electron density for 15 contoured at 2.5σ. Bottom left: 15 (green) adopts a
nearly identical binding pose as 6N (gray, PDB 6OQY), with the aniline moiety
reoriented in the opposite direction from the 6N styrene. Bottom right: Residues
proximal to the aniline group are made accessible by the novel binding mode
(sidechains shown as blue sticks). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
and small changes to the styrene, have revealed little in coregulator
recruitment and luciferase reporter assays15,19 and have been restricted
by limitations in the synthetic route. Although this group shows no
clear stabilizing interactions in crystal structures,16,19 removal of the
bridgehead group completely abolished activity in reporter assays.
Guided by computational docking and enabled by photoredox, a new
bridgehead moiety was installed that restored agonism while main-
taining high binding affinity. A crystal structure of one of the new
compounds, 15, in the LRH-1 LBD demonstrated that the general
binding pose was consistent with other 6HP agonists, though the new
5

J.L. Cornelison, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127293
N,N-dimethylaniline moiety rotated to a previously unaccessed region
of the binding pocket. Both the lack of critical contacts and the novel
orientation of the bridgehead group suggest promise for exploitation of
this novel binding mode in the development of more effective agonists
and novel antagonists, which are ongoing areas of research in our la-
boratories.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
15. Whitby RJ, Stec J, Blind RD, et al. Small molecule agonists of the orphan nuclear
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Acknowledgements
This work was supported by the National Institutes of Health
National Institute of General Medical Sciences [Grant T32-GM008367
to MLC], National Institutes of Health National Institute of Diabetes and
Digestive and Kidney Diseases [Grant F31-DK111171 to SGM; Grant
R01-DK115213 to EAO and NTJ], the National Science Foundation
[Grant DGE-1444932 to EHD], and an Emory Catalyst Grant to EAO
and NTJ. The authors thank the beamline staff at Southeast Regional
Collaborative Access Team, Argonne National Laboratory, for support
during remote crystal data collection. Additionally, the authors thank
Cameron Pratt (Emory) for helpful discussions and suggestions related
to the photoredox conjugate addition procedure.
17. Whitby Richard J, Dixon Sally, Maloney Patrick R, et al. Identification of Small
Molecule Agonists of the Orphan Nuclear Receptors Liver Receptor. J Med Chem.
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Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127293.
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