Luciferase-based HMG-CoA reductase degradation assay for activity and selectivity profiling of oxy(lano)sterols

Article abstract of DOI:10.1016/j.bmc.2019.115298

HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and is the target of cholesterol-lowering drugs, statins. Previous studies have demonstrated that the enzyme activity is regulated by sterol-induced degradation in addition to transcriptional regulation through sterol-regulatory-element-binding proteins (SREBPs). While 25-hydroxycholesterol induces both HMGCR degradation and SREBP inhibition in a nonselective manner, lanosterol selectively induces HMGCR degradation. Here, to clarify the structural determinants of selectivity for the two activities, we established a luciferase-based assay monitoring HMGCR degradation and used it to profile the structure-activity/selectivity relationships of oxysterols and (oxy)lanosterols. We identified several sterols that selectively induce HMGCR degradation and one sterol, 25-hydroxycholest-4-en-3-one, that selectively inhibits the SREBP pathway. These results should be helpful in designing more potent and selective HMGCR degraders.

Full text of DOI:10.1016/j.bmc.2019.115298

Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Luciferase-based HMG-CoA reductase degradation assay for activity and
selectivity profiling of oxy(lano)sterols
1
1
⁎
Ikuya Sagimori , Hiromasa Yoshioka , Yuichi Hashimoto, Kenji Ohgane
Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and is the
target of cholesterol-lowering drugs, statins. Previous studies have demonstrated that the enzyme activity is
regulated by sterol-induced degradation in addition to transcriptional regulation through sterol-regulatory-
element-binding proteins (SREBPs). While 25-hydroxycholesterol induces both HMGCR degradation and SREBP
inhibition in a nonselective manner, lanosterol selectively induces HMGCR degradation. Here, to clarify the
structural determinants of selectivity for the two activities, we established a luciferase-based assay monitoring
HMGCR degradation and used it to profile the structure-activity/selectivity relationships of oxysterols and (oxy)
lanosterols. We identified several sterols that selectively induce HMGCR degradation and one sterol, 25-hy-
droxycholest-4-en-3-one, that selectively inhibits the SREBP pathway. These results should be helpful in de-
signing more potent and selective HMGCR degraders.
HMG-CoA reductase
Degradation
Luciferase
Oxysterols
Lanosterols
SREBP
–13
1
. Introduction
without inhibiting the activity of the SREBP pathway.9
Similarly, a
class of synthetic small molecules with cholesterol-lowering activity,
including SR12813 and SR45023A (also known as apomine), also se-
HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in the
1
4–16
cholesterol biosynthetic pathway and is the target of cholesterol-low-
ering drugs, statins, which inhibit its catalytic activity via direct
binding to the catalytic domain. The activity of HMGCR is tightly
regulated by both transcriptional and post-transcriptional negative
lectively induces degradation of HMGCR.
The degradation induced
by lanosterols or the SR compounds seems to be mechanistically dif-
1
6
ferent from that induced by 25HC, but genetic screening studies have
only clarified the requirement of the membrane-spanning domain of
1
4,5,8
feedback mechanisms.
HMGCR and Insig, which presumably recruits E3 ligases to HMGCR.
2
5-Hydroxycholesterol (25HC), an oxidized product of cholesterol,
As the membrane domain of HMGCR contains a sterol-sensing domain
(SSD), which shares sequence similarity with some sterol-related/
sterol-sensitive proteins, including NPC1, Ptch1, and SCAP, it has been
inhibits transcription of the HMGCR gene by inhibiting the activation of
1
sterol-regulatory-element-binding proteins (SREBPs). Decades of re-
search have established that 25HC binds to an ER-resident oxysterol
postulated that lanosterol may directly bind to the membrane domain
2
,3
8,12,17
sensor, Insig, to prevent proteolytic activation of SREBPs.
It also
of HMGCR to induce complexation with Insig.
Recent studies on
exerts negative feedback regulation by inducing HMGCR degradation.
This degradation requires only the N-terminal membrane-spanning
domain of HMGCR (Fig. 1A), and genetic screening has demonstrated
NPC1 and Ptch1 have provided evidence of the role of SSD as a sterol-
18–25
binding domain,
supporting the idea that the SSD of HMGCR serves
as a lanosterol sensor, although direct binding has not yet been ex-
perimentally confirmed.
4
–6
that Insig is also required in this process.
The currently accepted
model is that Insig forms complexes with some E3 ubiquitin ligases, and
5HC-binding to Insig induces formation of a ternary complex, E3 li-
gase-Insig-HMGCR, leading to ubiquitination and proteasomal de-
Currently, only limited information is available on whether oxy-
sterols and lanosterols are differentially recognized by Insig and pos-
2
1
2,26
sibly HMGCR.
Such information would be useful for designing
7
,8
26
gradation of HMGCR.
more potent and selective HMGCR degraders. As direct binding assays
are notoriously difficult for membrane proteins such as Insig and
HMGCR, we sought to gain insight into this question by employing a
On the other hand, lanosterols, which are intermediates in the
cholesterol biosynthetic pathway, induce degradation of HMGCR
⁎
Corresponding author.
E-mail address: ohgane@iam.u-tokyo.ac.jp (K. Ohgane).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmc.2019.115298
Received 15 November 2019; Received in revised form 21 December 2019; Accepted 25 December 2019
0968-0896/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Ikuya Sagimori, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2019.115298

I. Sagimori, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 1. Design and basic characterization of HMGCR degradation assay with the HMGCR-dCat-ELuc cell lines. (A) Schematic representation of HMGCR structure and
HMGCR-dCat-ELuc constructs. (B) Luciferase-based monitoring of dose-dependent HMGCR degradation by previously reported small molecules (25HC, SR45023A).
Cells stably expressing HMGCR-dCat-ELuc were treated as indicated for 4 h and assayed for luciferase activity. The filled black circles represent SR45023A treatment,
and the open triangles represent 25HC treatment. Error bars denote standard deviation (SD) of triplicate experiments. (C) Kinetics of SR45023A-mediated HMGCR
degradation, and requirement of VCP as an ERAD component. The cells were treated with SR45023A (10 µM), CB5083 (10 µM), or both SR45023A (10 µM) and
CB5083 (10 µM) for the indicated time. (D) Importance of post-squalene cholesterol biosynthetic intermediates in the feedback regulation of HMGCR. The cells were
treated with an SQS inhibitor TAK-475 for 8 h, and the expression level of HMGCR-dCat-ELuc was examined by luciferase assay. The filled black circles and error bars
denote mean and SD. The gray crosses denote raw data from biological replicates (n = 8). (E) Degradation of HMGCR-dCat-ELuc induced by ketoconazole, and its
dependency on metabolic activity of SQS. Cells were treated with the indicated concentrations of ketoconazole in the absence or presence of 10 µM TAK-475 for 8 h
before luciferase activity measurement. The black circles and error bars denote mean and SD (biological replicates, n = 4) in the presence of TAK-475. The gray
triangles and error bars denote the mean and SD in the absence of TAK-475.
pharmacological approach, using cell-based assays that monitor
HMGCR degradation and SREBP pathway activity in living cells. Here,
we report the establishment and basic characterization of a luciferase-
based HMGCR degradation assay. We also present preliminary struc-
ture-activity relationships of oxysterols/lanosterols for both HMGCR
degradation and SREBP pathway activity.
(20HC), 6β-hydroxy-5α-cholestan-3β-ol (6bHC), 6α-hydroxy-5α-cho-
lestan-3β-ol (6aHC), 7β-hydroxycholesterol (7HC), 7-ketocholesterol
(7KC), 4β,5α-dihydroxycholestan-3β-ol (45HC), and 25-hydro-
xycholest-4-en-3-one (3K25HC). SR45023A was synthesized as pre-
3
0
viously reported . 25(R)-Cholest-5-en-3β,16β,26-triol (1626HC) was
synthesized by modified Clemmensen reduction of diosgenin (from
3
1
Tokyo Chemical Industries) according to the reported procedure
.
2
4,25-Epoxylanosterol
(2425EL)
and
24,25-dihydrolanosterol
2
. Materials and methods
(
(
2425DHL) were prepared from commercially available lanosterol
purchased from Tokyo Chemical Industries) containing > 50% 24,25-
2.1. Materials
32
dihydrolanosterol . 7,11-Diketolanosterol (711KL) was prepared by
3
3
ruthenium-catalyzed allylic oxidation from 24,25-dihydrolanosterol
Lipoprotein-deficient serum was prepared from FBS using Cab-O-sil
34
26
acetate . 4,4-Dimethylcholesterol was synthesized as reported pre-
2
7
(
Kodak) as previously reported. TAK-475 was from Sigma-Aldrich,
viously . 24,25-Dihydroxylanosterol (2425HL) was synthesized as re-
2
8
and CB5083 was from Cayman Chemicals. Other sterols were pur-
chased from the indicated sources: 22(S)-hydroxycholesterol (22SHC,
from Sigma-Aldrich), 22(R)-hydroxycholesterol (22RHC, from Sigma-
Aldrich), 6-ketocholesterol (6KC, from Sigma-Aldrich), 25-hydro-
xycholesterol (25HC, from Sigma-Aldrich), 19-hydroxycholesterol
33,35
ported previously
. Stock solutions of the sterols and other com-
pounds were basically prepared as 10 mM DMSO solutions and stored at
−
20 °C. For 25HC, which is poorly soluble in DMSO, the stock solution
was a 10 mM ethanol solution.
(
19HC, from Cayman Chemicals), and water-soluble cholesterol (from
FUJIFILM Wako Pure Chemicals). The following oxysterols were pre-
viously synthesized in our laboratory, and their characterization data
2.2. Expression vector for HMGCR-dCat-ELuc
2
0,29
was published in the previous reports
: 5α-hydroxy-6-ketocholes-
The coding sequence of human HMGCR (GenBank AB527413) was
PCR-amplified from pFN21A-HT-hHMGCR (Promega/Kazusa DNA
terol (5H6KC), 5α,6β-dihydroxycholesterol (56HC), 5α,6β,25-trihy-
droxycholesterol (5625HC), 7-keto-25-hydroxycholesterol (7K25HC),
3
6
Research Institute, FHC11622) with flanking SgfI and MluI sites by
7
β,25-dihydroxycholesterol
(7H25HC),
20(S)-hydroxycholesterol
using the following pair of primers: forward, AACGCGATCGCCATGTT
2

I. Sagimori, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
GTCAAGACTTTTTCGAATG; reverse, ATTACGCGTGGCTGTCTTCTTGG
TGCAAG. The PCR-amplified fragment was purified by agarose gel
electrophoresis followed by the use of MagExtractor (Toyobo), and the
fragment was digested with SgfI (Promega) and MluI (NEB) in NEBuffer
S2 and S3 can be found in the supplementary information.
General methods. All chemical reagents and solvents were pur-
chased from Sigma-Aldrich, Kanto Chemical Industry and Wako Pure
Chemical Industries, and used without further purification. Moisture-
sensitive reactions were performed under an atmosphere of argon,
unless otherwise noted, and monitored by thin-layer chromatography
(TLC; Merck silica gel 60 F254 plates). Bands were visualized using UV
light, iodine vapor, and/or acidic phosphomolybdic acid stain. Flash
chromatography was carried out with silica gel (Silica gel 60 N,
40–50 μm particle size) purchased from Kanto Chemical. NMR spectra
3
supplemented with BSA at 37 °C for 3 h, followed by inactivation of
SgfI at 65 °C for 20 min. A previously reported blank vector pCMV-AC-
3
7
FLAG-ELuc was similarly digested with SgfI and MluI, and depho-
sphorylated with calf intestine phosphatase. The obtained SgfI-
hHMGCR-MluI fragment and the vector fragment were ligated with
Ligation high ver 2 (Toyobo). The integrity of the plasmid was con-
firmed by DNA sequencing. To delete the catalytic domain of pCMV-
hHMGCR-FLAG-ELuc, PCR mutagenesis was performed using a KOD-
plus mutagenesis kit (Toyobo) with the following primers: forward,
ACGCGTACGCGGCCGCTCGAGGACTAC; reverse, TAGACATTCTTCAT
TAGGCCGAG. The deletion of the catalytic domain and the integrity of
the plasmid were confirmed by DNA sequencing.
1
were recorded on a JEOL JNM-ECA500 spectrometer at 500 MHz for H
1
3
NMR and at 125 MHz for C NMR. Proton and carbon chemical shifts
are expressed in δ values (ppm) relative to internal tetramethylsilane
1
(0.00 ppm), residual CHCl
3
(7.26 ppm) for H NMR and internal tet-
1
3
ramethylsilane (0.00 ppm) or CDCl (77.16 ppm) for C NMR. Data are
3
reported as follows: chemical shift, multiplicity (s, singlet: d, doublet; t,
triplet: q, quartet; m, multiplet: br, broad), coupling constants (Hz), and
integration. High-resolution mass spectra were recorded using a Bruker
micrOTOF II mass spectrometer.
2.3. Stable cell lines
HEK293 cells (ATCC) cultured in DMEM (Wako, product No. 044-
9765) supplemented 10% FBS and penicillin-streptomycin (Nacalai,
3β-acetoxy-25,26,27-trinorlanost-8-en-24-ol (S2). To a solution
of lanosterol 24,25-epoxide 3-O-acetate (S1, 727 mg, 1.50 mmol) in
2
product No. 26253-84) were transfected with pCMV-hHMGCR-dCat-
FLAG-ELuc using Lipofectamine LTX (Invitrogen). Selection pressure
was applied to the cells by adding 0.4 mg/mL G418 (Calbiochem).
Single clones were isolated by the limiting dilution method. Four clones
were tested for their response to 25HC treatment, and similar dose-
dependent degradation of HMGCR-dCat-ELuc was observed for all of
them. Of the four clones, the one with the highest expression level was
selected and used throughout this study.
dehydrated THF (140 mL) was added HIO
4
·2H O (513 mg, 2.25 mmol)
2
under an argon atomosphere. The mixture was stirred for 2 h, water
was added, and THF was removed under reduced pressure. The residue
was extracted with ethyl acetate, and the organic layer was washed
with brine, dried over sodium sulfate, and concentrated under reduced
pressure to give a colorless solid. The crude aldehyde was unstable, and
so was immediately used for the next reaction without purification. The
crude aldehyde was dissolved in 2-propanol (70 mL) and treated with
NaBH (170 mg, 4.50 mmol), and the reaction mixture was stirred
4
overnight at ambient temperature. After 18 h, the reaction was quen-
ched with saturated aqueous ammonium chloride solution at 0 °C, and
2.4. HMGCR degradation assay
2
-propanol was removed under reduced pressure. The residue was ex-
The stable cell line expressing HMGCR-dCat-ELuc was cultured on
6-well white plates (Greiner, product No. 655098) to about 90%
tracted with ethyl acetate, and the organic layer was washed with brine,
dried over sodium sulfate, and concentrated. Purification of the residue
by flash column silica gel chromatography (ethyl acetate: hexane: di-
9
confluency, and the cells were treated with a test compound for 4 h.
Luciferase activity and cytotoxicity were simultaneously assessed by
CytoRed-luciferase multiplex assay as reported previously.38
chloromethane = 1 : 2 : 2) afforded the title compound (382 mg, 57%)
1
as a colorless solid. H NMR (CDCl
3
) δ 4.50 (dd, J = 11.8, 4.3 Hz, 1H),
3
0
1
.67–3.62 (m, 2H), 2.05 (s, 3H), 1.00 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H),
2.5. Luciferase reporter assay for LDL receptor promoter
13
.88 (s, 6H), 0.87 (s, 3H), 0.69 (s, 3H); C NMR (CDCl
3
) δ 171.03,
34.41, 134.23, 80.91, 63.61, 50.46, 50.33, 49.78, 44.45, 37.78, 36.87,
The reporter gene assay for LDLr promoter was performed as de-
1
9
36.21, 35.24, 32.07, 30.93, 30.77, 29.56, 28.17, 27.89, 26.35, 24.22,
scribed previously using HEK293 cells. HEK293 cells on white 96-well
plates were transiently transfected with pGL4-LDLr-luciferase along
with pCMV-beta-galactosidase. After 24 h, the medium was replaced
with DMEM supplemented with 10% LPDS, test compounds were
added, and the cells were further cultured for 24 h. Luciferase activity
and beta-galactosidase activity were determined as described pre-
2
4.15, 21.33, 20.96, 19.17, 18.65, 18.09, 16.52, 15.75; HRMS (ESI)
+
calcd for C29
H
48
O
3
[M + Na] 467.3496, found 467.3483.
2
5,26,27-trinor-24-hydroxylanost-8-en-3β-ol (nor24HL, S3).
The acetate (80.1 mg, 0.180 mmol) was dissolved in THF (6 mL) and
MeOH (3 mL), and the solution was treated with 3.3 M aqueous KOH
(0.6 mL, 2.0 mmol). The reaction mixture was refluxed for 2 h, diluted
with water, and extracted with dichloromethane. The organic layer was
washed with brine, dried over sodium sulfate, and concentrated under
reduced pressure. The residue was purified by flash column chroma-
tography (ethyl acetate: hexane: dichloromethane = 1 : 2 : 2) to afford
viously.3
9–41
2.6. Synthesis of 25,26,27-trinor-24-hydroxylanost-8-en-3β-ol (nor24HL)
The title compound (S3, nor24HL) was synthesized as shown in
1
the title compound (63.5 mg, 0.158 mmol, 87%) as a colorless solid. H
Scheme 1 from lanosterol 24,25-epoxide 3-O-acetate S1, which was
prepared as reported.32 The 1H- and 13C NMR spectra of the compound
NMR (CDCl ) δ 3.64–3.61 (m,2H), 3.24 (dd, J = 11.8, 3.8 Hz, 1H), 1.00
3
Scheme 1. Synthesis of nor24HL.
3

I. Sagimori, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 2. Profiling the activities of oxysterols on induction of HMGCR degradation and inhibition of the SREBP pathway. (A) HMGCR degradation activity and SREBP
pathway-inhibitory activity of selected oxysterols. The data are the mean ± SD (n = 3 independent experiments). The black circles denote HMGCR degradation
activity, and the gray filled triangles denote inhibition of the SREBP pathway, as monitored by means of the LDLr-luciferase assay. (B) Structures of SR45023A and
the oxysterols used in this study. Group A is selective for HMGCR degradation. Group B is non-selective, consisting of sterols acting on both HMGCR degradation and
SREBP inhibition. Group C is selective for SREBP inhibition.
(
s, 3H), 0.98 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.81 (s, 3H),
29.58, 28.18, 27.94,27.82, 26.48, 24.23, 20.97, 19.13, 18.66, 18.23,
15.75, 15.41; HRMS (ESI) not detected in positive and negative ion
modes.
1
3
0
.69 (s, 3H); C-NMR (CDCl
3
) δ 134.39, 134.34, 78.97, 63.61, 50.37,
5
0.34, 49.79, 44.46, 38.88, 36.70, 36.21, 35.56, 32.08, 30.96, 30.81,
4

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Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 3. Dose-response analysis of selected oxysterols and SR45023A for HMGCR degradation activity and SREBP pathway-inhibitory activity. The black filled circles
and error bars represent mean ± SD (biological replicates, n = 3) for HMGCR degradation activity, and the open circles represent mean ± SD (biological
replicates, n = 3) for SREBP-inhibitory activity. The data are representative of two independent experiments, which gave similar results.
3
. Results and discussion
ELuc (Fig. 1D), confirming the importance of post-squalene cholesterol-
8,13
biosynthetic intermediates in the accelerated degradation process.
3
.1. Monitoring HMGCR degradation with stable cell lines expressing
Furthermore, we confirmed that treatment of the cells with ketocona-
zole, which leads to accumulation of endogenous lanosterols (lanosterol
and dihydrolanosterol) via inhibition of lanosterol demethylase
(CYP51A), induced degradation of HMGCR-dCat-ELuc in a dose-de-
pendent manner (Fig. 1E, gray triangles), again recapitulating previous
HMGCR-dCat-ELuc
The N-terminal transmembrane domain was reported to be suffi-
cient for the induction of accelerated degradation of HMGCR by sterols,
and previous studies have demonstrated that membrane domains of
HMGCR fused to beta-galactosidase or GFP respond similarly to the
9
–11
results.
This effect was abrogated in the presence of TAK-475, an
inhibitor of SQS, which lies upstream of lanosterol synthesis, further
supporting the importance of lanosterol accumulation for this effect
(Fig. 1E, black circles). Overall, the HMGCR-dCat-ELuc system essen-
tially reproduced findings previously obtained in studies using en-
dogenous, galactosidase-fused, or GFP-fused HMGCRs.1
2
6,42,43
sterol-induced degradation.
To further improve the assay sensi-
tivity and throughput, we replaced the catalytic domain of HMGCR
3
7,44–46
with emerald luciferase (ELuc),
and established a cell line stably
6,26,42,43
expressing HMGCR-dCat-ELuc (Fig. 1A). Notably, we could isolate
stable clones with only low expression levels of the HMGCR-dCat-ELuc
3
7
(
less than 1% luminescence compared to other ELuc-fused proteins) ;
3
.2. Profiling structure-activity relationships of oxysterols for HMGCR
the FLAG-tagged HMGCR-dCat-ELuc was hardly detectable by standard
western blotting analysis with anti-FLAG antibody, and indeed, was
only detectable after enrichment by immunoprecipitation (Fig. S1).
Despite such low expression, the high sensitivity of the luciferase assay
enabled quantitative evaluation of HMGCR-dCat-ELuc.
degradation and SREBP-pathway inhibition
To understand the structure features that dictate potency and se-
lectivity between HMGCR degradation and SREBP pathway inhibition,
we tested a panel of oxysterols with HMGCR degradation assay and LDL
receptor (LDLr) promoter assay, which monitors transcriptional activity
Treatment of this cell line with 25HC or SR45023A resulted in a
clear, dose-dependent reduction of luciferase activity, which reflects the
amount of HMGCR-dCat-ELuc proteins, and the half-maximal effective
concentrations (EC50 0.63 µM and 0.47 µM, respectively) were con-
1
9,48
through the SREBP pathway.
We focused on oxysterols bearing two
or more oxygen functionalities, as the poor aqueous solubility of non-
hydroxylated sterols and difficulty in delivering them to the ER could
potentially complicate analysis of the structure-activity/selectivity re-
lationships. As shown in Fig. 2, 25HC strongly induced HMGCR de-
gradation and also inhibited LDLr promoter activity, confirming the
1
6
sistent with data reported previously (Fig. 1B). Kinetic experiments
also confirmed that SR45023A induced the degradation of HMGCR
within several hours, as demonstrated before (Fig. 1C). Also, consistent
with the requirement of valosin-containing proteins (VCPs) for the
2
,7
2
8,47
non-selective nature of 25HC.
On the other hand, SR45023A ex-
proteasomal degradation of the HMGCR,
a VCP inhibitor CB-5083
hibited high selectivity for HMGCR degradation over SREBP inhibition,
blocked the SR45023A-induced degradation of HMGCR.
4
as demonstrated previously. Based on their activity profile, oxysterols
To further validate the HMGCR-dCat-ELuc assay, we next examined
if this assay allows monitoring of the acceleration of HMGCR de-
gradation by endogenous sterols. Treatment with TAK-475, an squalene
synthase (SQS) inhibitor, resulted in clear stabilization of HMGCR-dCat-
can be classified into three groups (Fig. 2B). Group A, which includes
5
6HC and 5625HC, selectively induces degradation of HMGCR without
clear inhibition of the SREBP pathway. Group B, including most of the
other oxysterols, indiscriminately induced HMGCR degradation and
5

I. Sagimori, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 4. Activity profile of selected lanosterol derivatives. (A) Structures of lanosterol derivatives and a lanosterol-related cholesterol derivative. (B) HMGCR de-
gradation activity and SREBP pathway-inhibitory activity of the lanosterol derivatives. The circles and error bars denote mean ± SD (biological replicates, n = 3)
from representative experiments independently performed twice. The filled circles represent HMGCR degradation activity and the open circles represent SREBP-
inhibitory activity.
inhibition of the SREBP pathway. Group C, whose sole member is
K25HC, selectively inhibited the SREBP pathway without causing
inhibition. 25-Hydroxylation increased the potency for both HMGCR
degradation and SREBP pathway-inhibitory activity, and 5,6-dihy-
droxylation conferred selectivity for HMGCR degradation over SREBP
inhibition. Interestingly, 3K25HC, which was selective for SREBP
pathway inhibition, also showed a sigmoidal response in the HMGCR
degradation assay, although the maximal effect was smaller. Overall,
these results clearly indicate that appropriate modifications of the sterol
structure should afford sterol derivatives that are selective for each
activity.
3
marked degradation of HMGCR. In group B, we observed a correlation
between HMGCR degradation activity and SREBP inhibition. Among
the oxysterols in group B, the presence of hydroxyl groups on the iso-
octyl side chain seems to contribute to the potency of both activities. In
contrast, hydroxylation or oxidation at the 5- or 6-position imparted
selectivity for HMGCR degradation over SREBP inhibition (for example,
compare 25HC, 56HC, and 5625HC). 19HC weakly induced degrada-
tion of HMGCR without inhibition of the SREBP pathway inhibition, in
accordance with a previous report showing both lack of binding of
3
.3. Selectivity of lanosterols for HMGCR degradation and SREBP-pathway
2
1
9HC to Insig and lack of SREBP inhibition. The result for 3K25HC
inhibition.
(
group C) indicated that the hydroxyl group at the 3 position is not
required for SREBP-inhibitory activity.
Lanosterols have already been shown to induce degradation of
To further validate the results obtained from the 10 µM screen, we
analyzed the dose-response relationships of selected compounds for
both HMGCR degradation activity and SREBP pathway-inhibitory ac-
tivity (Fig. 3). The results of the dose-response experiments were ba-
sically in accordance with those of the 10 µM screen. SR45023A showed
clear selectivity for HMGCR degradation over SREBP pathway
9,12
HMGCR without affecting SREBP pathway activity.
So far, 4,4-di-
and a recent study clarified
methyl substituents on lanosterol has been proposed to be the major
8,12
determinant of HMGCR degradation,
differences in the selectivity of endogenous, post-squalene cholesterol
biosynthetic intermediates, including lanosterol and 24,25-dihy-
drolanosterol (2425DHL), for HMGCR degradation and SREBP
6

I. Sagimori, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
inhibition.13 However, it remains unclear what determines the se-
lectivity and potency of lanosterols.
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1
146/annurev-biochem-062917-011852.
2
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.
.
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transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport
by binding to Insig. Proc Natl Acad Sci USA. 2007;104:6511–6518. https://doi.org/
To understand the structure-activity and structure-selectivity re-
lationships of lanosterol derivatives for HMGCR degradation and SREBP
inhibition, we prepared a small panel of (oxy)lanosterols and profiled
their effects on HMGCR degradation and SREBP inhibition (Fig. 4).
Exogenous addition of 2425DHL and 44MC, a 4,4-dimethylated cho-
lesterol, did not induce clear degradation of HMGCR or inactivation of
SREBP; as endogenous 2425DHL has been reported to induce HMGCR
1
0.1073/pnas.0700899104.
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3
degradation, this is probably due to insolubility and/or difficulty in
2
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8
entry of the compounds into cells. Three oxylanosterols, 2425EL,
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425HL, and 25HL, induced both degradation of HMGCR and inhibi-
2
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tion of SREBP in a nonselective manner, similarly to 25HC. Comparison
of 25HC and 25HL implies that 25-hydroxylation renders the sterol
nonselective and the difference in the steroid core structure (e.g., the
presence of 4,4-dimethyl groups, the 8,9-double bond, and 14-angular
methyl group) does not preclude interaction with Insig, which triggers
both HMGCR degradation and SREBP inhibition. Interestingly, 711KL
showed some selectivity for HMGCR degradation over SREBP inhibi-
tion, and nor24HL exhibited higher selectivity. These results point to
the possibility that appropriate modifications of sterols or lanosterols
could yield selective HMGCR degraders.
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In this study, we established a luciferase-based assay monitoring
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lano)sterols, including 56HC, 711KL, and nor24HL, that are selective
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2
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ture, as well as for designing chemical probes to test the hypothesis of
direct binding of (lano)sterols to the SSD of HMGCR. Overall, the lu-
ciferase-based HMGR assay reported here should be useful for studies
on the mechanisms of HMGCR degradation induced by small molecules,
and in identifying potential drug candidates to decrease cholesterol
synthesis by reducing the HMGCR protein level.
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Acknowledgements
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https://doi.org/10.1073/pnas.0405255101.
We thank the members of our laboratory for helpful discussions. The
work described in this article was supported in part by Grant-in-Aid for
Young Scientists B for K.O., Grants-in-Aid for JSPS Research Fellow for
H.Y., and Grants-in-Aid for Scientific Research B for Y.H. (JSPS
KAKENHI Grant number JP17K15487, JP18J14851, and JP17H03996).
19. Ohgane K, Karaki F, Dodo K, Hashimoto Y. Discovery of oxysterol-derived pharma-
cological chaperones for NPC1: implication for the existence of second sterol-binding
site. Chem Biol. 2013;20:391–402. https://doi.org/10.1016/j.chembiol.2013.02.009.
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relationships of oxysterol-derived pharmacological chaperones for Niemann-Pick
type C1 protein. Bioorg Med Chem Lett. 2014;24:3480–3485. https://doi.org/10.
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Declaration of Competing Interest
The authors declare no conflict of interest.
Appendix A. Supplementary material
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purified human NPC1. J Biol Chem. 2009;284:1840–1852. https://doi.org/10.1074/
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2. Lu F, Liang Q, Abi-Mosleh L, et al. Identification of NPC1 as the target of U18666A,
an inhibitor of lysosomal cholesterol export and Ebola infection. Elife. 2015;4:19316.
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3. Li X, Wang J, Coutavas E, Shi H, Hao Q, Blobel G. Structure of human Niemann-Pick
C1 protein. Proc Natl Acad Sci USA. 2016;113:8212–8217. https://doi.org/10.1073/
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The online version of this article contains supplementary materials
and methods (Immunoblotting and immunoprecipitation of HMGCR-
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1
13
dCat-FLAG-ELuc, H- and/or C NMR spectra for the compounds S2,
nor24HL (S3), 25HL, 2425HL, 711KL, and 2425EL). Supplementary
data to this article can be found online at https://doi.org/10.1016/j.
bmc.2019.115298.
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