Bioorg. Med. Chem.

36 (2021) 116095

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Withanolides from dietary tomatillo suppress HT1080 cancer cell growth

by targeting mutant IDH1
Yueying Yang a, 1, Ke Xiang a, 1, Dejuan Sun a, 1, Mengzhu Zheng b, Zhuorui Song a, Mingxue Li a,
Xuanbin Wang c, *, Hua Li a, b, *, Lixia Chen a, *
a
Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang
110016, China
b
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
c
Laboratory of Chinese Herbal Pharmacology, Oncology Center, Renmin Hospital, Biomedical Research Institute, Hubei Key Laboratory of Wudang Local Chinese
Medicine Research, Hubei University of Medicine, Shiyan 442000, China

A R T I C L E I N F O A B S T R A C T

Keywords: Isocitrate dehydrogenase (IDH) is one key rate-limiting enzyme in the tricarboxylic acid cycle, which is related to
Physalis ixocarpa various cancers. Tomatillo (Physalis ixocarpa), a special tomato, is widely consumed as nutritious vegetable in
Type-A withanolide Mexico, USA, etc. As a rich source for withanolides, the fruits of P. ixocarpa were investigated, leading to the
Antitumor
isolation of 11 type-A withanolides including 4 new ones (1 is an artificial withanolide). All these withanolides
Mutant IDH1
Physalin F
were evaluated for their inhibition on mutant IDH1 enzyme activity. Among them, physalin F (11) exhibited
HT1080 potent enzyme inhibitory activity and binding affinity with mutant IDH1. It inhibits the proliferation of HT1080
cells by selectively inhibiting the activity of mutant IDH1. Since Ixocarpalactone A, another major type-B
withanolide in this plant, could act on another energy metabolism target PHGDH, the presence of different
types of withanolides in tomatillo and their synergistic effect could make it a potential antitumor functional food
or drug.

1. Introduction
Isocitrate dehydrogenase (IDH), a key rate-limiting enzyme in the
tricarboxylic acid (TCA) cycle, catalyzes the oxidative decarboxylation
of isocitrate to α-ketoglutarate (α-KG) using NAD+/NADP+ as co­
factors.1,2 IDH family includes three isozymes (IDH1/2/3), among them,
IDH1 is located in the cytoplasm and peroxisome, and IDH2 and IDH3
are located in the mitochondria. IDH1/2 mutations have been detected
in a variety of cancers. Approximately, 70% of patients with grade II–III
gliomas and secondary glioblastomas were found IDH1 mutations.3-5
There are rarely IDH2 mutations in brain tumors, but about 20% of
patients with acute myeloid leukemia (AML) exhibited IDH mutations
and approximately half were IDH2 mutations.6,7 92.7% of IDH1 gene
mutants found in glioma cells are R132H mutations, and some low-
frequency mutations including R132Q, R132S, R132G, R132C and
R132L are also detected.5,8 The R132C mutations are more pervasive
than R132H in ALM.9 The mutant IDH1/2 is endowed with a new cat­
alytic activity that catalyzes the conversion of α-KG to R-2-hydroxy­
glutarate (R-2-HG) by consuming NADPH.8 R-2-HG is a competitive
inhibitor of α-KG-dependent enzymes, including DNA and histone
demethylases. R-2-HG significantly increased DNA and histone
methylation levels in cells carrying mutant IDH by inhibiting the

Abbreviations: IDH, Isocitrate dehydrogenase; H3K9me3, Histone 3 lysine 9 trimethylation; TCA, tricarboxylic acid; PHD, prolyhydroxylase; α-KG, α-ketogluta­
rate; AML, acute myeloid leukemia; R-2-HG, R-2-hydroxyglutarate; HIF-1α, hypoxia-inducible factor; MST, Microscale thermophoresis; LB, Luria-Bertani; IPTG,
isopropyl-β-D-1-thiogalactopyranoside; OD, optical density; PMSF, phenylmethylsulfonyl fluoride; CCK8, cell counting Kit-8; PDB, Protein Data Bank; Kd, equilibrium
dissociation constant; BBB, blood–brain barrier.
* Corresponding authors at: Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Phar­
maceutical University, Shenyang 110016, China (H. Li; L.X. Chen); Laboratory of Chinese Herbal Pharmacology, Oncology Center, Renmin Hospital, Biomedical
Research Institute, Hubei Key Laboratory of Wudang Local Chinese Medicine Research, Hubei University of Medicine, Shiyan 442000, China (X.B. Wang).
E-mail addresses: Wangxb@hbmu.edu.cn (X. Wang), li_hua@hust.edu.cn (H. Li), syzyclx@163.com (L. Chen).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.bmc.2021.116095
Received 23 January 2021; Received in revised form 19 February 2021; Accepted 20 February 2021
Available online 26 February 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.
Y. Yang et al.
activities of DNA and histone demethylases, showing that R-2-HG pro­
motes the occurrence of cancer by inducing epigenetic changes.10-13
A large number of small molecule inhibitors targeting mutant IDH
are being researched, and breakthrough progress has been made in the
past five years. FDA approved Enasidenib (Idhifa®, AG-221) and Ivosi­
denib (Tibsovo®, AGI-120) from Agios Pharmaceuticals for treatment of
relapsed/refractory AML (R/R AML) with mutant IDH2 and IDH1,14,15
respectively, in 2017 and 2018. Although inhibitors targeting mutant
IDH have been approved by the FDA, research on mutant IDH inhibitors
is far from adequate.
Natural products are rich source for the discovery of antitumor
drugs. Tomatillo (Physalis ixocarpa or Physalis philadelphica) is a special
green tomato, which grows wild along the Pacific Coast from California
to Nicaragua, and has been cultivated in central and western Mexico
since pre-Columbian times.16-18 The green fruits of Physalis ixocarpa
have been widely consumed as a kind of nutritious vegetable to make
salad or sauce in different regions of Mexico, USA, and Central Amer­
ica.19 Tomatillo belongs to the genus Physalis, a rich source for natural
withanolides with various bioactivities including antitumor, anti-
inflammatory, immunosuppressive, and antimicrobial effects.20 Ac­
cording to the difference of C-17 side chain, withanolides can be divided
into two types, namely, Type A withanolides with a side chain of
δ-lactone or δ-lactol and Type B with a γ-lactone or γ-lactol.20 Ixo­
carpalactone A, with a γ-lactone side chain (type-B withanolide), was
obtained as a major constituent from Physalis ixocarpa in our previous
study. It was determined to inhibit proliferation and growth of SW1990
pancreatic cancer cells by targeting NAD+-dependent enzyme 3-phos­
phoglycerate dehydrogenase (PHGDH) in vitro and in vivo.21 In addi­
tion, a natural product triptolide has also been reported to be a potential
candidate for treating malignant tumors with IDH1 mutations.22 In the
present study, 11 type-A withanolides (1–11) with a δ-lactone side chain
were isolated from this plant. Among them, physalin F (11), a major
seco-type-A withanolide was identified to be a potential inhibitor of
mutant IDH1. Although physalin F has been previously proved to
possess anti-tumor effects,22,23 the research on its potential targets and
mechanisms remains to be further studied. Herein, physalin F was found
to induce apoptosis and inhibit proliferation activity of HT1080 cells,
and down-regulate the production of R-2-HG by selectively inhibiting
the activity of mutant IDH1. In combination with our previous study,21
type-A and type-B withanolides act on different targets of energy
metabolism to exert antitumor effects, suggesting tomatillo could be
used as potential antitumor functional food or drug through the syner­
gistic effect of different types of withanolides.
2. Materials and methods
2.1. General experimental procedures
Optical rotations were recorded on a PerkinElmer 241 polarimeter.
ECD spectra were recorded on a Bio-Logic Science MOS-450 spectrom­
eter. NMR spectra were measured on an AV-600 spectrometer. Chemical
shift values are depicted in δ (ppm) by using the resonances of the sol­
vent CD3OD (δH 4.87, 3.31 and δC 49.15) or pyridine–d5 (δH 7.58 and δC
135.9) as references, and the coupling constants are expressed as J in Hz.
HRESIMS data were collected on an Agilent 6210 TOF mass-
spectrometer. Silica gel (200–300 mesh, Qingdao Marine Chemical
Factory) and octadecyl silica gel (Merck Chemical Company Ltd., Ger­
many) were applied for column chromatography (CC). Silica gel GF254
for TLC was purchased from Qingdao Marine Chemical Factory (Qing­
dao, China). RP-HPLC was performed on an LC-6AD liquid chromatog­
raphy system equipped with an ODS column (C18, 250 × 20 mm, 120 Å,
5 μm, YMC Co. Ltd.) and SPD-20A UV detector (Shimadzu, Kyoto,
Japan).
2
Bioorganic & Medicinal Chemistry 36 (2021) 116095
2.2. Plant material
The fruits of Physalis ixocarpa were purchased from Irvine, Carli­
fornia, America, in June 2015, and were authenticated by Dr. Jing-Ming
Jia, Shenyang Pharmaceutical University. A specimen (NSJ. 20150021)
was preserved in the herbarium of Shenyang Pharmaceutical University.
2.3. Extraction and isolation
The air-dried fruits of Physalis ixocarpa (18 kg) were extracted with
75% EtOH (3 × 90 L × 3 h) to give a total extract after removing solvent
in vacuo. The extract (1.8 kg) was suspended in water (5.0 L) and
extracted with petroleum ether and EtOAc (4 × 5.0 L) successively. The
EtOAc extract (115 g) was subjected to silica gel column chromatog­
raphy (CC) (8 × 120 cm) eluted with CH2Cl2-CH3OH (100:0, 100:1,
70:1, 40:1, 20:1, 10:1, 8:1, 5:1, 3:1, 1:1, 0:100, v/v) to afford eight
fractions (E1 − E8). Fraction E3 (13.2 g) was separated by silica gel CC
(5 × 80 cm) eluted with petroleum ether-acetone (100:0, 70:1, 50:1,
30:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:100) to afford eight subfractions (E31-
E38). E33 (798 g) was chromatographed on an ODS column eluting with
CH3OH-H2O (30:70, 50:50, 70:30), and then purified with preparative
HPLC (CH3OH-H2O, 60:40) to yield compounds 5 (40.7 mg,
tR = 47.4 min) and 8 (13.5 mg, tR = 35.2 min). Fraction E34 (2.4 g) was
recrystallized with acetone to afford compound 9 (135 mg). Fraction
E35 (1.7 g) was recrystallized with acetone to afford compound 10
(103 mg). E36 (2.3 g) was purified with silica gel CC (8 × 120 cm) eluted
with CH2Cl2-acetone (80:1, 50:1, 20:1, 10:1, 7:1, 4:1) to give 11
(110 mg). E38 (205 mg) was purified by preparative HPLC (CH3OH-
H2O, 60:40) to afford compound 1 (9.2 mg, tR = 35.2 min). Fraction E4
(44 g) was isolated by silica gel CC (3.5 × 80 cm) with an increasing
acetone percentage in petroleum ether from 10 to 100% to give six
subfractions (E41 − E46). Subfraction E46 (5.8 g) was subjected to a
ODS CC using CH3OH-H2O (30:70, 50:50, 70:30) as eluent to afford
eight fractions (E461 − E468). Fraction E466 (45.6 mg) was separated
by HPLC (MeOH-H2O, 60:40) to obtain compounds 6 (21.2 mg,
tR = 13.4 min) and 7 (17.2 mg, tR = 15.9 min). Fraction E7 (15.4 g) was
applied to a silica gel column (3.5 × 80 cm) using CH2Cl2-CH3OH (0:1,
20:1, 10:1, 8:1, 5:1, 3:1, 1:1, 0:100) as eluent to yield six fractions
(E71 − E76). Subfraction E74 (921.7 mg) was separated on an ODS CC
(2 × 70 cm) eluted with CH3OH-H2O (30:70, 50:50, 70:30) to obtain
E741-E744. Separation of E662 (365.4 mg) through preparative HPLC
(MeOH-H2O, 50:50) yielded compounds 2 (10.5 mg, tR = 77.8 min), 3
(20.2 mg, tR = 95.2 min), and 4 (11.5 mg, tR = 39.2 min).
2.4. Enzymatic hydrolysis and HPLC analysis of compounds 2–4
Compounds 2–4 (each 3.0 mg), were dissolved in 3.0 mL of water,
respectively, then adding about 12.0 mg of snailase, shaking the mixture
in a 37 ◦ C constant temperature shaker, and monitoring the progress of
the reaction by TLC every 2 h until the reaction was finished (10 h). The
reaction was finally quenched with ethyl acetate. The hydrolysate was
extracted with 5 mL ethyl acetate for five times. The solvent of organic
phase was evaporated to obtain the corresponding aglycone, and the
aqueous phase was centrifuged to remove the enzyme, and then freeze-
dried. The obtained glucose was dissolved in an acetonitrile–water so­
lution (75:25), filtered through a 0.45 μm filter, and then analyzed by
HPLC, using a JASCO LC-NetII/ADC system, equipped with a JASCO OR-
4090 detector and a gradient pump. The column was the Shodex Asa­
hipak NH2P-50 4E (250 × 4.6 mm, 5 μm). The HPLC-OR spectrum of d-
glucose showed a positive peak at 8.52 min and the glucose from com­
pounds 2–4 showed a positive peak at 8.45 min (Supplementary infor­
mation Figs. S30–S31).
2.5. X-ray crystallographic analysis of compound 5
Colorless crystal of 5 was obtained from CH2Cl2 and MeOH mixed
Y. Yang et al.
solvent system. The crystallography data were collected on a Super­
Nova, Dual, Cu at zero, AtlasS2 diffractometer using monochromatized
Cu Kα (λ = 1.54184 Å) radiation. The crystal was kept at 150.00(10) K
during the data collection process. Structure determination and refine­
ment were executed by using the SHELXL program. Crystallographic
data were deposited at the Cambridge Crystallographic Data Centre
(CCDC No. 2049960) and can be obtained free of charge from the CCDC
Web site (www.ccdc.cam.ac.uk). Crystal Data of 5: C28H40O6
(M = 472.60 g/mol): orthorhombic, space group P212121 (no. 19),
a = 8.7385(4) Å, b = 11.0507(8) Å, c = 25.0443(16) Å, V = 2418.4(3)
Å3, Z = 4, T = 150.00(10) K, μ (Cu Kα) = 0.722 mm− 1, Dcalc = 1.298 g/
cm3, 8885 reflections measured (7.06◦ ≤ 2Θ ≤ 147.628◦ ), 4213 unique
(Rint = 0.0561, Rsigma = 0.0828) which were used in all calculations. The
final R1 was 0.0632 (I > 2σ(I)) and wR2 was 0.1827 (all data). The Flack
parameter was − 0.05(3).
2.6. Cell and antibodies
HT1080 human fibrosarcoma cells carrying IDH1R132C mutation was
from the American Type Culture Collection (ATCC, USA). Primary an­
tibodies including β-Actin, HIF-1α, p53, H3K9me3, IDH and secondary
antibodies having a source corresponding to the primary antibody were
obtained from Proteintech (Rosemont, Illinois, USA).
2.7. Cloning, expression, and purification of IDH1, IDH1R132H
The 132-arginine (R) of wild-type IDH1 plasmid with a C-terminal
6 × His tag was mutated to histidine (R132H) using Quik-change site-
directed mutagenesis kit (Agilent). The IDH1R132H gene was constructed
into the pET28a vector and the recombinant plasmid was transduced
into E. coli strain BL21 (DE) (Novagen). The strains selected on 60 mM
kanamycin plates were cultured in Luria-Bertani (LB) medium at 37 ◦ C
to an OD600 value of 0.8, and then cells was induced to express protein
via 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 18 h at
18 ◦ C. The harvested cells were resuspend in a lysis buffer (20 mM Tris,
pH 8.0, 250 mM NaCl, 5 mM β-mercaptoethanol, 0.1% TritonX-100, and
5% glycerol) supplementing protease inhibitors phenylmethylsulfonyl
fluoride (PMSF), and lysed by ultrasonication on ice. IDH1 and
IDH1R132H carrying the polyhistidine peptide were bound to Niagarose
affinity resin (Qiagen), washed with buffer (20 mM Tris, pH 8.5, 250 mM
NaCl, and 10 mM imidazole), and eluted with elution buffer (20 mM
Tris, pH 8.5, 250 mM NaCl, and 150 mM imidazole). Further purifica­
tion of IDH1 and IDH1R132H by size exclusion chromatography (Super­
dexTM 200, GE) and anion exchange chromatography (HiTrap QFF, GE)
using the ÄKTATM Pure (GE) Protein Purification System.
2.8. In vitro enzyme inhibition assay
The inhibition activity of IDH1R132H was determined by measuring
the consumption of NADPH using a BioTek Synergy HT microplate
reader. The 50 μL reaction system contained 2 μM IDH1R132H, 4 mM
MgCl2, 2 mM α-KG, 100 μM NADPH (for NADPH ≫ km), and 50 mM
HEPES buffer (pH = 7.5). The compound was incubated with IDH1R132H
for 10 min prior to adding the 2 mM α-KG into the 96-well microplate to
trigger reaction, which was used to determine the IDH1R132H enzyme
inhibitory activity of the compounds. The absorbance was monitored
every 1 min at 340 nm for 20 min to determine the NADPH consump­
tion. Wild-type IDH1 activity and sensitivity to compounds were
determined by monitoring NADPH production at 340 nm. The 50 μL
reaction system, contained 43 nM purified wild-type IDH1, 50 μM so­
dium (D)-isocitrate, 4 mM MgCl2, 1 mM NADP+ (≫km for NADP), and
50 mM HEPES buffer (pH = 7.5). Each value was presented as the
mean ± SD of three independent assays, and the data was imported into
GraphPad Prism 5.01 (GraphPad Software) to calculate the IC50 of
compounds for IDH1R132H or IDH1.
3
Bioorganic & Medicinal Chemistry 36 (2021) 116095
2.9. Microscale thermophoresis (MST) assay
Small molecule-protein interactions were determined by the Micro­
scale thermophoresis method25 and specific experimental methods and
details were referenced to manufacturer’s protocol. IDH1R132H was
labeled with the Monolith NTTM Protein Labeling Kit RED (Cat # L001).
The labeled IDH1R132H was diluted to 20 nM by a 20 mM HEPES (pH 7.5)
and 0.05% (v/v) Tween-20. Physalis F was diluted 1:1 into different
concentrations of working solution and incubated with IDH1R132H for
20 min at room temperature, and then samples were added into Mono­
lithTM standard-treated capillaries. After incubation for 10 min at 25 ◦ C,
20% laser power and 100% LED power were used to measure the ther­
mophoresis on a Monolith NT.115 instrument (NanoTemper Technolo­
gies). Kd value was fitted by the NT Analysis software of the Monolith
NT.115 instrument.
2.10. Cell culture
HT1080 cells were cultured in high Glucose Dulbecco’s modified
Eagle’s medium (DMEM) with 5% CO2 (v/v) at 37 ◦ C in a humidified
tissue culture incubator according to ATCC instructions. DMEM media
was supplemented with 10% fetal bovine serum (FBS) (Sigma) and
penicillin/streptomycin (100 unit/mL, Invitrogen).
2.11. R-2-Hydroxyglutarate measurement
After normal adherently grown HT1080 cells were incubated with
physalis F (0.94–30 μM) or DMSO for 48 h, the cell culture medium was
collected and diluted with methanol. The diluted medium was centri­
fuged to remove the precipitate, and the supernatant was quantitatively
analyzed for the content of R-2-HG by LC-MS/MS using an API4000
triple quadrupole mass spectrometer (AB Sciex, USA) equipped with an
electrospray ionization (ESI) source. The mass spectrometer was
coupled to a Shimadzu Prominence UFLC system (Shimadzu Corpora­
tion, Japan) with a Welch Ultimate® XB-C18 column
(100 mm × 2.1 mm, 3.0 μm; Welch Materials, China) Mobile phase B
(methanol) was raised from 30% to 90% in 90 s and then mobile phase B
was held at 90% for 30 s. After that, the mobile phase B was instantly
reduced from 90% to 30% and held for 60 s. Mobile phase A was 10 mM
ammonium acetate dispersed in water (pH 7.4). Mass spectrometry was
performed under multiple reactions monitoring (MRM) to monitor the
transition of R-2-HG precursor to product ions with m/z of
146.8 → 128.6. Purchased R-2-HG (Sigma) for LC-MS/MS analysis, data
acquisition and processing by Analyst 1.6.1 software (AB Sciex) and
calibration curve established. The peak area of R-2-HG was normalized
using the peak area of the internal standard. IC50 was calculated from
the dose–response curve by GraphPad Prism 5.01.
2.12. Cytotoxicity test
The cells were seeded in 96-well cell culture plates and cultured in
DMEM medium containing 10% FBS until the cells were fully adherent.
Physalin F was dissolved in DMSO to make a 50 mM stock solution and
stored at 4 ◦ C, and the concentration of DMSO was maintained below
0.5% in all cell cultures. Adherent cells were continuously cultured with
fresh medium containing different concentrations of physalis F for 48 h
at 37 ◦ C in a humidified incubator supplemented with 5% CO2. After
48 h, cell viability was determined by the cell counting Kit-8 (CCK8)
assay. The cells were treated and incubated according to the manufac­
turer’s instructions, and then the absorbance was measured at 450 nm
using a microplate reader (Thermo Scientific Varioskan Flash). All ex­
periments were repeated for three times.
2.13. EdU and DAPI double staining
Logarithmic growth phase HT1080 cells were seeded into 24-well
Y. Yang et al.
plates and cultured to normal growth stage. Cells were treated with
3.25 μM, 6.5 μM physalin F and DMSO (control group) for 24 h, and then
the HT1080 cells were incubated with the serum-free DMEM medium
containing 50 μM EdU (Yefluor 594 Edu Imaging Kits, Yeasen, Cat #
40276ES60). After 2 h, the medium was discarded and washed three
times with phosphate buffer saline (PBS). The cells in the 24-well plate
were incubated with 4% paraformaldehyde for 30 min at room tem­
perature, and then incubated with 2 mg/mL glycine for 5 min on a
horizontal shaker. 0.5% TritonX-100 was treated to the cells and incu­
bated for 10 min in a horizontal shaker. Apollo® fluorescent dye was
added to each well and incubated for 30 min at room temperature in a
horizontal shaker for the purpose of specifically reacting with EdU.
Nuclei were counterstained with DAPI. Cells were imaged using a
fluorescence microscopy (TCS SP2 AOBS, Leica). All experiments were
repeated for three times.
2.14. Flow cytometric analysis
After physalin F was incubated with normal adherently grown
HT1080 cells for 48 h, the collected cells were dispersed into
1 × Annexin V Binding Buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM
NaCl and 2.5 mM CaCl2). HT1080 cells were incubated with AnnexinV-
FITC/PI double staining for 15 min in the dark according to the in­
structions of the Annexin V-FITC/PI Apoptosis Detection Kit (Nanjing
KeyGen Biotech. Inc.). Apoptosis was detected by flow cytometry (BD
Biosciences, FACSCalibur).
2.15. Western blot assay
HT1080 cells were seeded in 6-well plates and treated with physalin
F for 48 h. Cells were collected and ruptured in the RIPA lysis buffer
(Beyotime, China). The total protein content was determined using BCA
Protein Quantitation Kit (Beyotime, China). Lysate was separated by
12% SDS-PAGE, electrophoretically transferred onto a PVDF membrane
(Millipore), blocked in 5% non-fat milk for 1 h. The membranes were
incubated with the specific primary antibody for 2 h at room tempera­
ture, incubated overnight at 4 ◦ C, and incubated with horseradish
peroxidase-conjugated secondary antibody for 2 h at room temperature
followed by chemiluminescence detection. Band density was quantified
using ImageJ software.25 The Nuclear and Cytoplasmic Protein Extrac­
tion Kit (Beyotime, China) was used for the extraction of nuclear pro­
teins. The extraction process is based on manufacturer’s instructions.
2.16. Immunofluorescence staining
HT1080 cells were seeded onto 6-well plate slides until the cells were
fully adherent and incubated with fresh medium containing different
concentrations (3.25 μM, 6.5 μM) of physalin F. After 48 h, cells were
fixed with 4% paraformaldehyde for 15 min and then permeabilized
with 0.1% Triton X-100 for 5 min at 4 ◦ C. HT1080 cells were blocked
with 5% bovine serum albumin for 2 h at room temperature, and then
incubated overnight at 4 ◦ C with 1:100 diluted primary antibodies
(H3K9me3, HIF-1α, p53). After washing with PBS for three times, 1:500
diluted fluorescent secondary antibody (Molecular Probes, Eugene, OR)
was added to the cells and incubated for 1 h in the dark. Nuclei were
counterstained with DAPI and the cells were imaged using a fluores­
cence microscopy. For the Hoechst 33,258 staining, HT1080 cells are
blocked for 2 h and then stained with Hoechst 33,258 fluorescent dye for
30 min at temperature.
2.17. shRNA transfection.
The hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector was used to
infect HT1080 cells. The sequences of oligo shRNA were as follows: 5′ -
GGACTTGGCTGCTTGCATT-3′ for IDH1 (gene accession number: NM
005896). Inoculate HT1080 cells into a 6-well plate and continue to
4
Bioorganic & Medicinal Chemistry 36 (2021) 116095
culture until the cell confluence is above 90%. Then transfection, 2 µg
plasmid per well and 4 µL Hieff Trans™ Liposomal Transfection Reagent
(Yeasen, Cat # 40802ES02) are incubated for 20 min at room temper­
ature in DMEM without FBS. After 6 h of transfection, change to fresh
DMEM medium containing 10% FBS and continue to culture for 48 h.
2.18. Molecular docking
Physalin F was input as the mol file format. The crystal structures of
wild-type IDH1 (PDB ID: 1T0L) and IDH1R132H (PDB ID: 6B0Z) were
downloaded from the protein data bank (PDB http://www.rcsb.org/p
db).1,26 The Surflex-Dock GeomX docking mode of SYBYL-X 2.0 soft­
ware (Tripos Associates, St. Louis, MO, USA) was selected for ultra-high
precision molecular docking.27,28 The wild-type IDH1 and IDH1R132H
were optimized before docking, including extracting the original ligand
of the protein, removing unnecessary molecules, adding hydrogens to
amino acid residues, etc. The “protomol” was generated using a ligand-
based model, with the threshold and bloat parameters set to default
values of 0.50 and 0 Å. The maximum number of poses for the ligand
physalin F was set to 20. The lowest-energy and most available orien­
tation of physalin F was selected for subsequent investigation.
2.19. Statistical analysis
Data analysis using a statistical analysis software GraphPad Prism
5.01. Data were expressed as mean ± SD. Values were analyzed by one-
way analysis of variance (ANOVA) using SPSS version 12.0 software
(SPSS Inc., Chicago, IL, USA), and P < 0.05 was considered statistically
significant.
3. Results and discussion
3.1. Structure elucidation of the isolated compounds
The investigation of the constituents from EtOAc fraction of fruits of
Physalis ixocarpa led to the isolation of four new type A-withanolides
(1–4), and seven known analogues (Fig. 1), which were identified as
ixocarpanolide (5),29 2,3-dihydro-3β-methoxywithaphysacarpin (6),30
withasomniferol B (7),31 (20R,22R,24S,25R)-4β,20β-dihydroxy-5β,6β-
epoxy-3β-methoxy-1- oxowithanolide (8),32 physalin D (9),33 physalin B
(10),34 physalin F (11),35 by comparing their 1H and 13C NMR data with
those reported in literatures.
Compound 1 was obtained as white powder. The molecular formula
was determined to be C29H44O8 by its HRESIMS at m/z 519.2973
[M − H]− (calcd for C29H43O8, 519.2963), indicating eight indices of
hydrogen deficiency. The 1H NMR spectrum (Table 1) of compound 1
showed five methyl groups at δH 1.76, 1.53, 1.21 (each 3H, s), 1.33 (3H,
d, J = 6.7 Hz) and 1.23 (3H, d, J = 6.7 Hz), and four proton signals on
oxygenated carbons at δH 3.37 (1H, br s), 3.91 (1H, s), 3.93 (1H, m) and
5.04 (1H, dd, J = 10.9, 3.5 Hz). The 13C NMR spectrum (Table 1)
involved 29 carbon signals including one ketone carbonyl signal at δC
210.0, one ester carbonyl signal at δC 176.3, five oxygen-bearing carbon
signals at δC 87.9, 80.3, 79.2, 78.4 and 75.1, and two carbon resonances
of a three-membered epoxy ring at δC 65.4 and 58.5. The above NMR
data revealed a typical withanolide of compound 1. Comparison of its
NMR data with those of the known philadelphicalactone A,36 showed
the absence of one Δ2,3 double bond (δH 6.42, 7.21; δC 132.4, 144.9) and
the presence of two saturated carbons (δH 3.31, 2.94; δC 41.5, 79.2) and
an extra methoxyl [δH 3.31 (3H, s); δC 57.1], suggesting the reduction of
Δ2,3 double bond and the linkage of a methoxyl on C-3 in 1. This sup­
position was confirmed by the detailed interpretation of its HMBC cor­
relations (Fig. 2) from H2-2 to C-1/C-3/C-4/C-6/C-10, H-4 to 3-OCH3/C-
2/C-3/C-5/C-10, H-3 to 3-OCH3/C-5, and 3-OCH3 to C-3. Based on
biosynthetic considerations and comparison with a single-crystal X-ray
diffraction (XRD) data of compound 5 (Fig. 4),20 the absolute configu­
rations of two stereogenic carbon centers C-24 and C-25 in the saturated
Y. Yang et al.
Fig. 1. Structures of compounds (1-11) from Physalis ixocarpa.
six-membered lactones were defined to be R and S. In the NOESY
spectrum (Fig. 3), CH3-21 was correlated with CH3-28, H-3 was corre­
lated with H-6, indicating an α-orientation for CH3-21 and H-3. The ECD
spectrum gave positive Cotton effect at 250 nm, demonstrating that C-22
was R configuration.32 Therefore, the structure of 1 was determined as
2,3-dihydro-3β-methoxyphiladephicalatone A. Compound 1 was hy­
pothesized to be intermolecular Michael-type addition adducts of polar
protic solvent nucleophiles like methanol.37
Compound 2, amorphous powder, possesses a molecular formula of
C36H56O12 based on its HRESIMS ([M+Na]+ m/z 703.3656) and 13C
NMR data, indicating nine indices of hydrogen deficiency. The 1H NMR
spectrum (Table 1) of compound 2 showed six methyl groups at δH 2.0,
1.53, 1.22, 1.04 (each 3H, s), 1.27 (3H, d, J = 6.6 Hz) and 1.20 (3H, d,
J = 6.6 Hz), one olefinic proton at δH 5.56 (1H, m), two proton signals on
oxygenated carbons at δH 5.12 (1H, m) and 4.03 (1H, m), and an
anomeric proton at δH 5.15 (1H, d, J = 7.7 Hz) on a sugar. The 13C NMR
spectrum (Table 1) of 2 showed 36 carbon signals, among them, δC 176.8
and 170.5 were two ester carbonyl signals, δC 137.9 and 124.8 belonged
to two olefinic carbon signals, δC 102.7, 79.0, 75.5, 72.1, 71.9 and 63.1
were assigned to a group of sugar signals, and δC 81.7, 78.9, 75.7, 75.3
and 73.1 were five oxygen-bearing carbon signals. Enzymatic hydrolysis
of 2 with snailase liberated d-glucose, which was identified by HPLC
with an optical rotation detector (HPLC-OR). In combination with the
coupling constant (7.7 Hz), the sugar moiety was determined as β-d-
glucose. The data of 2 were in accordance with those of (20R,22R)-lα-
acetoxy-14α,20β-dihydroxywitha-5,24-dienolide-3β-(O-β-D- glucopyr­
anoside),38 except for the absence of the Δ23,24 double bond signals (δC
148.9 and 122.0) and the presence of a saturated single bond signals (δC
41.3 and 32.6; δH 2.22 and 1.70), as well as the deshielded chemical shift
value of C-16 in 2 from δC 20.8 to δC 78.9, and the shielded chemical shift
value of C-14 from δC 84.7 to δC 54.8. The HMBC observed from H-14 to
C-13/C-16 and H-15 to C-13/C-14, demonstrated the linkage of a hy­
droxyl group on C-16. The HMBC correlations from CH3-27/CH3-28/H-
24/H-23 to C-25 and CH3-27/CH3-28 to C-24 further confirmed the
presence of single bond between C-24 and C-25. The anomeric proton H-
1′ correlated to C-3, and H-3 to the anomeric carbon C-1′ , corroborated
that the β-d-glucopyranosyl was linked at C-3 of the aglycone. In the
NOESY spectrum (Fig. 3), H-1 correlated to H-9/H-3, CH3-19 correlated
to H-16, CH3-27 correlated to CH3-21, demonstrating an α-orientation
5
Bioorganic & Medicinal Chemistry 36 (2021) 116095
for H-1/H-3, and a β-orientation for H-16/CH3-21. Therefore, the
structure of 1β-acetoxy-16α,20α-dihydroxywitha-5-en-26,22-olide-3β-
O-β-d-glucopyranoside was established.
Compound 3 was isolated as a white powder. Its molecular formula
was determined to be C36H56O12 according to positive HRESIMS at m/z
681.3814 ([M+H]+, C36H57O12, 681.3844), which indicated nine de­
grees of unsaturation. The NMR data (Table 1) of compound 3 were
closely similar to those of 2, except for the obvious differences of C-17
(δC 57.9) and C-20 (δC 78.4) in 3, but C-17 (δC 64.6) and C-20 (δC 75.7) in
2, demonstrating that 3 should be the epimer at C-16 of 2. The HMBC
correlations (Fig. 2) from H-17 to C-22/C-16/C-13/ C-21/C-18 further
supported the presence of OH-16. In the NOESY spectrum (Fig. 3), H-14
was correlated with H-16, indicating that H-16 was α-orientated, and
correspondingly OH-16 was β-orientated. Thus, the structure of 3 was
determined as 1β-acetoxy-16β,20α-dihydroxywitha-5-en-26,22-olide-
3β-O-β-d-glucopyranoside.
The formula of 4 was deduced as C42H66O16 via the HRESIMS ion at
m/z 849.4436 [M+H]+ (calcd for C42H67O16, 849.4423) and 13C NMR
data. Its NMR data (Table 1) are highly similar to those of the known
24,25-dihydrowithanolide VI,39 except for the appearance of an extra
acetoxy group [δC 172.2,δH 2.04(3H, s)] in 4. The HMBC correlations
from the methyl proton at δH 2.04 (3H, s) to the ester carbonyl signal at
δC 172.2, and H-1 to the ester carbonyl signal at δC 172.2/C-5/C-3
(Fig. 2), demonstrated that the acetoxy group was linked to C-1. The
1-acetoxy and OH-21 were deduced to be α- and β- oriented, respec­
tively, based on the NOESY correlations of H-1 with CH3-19 and of CH3-
21 with CH3-28. Thus, the structure of 4 was identified as 1α,3β,20β-
trihydroxy-witha-5-en-3-O-[β-d-glucopyranosyl(1 → 6)]-β-d-
glucopyranoside.
3.2. IDH1R132H enzyme inhibition assay in vitro
Recombinant IDH1 and IDH1R132H proteins were overexpressed and
purified, and enzyme reaction systems were constructed to screen all
these isolated compounds with potent inhibitory activity of IDH1R132H.
Ultimately, physalin B (10) and physalin F (11) were found to selectively
inhibit the activity of IDH1R132H in vitro with IC50 values of
1.26 ± 0.12 μM and 0.63 ± 0.12 μM, respectively. While, physalin B and
physalin F exhibited almost no inhibitory effect on wild-type IDH1, with
Y. Yang et al. Bioorganic & Medicinal Chemistry 36 (2021) 116095

Table 1
1
H-NMR and 13C-NMR spectroscopic data for compounds 1-4 (δ in ppm, J in Hz).
No 1a 2a,c 3a,c 4a,b

δC δH δC δH δC δH δC δH

1 210.0 75.3 5.35 br s 75.4 5.31 br s 76.6 5.07 br s

2 41.5 3.31 dd (15.3, 7.7) 34.7 2.61 m 34.8 2.54 m 34.8 2.17 o
2.94 dd (15.4, 3.5) 2.09 m 2.03 m 1.88 m
3 79.2 3.93 m 73.1 4.57 m 73.1 4.54 m 74.7 3.94 m
4 75.1 3.91 s 38.8 2.93 m 38.8 2.91 m 38.9 2.57 m
2.66 m 2.63 m 2.41 m
5 65.4 137.9 138.0 138.7 -
6 58.5 3.37 br s 124.8 5.56 m 124.8 5.52 m 125.2 5.57 m
7 30.3 1.69 m 32.3 1.40 m 32.1 1.86 m 32.8 2.04 o
1.26 m 1.57 m 1.74 m
8 32.1 2.29 m 31.2 1.53 o 32.1 1.66 m 32.3 1.59 o
9 43.5 1.44 m 42.9 1.53 o 42.9 1.49 o 43.8 1.36 o

10 51.6 41.1 41.3 41.9 -

11 21.9 1.64 m 20.8 1.61 o 20.9 1.55 m 21.5 1.21 o
1.50 m
12 34.2 3.06 m 41.0 2.14 m 40.9 2.14 m 41.4 2.17 o
2.16 m 1.67 m 1.16 m 1.23 m
13 49.5 44.9 44.1 43.7 -
14 51.3 2.06 m 54.8 54.9 0.79 m 55.8 0.94 m
15 24.6 1.25 m 37.9 1.92 m 38.4 2.27 m 38.3 2.29 m
1.61 m 1.39 m
16 32.9 2.21 m 78.9 4.03 m 78.9 3.99 m 21.4 1.42 m
1.92 m
17 87.9 64.6 1.86 d (5.8) 57.9 1.34 d (7.3) 58.5 1.36 o
18 15.8 1.21 s 15.4 1.22 s 15.6 1.44 s 15.4 1.20 s
19 15.9 1.76 s 19.7 1.04 s 19.8 0.98 s 19.9 1.16 s
20 78.4 75.7 78.4 79.3 -
21 21.1 1.53 s 21.3 1.53 s 22.2 1.49 s 21.3 1.33 s
22 80.3 5.04 dd (10.9, 3.5) 81.7 5.12 m 81.3 4.95 m 82.2 4.66 m
23 32.4 2.24 m 32.2 1.92 o 31.4 1.66 m 33.2 1.94 m
1.39 m 1.66 m
24 31.9 41.3 2.22 m 40.8 2.12 m 41.5 2.34 m
25 40.7 2.12 m 32.6 1.70 m 33.1 1.95 o 32.7 1.59 o
26 176.3 176.8 176.6 179.1
27 14.6 1.33 d (6.7) 21.4 1.20 d (6.6) 21.5 1.04 d (6.6) 14.6 1.22 d (6.2)
28 21.3 1.23 d (6.7) 14.8 1.27 d (6.6) 14.8 1.22 d (6.6) 17.9 1.30 d (6.2)
29 170.5 170.5 172.2
30 21.2 2.0 s 21.2 1.95 s 21.2 2.04 s
1′ 102.7 5.15 d (7.7) 102.7 5.12 d (7.7) 102.4 4.42 d (7.8)
2′ 75.5 4.36 m 75.7 3.97 m 75.4 3.21 m
3′ 79.0 4.14 m 79.0 4.11 m 76.9 3.48 m
4′ 71.9 5.25 m 72.1 4.29 m 72.6 3.86 m
5′ 72.1 4.35 m 73.4 4.82 m 74.2 3.33 m
6′ 63.1 4.60 m 63.1 4.57 m 70.8 3.99 m
4.45 m 4.39 m
1′ ′ 103.1 4.88 d (7.7)
2′ ′ 73.9 3.56 m
3′ ′ 79.8 3.38 m
4′ ′ 72.3 3.66 m
5′ ′ 76.8 4.57 m
6′ ′ 62.1 3.82 m
3.69 m
3-OCH3 57.1 3.31 s
a 1
H-NMR spectrum recorded at 600 MHz, 13C-NMR spectrum recorded at 150 MHz, in C5D5N.
b 1
H-NMR spectrum recorded at 600 MHz, 13C-NMR spectrum recorded at 150 MHz, in CD3OD.
c
“o” represents overlapped.

IC50 > 200 μM (Fig. 5A, B). physalin B. Therefore, physalin F was selected for further investigation.

3.3. Microscale thermophoresis (MST) assay
Microscale thermophoresis (MST) is a technology based on fluores­
cence detection and thermophoresis to assay interactions of protein–­
protein or protein-small molecule.40 To further validate the results of
enzyme activity assay, MST method was applied to evaluate the binding
affinity of IDH1R132H with physalin B and physalin F. The equilibrium
dissociation constant (Kd) of physalin B and physalin F were
45.30 ± 4.84 µM and 15.30 ± 2.12 µM, respectively (Fig. 5C), showing
that physalin F has a stronger binding affinity with IDH1R132H than
3.4. Physalin F inhibited HT1080 cell proliferation and induced HT1080
cell apoptosis
Cell viability was determined by CCK8 assay after treatment of cells
with different concentrations of physalin F. It presented conspicuous
growth inhibitory activity on HT1080 cells with an IC50 of
2.41 ± 0.04 μM (Fig. 6A). To further verify the above results, we per­
formed EdU and DAPI double staining assays on HT1080 cells to testify
the effect of physalin F on HT1080 cell proliferation. When HT1080 cells
were treated with different concentrations of physalin F, the number of

6
Y. Yang et al. Bioorganic & Medicinal Chemistry 36 (2021) 116095

Fig. 2. Key HMBC correlations of compounds 1-4.

Fig. 3. Key NOESY correlations of compounds 1-4.

EdU-positive cells showed a dramatically dose-dependent decrease
compared to the control group, elucidating that physalin F can strikingly
inhibit the proliferation of HT1080 cells (Fig. 6B). To validate whether
physalin F has a pro-apoptotic activity on HT1080 cells, cells treated
with 3.25 μM and 6.5 μM physalin F were stained by Hoechst 33258,
respectively. After treatment with physalin F, HT1080 cells exhibited
fragmentation and densely stained nuclei compared to the control group
(Fig. 6C). The activity of physalin F on apoptosis of HT1080 cells was
further testified by flow cytometry assay. 6.5 μM physalin F can induce
23.9% HT1080 cells to undergo apoptosis much higher than the control
group with DMSO (3.2%) (Fig. 6D).
3.5. Physalin F can selectively inhibit mutant IDH1 catalytic activity in
HT1080 cells
The hallmark of canceration of cells carrying mutation IDH1 is the
production and accumulation of a large amount of R-2-HG in cancer
cells and peripheral blood.41,42 Since IDH1R132H and IDH1R132C have the
same catalytic activity that reduces α-KG to R-2-HG by consuming
NADPH,9 the most common cell line HT1080 with IDH1R132C mutation
was selected to further investigate the biological activity of physalin F in
vitro. After HT1080 cells were administrated with different concentra­
tions of physalin F, the concentration of R-2-HG in the cell culture me­
dium was determined by LC-MS/MS to reflect the inhibition effect of
physalin F on the mutant IDH1. The results showed that physalin F was
able to dramatically reduce the concentration of R-2-HG in HT1080 cells
in a dose-dependent manner (Fig. 7A).

7
Y. Yang et al. Bioorganic & Medicinal Chemistry 36 (2021) 116095

Fig. 4. X-ray ORTEP drawing of compound 5.

Fig. 5. IDH1R132H inhibitory activity evaluation and binding affinity assay of physalin B and physalin F. (A) IC50 of physalin B against IDH1R132H and wild-type IDH1.
(B) IC50 of physalin F against IDH1R132H and wild-type IDH1. (C) Measurements of binding affinity of physalin B with IDH1R132H by MST, and the resulting binding
curve was shown. (D) Measurements of binding affinity of physalin F with IDH1R132H by MST, and the resulting binding curve was shown.

The level of histone hypermethylation and the accumulation of
hypoxia-inducible factor (HIF-1α) are the result of the massive produc­
tion of R-2-HG in IDH1 mutant cells, which can induce tumorigenesis,
invasion and metastasis to a certain extent.43,44 In our experiments, we
further evaluated the effect of physalin F on the level of histone
methylation and the content of HIF-1α in HT1080 cells when physalin F
inhibits the activity of the mutant IDH1 enzyme. Western blot analysis
and immunofluorescence staining confirmed that physalin F could
down-regulate the level of H3K9me3 in HT1080 cells (Fig. 7B). What’s
more, immunofluorescence staining results elucidated that physalin F
can dramatically reduce the accumulation of HIF-1α (Fig. 7C). These
results indicate that the histone hypermethylation and the accumulation
of HIF-1α are avoided due to physalin F reducing the amount of R-2-HG
in HT1080 cells.
The high expression of R-2-HG activates the expression of miR-380-
5p by stabilizing the accumulation of HIF-2α. MiR-380-5p is a charac­
terized microRNA against p53 expression.45 Therefore, the effect of
physalin F on the p53 expression was investigated in our research.
Immunofluorescence staining results indicated that physalin F rescued
p53 expression (Fig. 7D).
3.6. Knockdown of IDH1R132C weakens the inhibitory activity of physalin
F on HT1080 cell growth
To investigate the role of IDH1R132C in inhibition of physalin F on
cancer cell viability, transfection of HT1080 cells with adenovirus to
knock down the expression of endogenous IDH1R132C, and the highest
fluorescence was achieved after transfection for 24 h (Fig. 8A). The

8
Y. Yang et al. Bioorganic & Medicinal Chemistry 36 (2021) 116095

Fig. 6. Effect of physalin F on proliferation and apoptosis of HT1080 cells. (A) The inhibition effect of physalin F on cell viability of HT1080 was determined by
CCK8. (B) The effects of different concentrations of physalin F on the proliferation of HT1080 cells were investigated by EdU and DAPI double staining. ***P < 0.001
vs control group. (C) Physalin F induced apoptotic effect on HT1080 cells by Hoechst 33258 staining and fluorescence microscope observation (×200). Bar = 20 μm.
(D) HT1080 cells were treated with physalin F, double stained with Annexin V-FITC/PI, and then assayed by flow cytometry.

Fig. 7. Physalin F can selectively inhibit mutant IDH1 catalytic activity in HT1080 cells. (A) After physalin F treatment, the production of R-2-HG in HT1080 cells
was down-regulated with increasing concentrations of physalin F using LC-MS/MS. (B) Physalin F can dramatically reduce the level of H3K9 methylation in HT1080
cells. ***P < 0.001 vs control group. (C) Immunofluorescence staining was used to determine that physalin F significantly abolished HIF-1α accumulation in HT1080
cells. (D) Physalin F was able to up-regulate the expression of p53 protein.

expression of IDH1R132C was significantly decreased in HT1080 cells
transfected with shIDH1R132C compared to the control group shMock
(Fig. 8B, C). Cell viability was determined by CCK8 after HT1080 cells
transfected with shMock or shIDH1R132C were administrated with
different concentrations of physalin F, respectively. The results showed
that knockdown of IDH1R132C expression decreased the inhibition
sensitivity of physalin F to HT1080 cells (Fig. 8D), further confirming
that the anti-tumor activity of physalin F was dependent on the
expression of IDH1R132C.
3.7. Molecular docking analysis of the interaction mode of physalin F and
IDH1R132H
According to the evaluation of enzyme activity, binding affinity and
cell-based inhibition activity of physalin F, it has been known that there
is a strong interaction between physalin F and IDH1R132H and this
interaction selectively inhibits the catalytic activity of IDH1R132H. The
interaction between physalin F and IDH1R132H was visualized through
molecular docking to facilitate the understanding of its molecular

9
Y. Yang et al.
Fig. 8. After knocking down the expression of IDH1R132H, the effect of physalin F on cell viability of HT1080 was significantly decreased. (A) Adenovirus (diluted
100-fold) infected HT1080 cells for 24 h to obtain the strongest fluorescence. (B, C) HT1080 cells were transfected with shMOCK and shIDH1, respectively, and
Western blot was used to detect mutant IDH1 expression. (D) Compared with shMOCK cells, physalin F had less effect on cell growth of shIDH1-transfected
HT1080 cells.
mechanism. The lowest-energy binding conformation of physalin F was
shown in Fig. 9A and B. IDH1R132H allosteric pocket is a highly hydro­
phobic envelop formed from abundant hydrophobic amino acid resi­
dues.46 The docking results indicate that physalin F forms strong
hydrophobic interactions with several amino acids in hydrophobic
envelop, including Ala111, Leu120, Ile130, Trp267, Val281, Tyr285,
and Met291. At the same time, hydrogen bonds with Ile128, Ser278, and
Ser287, and π-σ interaction with Try124 are formed between physalin F
and IDH1R132H (Fig. 9A, B). α10 (residues 271–286, also called “regu­
latory segment”), a key α helix segment of IDH1R132H, is also part of the
allosteric pocket. The conformational change of α10 is directly related to
the enzyme catalytic activity.1 In our study, physalin F can interact with
some amino acid residues of α10, thereby preventing its conformational
change. This interaction locks IDH1R132H in a catalytic inactive confor­
mation. This may be a potential molecular mechanism by which phys­
alin F inhibits the enzyme activity of IDH1R132H.
In order to better understand the stronger selectivity of physalin F for
IDH1R132H compared to wild-type IDH1, we also docked physalin F with
wild-type IDH1 and set a binding pocket in the same position as
IDH1R132H for docking. Due to the strong steric hindrance, physalin F
cannot enter the allosteric pocket. We carried out a protein conforma­
tion superposition of wild-type IDH1 and IDH1R132H, and found that the
IDH1R132H allosteric pocket has a very spacious “door” that allows
physalin F to enter the allosteric pocket compared to wild-type IDH1
(Fig. 9C). Regulatory segment α10 in wild-type IDH1 blocked the
binding site of physalin F, and the same situation also appeared in the
study of X. Xie et al.47 Mutation of Arg132 to His132 disrupts the
interaction between Arg132 and Asn271 resulting in a significant in­
crease in the flexibility of α10,48 which is necessary for physalin F to
enter the allosteric pocket.
In summary, 11 type-A withanolides including 4 new ones (1 is an
artificial withanolide) were obtained from the dietary tomatillo (Physalis
ixocarpa). All these isolated withanolides were evaluated for their in­
hibition on mutant IDH1 enzyme activity. And physalin F, as a major
10
Bioorganic & Medicinal Chemistry 36 (2021) 116095
type-A withanolide isolated from Physalis ixocarpa, exhibited the most
potent selective inhibition on the activity of mutant IDH1 in vitro. The
binding affinity between physalin F and mutant IDH1 was verified by
MST quantitative analysis. Physalin F inhibits HT1080 cell proliferation
and induces cell apoptosis. Physalin F can reduce the level of histone
methylation and block the accumulation of HIF-1α by selectively
reducing the concentration of R-2-HG in HT1080 cells. In addition, the
targeting effect of physalin F on mutant IDH1 was verified by shRNA
knockdown of mutant IDH1 expression in HT1080 cells. Because the
expression of mutant IDH1 didn’t be completely knocked out, physalin F
can still inhibit the remaining mutant IDH1 to reduce cell viability. p53
protein expression was up-regulated by physalin F, which may be
another reason for the decreased cell viability. However, the cell
viability of the shIDH1 group was still higher than the control group,
implying that physalin F could selectively target at mutant IDH1. Mo­
lecular docking indicated that physalin F can bind to allosteric sites to
prevent the conformational change of α10, further locking IDH1R132H in
the catalytic inactive conformation. All of these data indicate that
physalin F might act as a mutant IDH1 inhibitor with good efficacy and
selectivity in tumor therapy. Therefore, tomatillo could be used as po­
tential antitumor functional food or drug through the synergistic effect
of type-A and type-B withanolides acting on different targets of energy
metabolism. Previous studies have reported that Licochalcone A from
Glycyrrhiza rhizome48 and cyathisterol from fruit body of Ganoderma
sinense49 can also be used as inhibitors of mutant IDH1 to inhibit the
proliferation of HT1080 cells. The structural diversity and richness of
natural products make them as an important source for the discovery of
potential targeted anti-tumor drugs.
Further in vivo experiments to verify the anti-tumor effect of physalin
F and explore the underlying action mechanism will be carried out in our
future study. Reasonable design of compounds with higher selectivity
using physalin F as the lead compound base on the interaction of
physalin F and active pocket of mutant IDH1 could also attract great
interest of medicinal chemists.
Y. Yang et al. Bioorganic & Medicinal Chemistry 36 (2021) 116095

Fig. 9. Visual analysis of the interaction mode between physalin F and IDH1R132H by molecular docking. (A) Detailed view of the interaction between physalin F and
the allosteric pocket amino acid residues of IDH1R12H. These pictures were plotted with Discovery Studio 2017 R2 (BIOVIA, USA). (B) Ligand interaction diagram of
physalin F with IDHR132H. (C) Wild-type IDH1 and IDH1R132H protein conformations are superimposed. Wild-type IDH1 was displayed as cartoon mode in pink, and
IDH1R132H was displayed as cartoon mode in gray. Amino acid residues (residues 120-134, 250-263, 270-291) around the wild-type IDH1 allosteric pocket are
marked in green and amino acid residues (residues 120-134, 250-263, 277-291) around the IDH1R132H allosteric pocket are marked in blue.

Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116095.
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