Discovery of a series of 5-phenyl-2-furan derivatives containing 1,3-thiazole moiety as potent Escherichia coli β-glucuronidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.105306

Gut microbial β-glucuronidases have drawn much attention due to their role as a potential therapeutic target to alleviate some drugs or their metabolites-induced gastrointestinal toxicity. In this study, fifteen 5-phenyl-2-furan derivatives containing 1,3

Full text of DOI:10.1016/j.bioorg.2021.105306

Bioorganic Chemistry 116 (2021) 105306
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Bioorganic Chemistry
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Discovery of a series of 5-phenyl-2-furan derivatives containing 1,3-thiazole
moiety as potent Escherichia coli β-glucuronidase inhibitors
,
,
,
Tao-Shun Zhoua 1, Lu-Lu Heb 1, Jing Hea, Zhi-Kun Yanga e, Zhen-Yi Zhoua, Ao-Qi Dua,
Jin-Biao Yua, Ya-Sheng Lia, Si-Jia Wanga c, Bin Weia *, Zi-Ning Cuib *, Hong Wanga
,
,
d
,
,
,d,*
a College of Pharmaceutical Science & Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology,
Hangzhou 310014, China
b State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Lingnan Guangdong Laboratory of Modern Agriculture, Integrative
Microbiology Research Centre, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642,
China
c Center for Human Nutrition, David Geffen School of Medicine, University of California, Rehabilitation Building 32-21, 1000 Veteran Avenue, Los Angeles, CA 90024,
USA
d Key Laboratory of Marine Fishery Resources Exploitment & Utilization of Zhejiang Province, Hangzhou 310014, China
e Innovation Center of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
A R T I C L E I N F O
A B S T R A C T
Keyword:
Gut microbial β-glucuronidases have drawn much attention due to their role as a potential therapeutic target to
alleviate some drugs or their metabolites-induced gastrointestinal toxicity. In this study, fifteen 5-phenyl-2-furan
derivatives containing 1,3-thiazole moiety (1–15) were synthesized and evaluated for their inhibitory effects
against Escherichia coli β-glucuronidase (EcGUS). Twelve of them showed satisfactory inhibition against EcGUS
5-phenyl-2-furan derivatives
1,3-thiazole moiety
β-glucuronidase inhibitors
Docking
with IC50 values ranging from 0.25
μ
M to 2.13
μ
M with compound 12 exhibited the best inhibition. Inhibition
M) was an uncompetitive inhibitor for EcGUS
Structure-inhibitory activity relationship
kinetics studies indicated that compound 12 (Ki = 0.14 ± 0.01
μ
and molecular docking simulation further predicted the binding model and capability of compound 12 with
EcGUS. A preliminary structure-inhibitory activity relationship study revealed that the heterocyclic backbone
and bromine substitution of benzene may be essential for inhibition against EcGUS. The compounds have the
potential to be applied in drug-induced gastrointestinal toxicity and the findings would help researchers to design
and develop more effective 5-phenyl-2-furan type EcGUS inhibitors.
1. Introduction
source. This process leads to the accumulation of the active metabolite
SN-38 in the intestinal lumen, thereby resulting in severe diarrhea and
β-Glucuronidase (EC 3.2.1.31) is a kind of glycosidase that can hy-
drolyze β-linked glucuronides of complex glycosides [1–3]. In the
gastrointestinal tract, many bacteria secret β-glucuronidases, including
Salmonella, Klebsiella, Yersinia, Shigella, and especially Escherichia [4].
Extensive studies have confirmed that gut microbial β-glucuronidases
play key roles in the gastrointestinal toxicity caused by some drugs or
their metabolites. For example, CPT-11 (irinotecan) is a clinically
commonly used chemotherapeutic drug for the treatment of various
cancers [5–7]. When SN-38 glucuronide (SN-38G), the inactive metab-
olite of CPT-11, was excreted into the intestinal lumen, gut bacteria can
catalyze the hydrolysis of SN-38G and obtain aglycone as a carbon
mucosal damage [8–10]. Many carboxylic acid-containing nonsteroidal
anti-inflammatory drugs (NSAIDs) also have similar metabolic path-
ways, thus causing serious lower gastrointestinal ulcers or gastrointes-
tinal bleeding and anemia [11–13]. The side effects directly restrict the
clinical dose intensification and efficacy of CPT-11 or NSAIDs.
Since the mechanism mentioned above has been accepted for de-
cades, the inhibition of gut bacteria β-glucuronidase activity is a
reasonable strategy to alleviate intestinal toxicity causing by CPT-11 or
NSAIDs [14]. Escherichia coli is ubiquitous in the intestinal environment
and EcGUS has been widely recognized as the culprit to chemotherapy-
induced diarrhea and colorectal carcinogenesis [9,15]. So far, various
* Corresponding authors at: College of Pharmaceutical Science & Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang
University of Technology, Hangzhou 310014, China (B. Wei and H. Wang).
E-mail addresses: binwei@zjut.edu.cn (B. Wei), zinningcui@scau.edu.cn (Z.-N. Cui), hongw@zjut.edu.cn (H. Wang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.105306
Received 9 June 2021; Received in revised form 16 August 2021; Accepted 24 August 2021
Available online 27 August 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

T.-S. Zhou et al.
Bioorganic Chemistry 116 (2021) 105306
natural and synthetic EcGUS inhibitors have been identified and re-
ported. For example, our previous studies found that sanggenon C and
kuwanon G exhibited strong mixed-type inhibition against EcGUS and
caffeic acid ethyl ester acted as a potent competitive inhibitor for EcGUS
[16,17]. Furthermore, synthetic compounds account for a much larger
percentage of reported EcGUS inhibitors. For example, our research
group has already reported that thirteen thiazolidin-2-cyanamide de-
rivatives (Fig. 1) and thirty 1,3,4-thiadiazole derivatives (Fig. 1) were
potent EcGUS inhibitors and the IC50 value of the most active inhibitor is
Table 1
The inhibitory activity against EcGUS of AMX, DSL, and compounds 1-15a.
No
R
Molecular weight (Da)
Inhibition rate 10
μM
IC50 (μM)
1
2-Cl
321.79
321.79
321.79
305.34
305.34
305.34
323.33
323.33
332.35
332.35
332.35
366.25
301.38
317.38
287.35
313.78
210.14
88.8 ± 0.7%
25.0 ± 3.8%
89.7 ± 1.1%
22.0 ± 0.4%
88.4 ± 0.3%
88.3 ± 0.5%
89.3 ± 0.3%
63.0 ± 0.5%
85.7 ± 1.8%
25.4 ± 1.3%
86.7 ± 2.7%
90.2 0.9%
89.5 ± 1.2%
87.9 ± 1.3%
88.7 ± 0.8%
98.5 ± 1.2%
13.3 ± 1.5%
0.33 ± 0.03
2
3-Cl
NDb
3
4-Cl
0.32 ± 0.01
NDb
4
2-F
5
3-F
0.63 ± 0.05
1.36 ± 0.02
0.30 ± 0.01
2.13 ± 0.10
0.31 ± 0.01
NDb
6
4-F
7
2,4-di-F
2,6-di-F
2-NO2
3-NO2
4-NO2
4-Br
up to 0.082 μM [18,19]. Remarkably, these above synthetic compounds
8
contain 5-phenyl-2-furan and thiazole moieties which can easily form
intermolecular hydrogen bonds with different kinds of biological en-
zymes [20,21]. Therefore, those heterocyclic compounds containing
atoms like oxygen, nitrogen, sulfur always become a hot-spot in the eyes
of chemists due to their broad-spectrum biological activity [22,23].
Based on our previous study, fifteen 5-phenyl-2-furan derivatives
containing 1,3-thiazole moiety were selected for the screening of potent
EcGUS inhibitors. Then the inhibitory behavior and structure-inhibitory
activity relationships (SAR) of the compounds against EcGUS were
further characterized. Finally, a molecular docking study was performed
to visualize the molecular determinants of EcGUS inhibition.
9
10
11
12
13
14
15
AMX
DSL
0.36 ± 0.03
0.25 0.02
0.51 ± 0.02
0.64 ± 0.04
0.67 ± 0.02
0.10 ± 0.004
65.69 ± 6.01
4-Me
4-OMe
H
a all data were determined in three independent reactions.
b ND means not determined.
μ
M than DSL (IC50 = 65.69 ± 6.01
found to have the most potent inhibitory effect (IC50 = 0.25 ± 0.02
and compound 8 exhibited the worst inhibitory effect (IC50 = 2.13 ±
0.10 M).
μ
M). Among them, compound 12 was
μM),
2. Results
μ
2.1. Screening of potent EcGUS inhibitor
The inhibitory potency of the fifteen 5-phenyl-2-furan derivatives
containing 1,3-thiazole moiety (Fig. 1) was determined and shown
(Table 1). DSL (Fig. 1) and AMX (Fig. 1) were used as positive controls.
These fifteen test compounds had stronger inhibitory activity than DSL
(13.3 ± 1.5%) but the activity was weaker than AMX (98.5 ± 1.2%) at
2.3. SAR of test compounds against EcGUS
As shown in Table 1, the test compounds 1–15 have different
substituted phenyl moieties and demonstrated varied inhibitory effects
against EcGUS. Compounds bearing a chloro- or nitro-substituted phenyl
moiety, 1 (ortho-chloro), 3 (para-chloro), 9 (ortho-nitro), and 11 (para-
10 μM. Three compounds showed weak inhibition (2: 25.0 ± 3.8%; 4:
22.0 ± 0.4%; 10: 25.4 ± 1.3%) against EcGUS with the inhibition rate
less than 50% and the rest compounds showed much potent inhibitory
effects (63.0%-90.2%) with compound 12 showing the highest inhibi-
tory potency (90.2 ± 0.9%).
nitro) shared the equal value of IC50 (about 0.3 μM) and they appeared
more active than compounds 2 (meta-chloro) and 10 (meta-nitro),
indicating that chlorine and nitro substitutions made the same inhibi-
tory difference against EcGUS and meta substitutions of them decreased
the inhibitory activity. Interestingly, when ranked the IC50 values of
compounds 4, 5, 6, 7, and 8, we could find that meta-fluorine substi-
tution is more prominent than ortho- or para-fluorine substitutions on
the phenyl moiety for EcGUS inhibition, and fluorine substitution at the
4- or 6-position of the phenyl moiety of compound 4 is beneficial for
EcGUS inhibition and the former is more favorable than the latter.
Moreover, there were many subtle relationships about the substitution
2.2. IC50 values of test compounds against EcGUS
Twelve compounds were selected to further assess the IC50 values
(the concentration of the inhibitor to show half-maximal inhibition).
Fig. 2A, B, and Table 1 illustrated that the twelve compounds displayed
more potent inhibitory effects with IC50 values range from 0.25 to 2.13
Fig. 1. Structures of representative inhibitors and test compounds.
2

T.-S. Zhou et al.
Bioorganic Chemistry 116 (2021) 105306
Fig. 2. The dose-dependent inhibition curves of inhibitors on PNPG-hydrolyzing activity of EcGUS. (A) Compounds 1–7 and two positive controls against EcGUS; (B)
Compounds 8–15 against EcGUS. All data were expressed as mean ± standard deviation of triplicate reactions.
of benzene that could be discovered. For example, by comparing analogs
with a para-substituted phenyl moiety, including compounds 3 (IC50
0.32 ± 0.01 M), 6 (IC50 = 1.36 ± 0.02 M), 11 (IC50 = 0.36 ± 0.03 M),
12 (IC50 = 0.25 ± 0.02 M), 13 (IC50 = 0.51 ± 0.02 M), 14 (IC50 = 0.64
± 0.04 M) and 15 (IC50 = 0.67 ± 0.02 M), the relationships against
determinants of compound 12 in inhibiting EcGUS were explored by
molecular docking simulation. The detailed binding modes of DSL, AMX,
and compound 12 with the active pocket of EcGUS were plotted in
Fig. 4. According to relevant reports that the active pocket of EcGUS
includes two pivotal residues Glu413, Glu504, and two adjacent
monomeric bacterial loops (Ser360-Glu367)[14]. From the 2D figures,
we could find that DSL formed hydrogen bond interactions in the B chain
with Arg562 (2.11 Å), Glu413 (2.13 Å), Glu504 (2.13 Å), Asn566 (2.22
Å), Trp549 (2.55 Å), and Lys 568 (2.51 Å) (Fig. 4A) In addition to
generating hydrogen bonds with key residues Glu504 (2.00 Å) and
Glu413 (2.14 Å), AMX also have strong hydrophobic interactions with
Tyr472 and Leu361 in the B chain (Fig. 4B). Similarly, compound 12
was found to have hydrogen interactions with Arg562 (2.63 Å) and
Glu413 (3.54 Å), and also developed hydrophobic interactions with
Phe448, Tyr472, Leu361 in B chain and Phe365 from A chain (Fig. 4C).
Moreover, compound 12 could be well docked to the active pocket
by hydrogen bonds (sulfur substituents of thizole) and Van der Waals
(benzene and furan rings) interactions with the bacterial loops (Fig. 4F),
and AMX shared a similar docking pose because of carrying aromatic
structures (Fig. 4E). while DSL barely interacted with residues of the
bacterial loops (Fig. 4D), which may account for its corresponding weak
inhibition of EcGUS, although it developed more hydrogen bonding
interactions with EcGUS than compound 12 and AMX.
=
μ
μ
μ
μ
μ
μ
μ
EcGUS can be listed as follows: Br > Cl ≈ NO2 > Me > OMe ≈ H > F. It
also could be summarized that fluorine substitution at the para position
of the phenyl moiety are adverse for EcGUS inhibition and bromine,
chlorine, nitro, and methyl substitutions are advantageous.
2.4. Inhibition behaviors of compound 12 against EcGUS
The inhibition behaviors of compound 12 and two controls were
further explored with different concentrations of inhibitor and substrate.
As shown in Fig. 3, compound 12 and AMX were uncompetitive in-
hibitors for EcGUS with Ki values of 0.14 ± 0.01 μM and 0.09 ± 0.01 μM,
respectively. DSL was identified as a mix-type inhibitor for EcGUS (Ki =
32.16 ± 5.28 μM). These results imply that similar to AMX, compound
12 only binds to the enzyme-substrate complex of EcGUS and PNPG.
2.5. Molecular docking simulation
As mentioned above, compound 12 showed superior inhibitory ac-
tivity against EcGUS among these test compounds, and the molecular
Fig. 3. The Lineweaver-Burk plots of (A) DSL, (B) AMX, (C) Compound 12 against EcGUS. All data were expressed as mean ± standard deviation of tripli-
cate reactions.
3

T.-S. Zhou et al.
Bioorganic Chemistry 116 (2021) 105306
Fig. 4. Ligand interactions of (A) DSL, (B) AMX, and (C) compound 12 with EcGUS. The binding modes of (D) DSL (orange), (E) AMX (magenta), and (F) compound
12 (green) in the active site of EcGUS, respectively. The dark green belts represent the bacterial loops (Ser360-Glu367). The A and B chains are from the upper and
lower adjacent monomers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Discussion
benzene ring and nitrogen or oxygen heterocycle, and showed excellent
EcGUS inhibitory activity. The in vivo potency of compound 12 is
promising and needs further study.
Gut bacterial β-glucuronidases have been proven to play crucial roles
in intestinal toxicity induced by CPT-11/NSAIDs. According to relevant
researches, a growing number of intractable diseases have been reported
to be inextricable with gut bacterial β-glucuronidases, the over-
expression of which is a trigger of several pathologies such as cancer
[24] and inflammation in joints [25]. Thiazole derivatives were
frequently used to screen for β-glucuronidase inhibitors in recent
studies. For example, Khan et al synthesized twenty-eight thiazole de-
rivatives (Fig. 1) and found that four analogs showed single-digit IC50
values as β-glucuronidase inhibitors [26]. Taha et al. screened a series of
oxadiazole coupled-thiadiazole derivatives and found the most active
Although many gut microbes secret β-glucuronidases, there is no
direct evidence that β-glucuronidases from other gut microbes play
essential roles in the toxicity of CPT-11. Therefore, in the present study,
we only studied the inhibitory activity of the 5-phenyl-2-furan de-
rivatives on EcGUS. Moreover, some reports indicated that CPT-11 can
induce shifts in gut microbial composition and influence the abundance
of β-glucuronidase in other gut bacterial phyla [30–32]. The inhibitory
potential of these derivatives against β-glucuronidases from other gut
bacterial isolates warrants further investigation, which would be helpful
to develop drug leads with better in vivo effects in alleviating CPT-11-
induced toxicity.
candidate 26 with an IC50 value of 0.96 μM [27]. In the present study,
our previously synthesized 5-phenyl-2-furan derivatives containing 1,3-
thiazole moiety were screen for EcGUS inhibitors. Twelve out of fifteen
compounds showed more potent inhibitory effects against EcGUS than a
commonly used β-glucuronidase inhibitor, DSL, with IC50 values ranging
In our recent report, we found that substitutions on the phenyl ring of
5-phenyl-2-furan derivatives containing 1,3,4-thiadiazole moiety
significantly affect the inhibitory effects of test compounds against
EcGUS and meta-fluoro-substitution on the phenyl ring (IC50 = 0.082
from 0.25
μ
M to 2.13
μM. Inhibition behavior analysis indicated that
μM) was more favorable than other tested substituent groups [19].
compound 12 was an uncompetitive EcGUS inhibitor with a Ki value of
0.14 M and SAR analysis suggested that bromine substitution at the
Therefore, in the present study, we mainly investigated the inhibitory
effects of a series of 5-phenyl-2-furan derivatives with different sub-
stitutions on the phenyl ring and found that para-bromo-substitution on
the phenyl ring was most beneficial for EcGUS inhibition among all
substitution groups tested. Comparing these two studies we can find a
common characteristic for 5-phenyl-2-furan derivatives containing
thiadiazole moiety, in terms of fluorine substitution, meta-substitution
favors ortho- and para-substitution for EcGUS inhibition. The findings
further deepen our understanding about the effects of the phenyl sub-
stitution of these derivatives on EcGUS inhibition. However, sub-
stitutions on the 1,3-thiazole moiety may also affect their inhibitory
activity against EcGUS, which need further investigation.
μ
para position of the phenyl moiety is most helpful for EcGUS inhibition
among these compounds. Besides, molecular docking simulation also
demonstrated that compound 12 formed more hydrophobic interactions
with the bacterial loops, which is more favorable for EcGUS inhibition.
Since the crystal structure was determined in 2010, EcGUS has
become a frequently-used target for screening β-glucuronidase in-
hibitors. For instance, Ahmad’s research group firstly found that three
old-drugs (nialamide, isocarboxazid, and AMX) showed high cell-free
inhibition (IC50: 71, 128, and 388 nM, respectively) with no signifi-
cant activity against purified mammalian β-glucuronidase [28]. After
two years, the less toxic drug AMX was designed to use along with CPT-
11 to treat tumor-bearing mice and the results revealed that AMX ach-
ieved the significant anti-diarrhea effect and suppression of tumor
growth [29]. Compound 12 shares similar moieties with AMX, including
In conclusion, twelve 5-phenyl-2-furan derivatives containing 1,3-
thiazole moiety are identified as potent EcGUS inhibitors with IC50
values ranging from 0.25 μM to 2.13 μM, which have the potential to be
developed as prodrugs to alleviate the toxicity induced by CPT-11.
4

T.-S. Zhou et al.
Bioorganic Chemistry 116 (2021) 105306
Preliminary structure-inhibitory activity relationship study indicates
that the heterocyclic structure and bromine substitution at the C4-
position of the phenyl moiety are beneficial for EcGUS inhibition,
which helps develop more powerful EcGUS inhibitors from 5-phenyl-2-
furan derivatives.
16 ◦C to induce the expression of β-glucuronidase. Thirdly, the cells were
collected by centrifugation at 5000g for 20 min at 4 ℃ and suspended
using PBS buffer (pH 7.4) and then sonicated for 5 min and 4 cycles in an
ice bath. Fourthly, the crude EcGUS solution was loaded onto a Ni-NTA
column (GE Healthcare Biosciences UK Ltd.) eluting with 0, 20, 250 mM
imidazole, and eluants of different concentrations were analyzed for
β-glucuronidase activity using PNPG as the substrate. Finally, these el-
uants showing marked activity were combined and concentrated by
ultrafiltration (Amicon Ultra 50 kDa, Merck Millipore, Darmstadt, Ger-
many). Protein concentration was measured using the BCA Protein
Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s
instruction and purity was determined by SDS-PAGE.
4. Experimental
4.1. Materials
Fifteen novel 5-phenyl-2-furan derivatives containing 1,3-thiazole
moiety were synthesized in the Prof. Zi-Ning Cui research group from
South China Agricultural University (Guangzhou, China). Recombinant
E. coli BL21 (DE3) was kindly provided by Prof. Ru Yan from the Uni-
versity of Macau (Macau, China). p-Nitrophenyl-β-D-glucuronide acid
(PNPG), D-saccharic acid-1,4-lactone (DSL), amoxapine (AMX), and
dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA). Dul-
becco’s phosphate-buffered saline (PBS) (Life Technologies, Carlsbad,
CA, USA). Imidazole, kanamycin, and isopropyl β-D-1-thiogalactopyr-
anoside (IPTG) (Biosharp, Hefei, China). Deionized water was obtained
by a Milli-Q purification system (Millipore, Bedford, MA, USA). Purities
of agents were >98%.
4.4. β-Glucuronidase inhibition assay in vitro
The method of β-glucuronidase inhibition assay in vitro had been
mentioned in our previous studies [16,18]. Briefly, the assays were
performed in 96-well plates (Nunc, Denmark) and every well with a total
volume of 100
mL), 10
L PNPG (final concentration, 250
μ
L including 10
μ
L EcGUS (final concentration, 2.0
M), 10
L PBS buffer (pH 7.4).
μg/
μ
L test compound or DSL/AMX (final concentration, 10
μ
μ
μ
M) and 70
μ
All reactions were performed in triplicate and the β-glucuronidase ac-
tivity was measured by detecting the formation of PNP at OD405 nm
after incubating at 37 ◦C for 30 min. The PNP concentration was further
determined by setting a standard curve (0, 10, 20, 40, 60, 80, 100, and
4.2. General synthetic procedure
The synthetic route of test compounds was shown in Fig. 5, which
was similar to a previous report [22] with a minor modification. Firstly,
the intermediate I was synthesized with substituted aniline and furoic
acid by Meerwein arylation reaction [33,34]. Secondly, at room tem-
perature, chloracetaldehyde, and hydrochloric acid were added to a
round-bottomed flask to react for 2 h, then the above reaction solution
was stirred and mixed with ammonium dithiocarbamate for 1 h, and
then increasing the reaction temperature to 74 ℃, the mixture reacted to
produce the intermediate II for 3 h. Thirdly, the intermediate I and
dichlorosulfoxide were stirred and refluxed in anhydrous toluene at
80 ◦C for 3 h to afford the 5-phenyl-2-furancarbonyl chloride, which was
added to a mixture of acetonitrile, triethylamine, 4-dimethylaminopyr-
idine, and the intermediate II to obtain the final compounds by stir-
120 μM PNP) in PBS buffer (pH 7.4). The relative activity of experi-
mental groups was calculated by comparing the generated PNP con-
centration with the blank control group.
To further determine IC50 values of these compounds against EcGUS
in vitro, the reaction conditions are as follows: 10 μL EcGUS (final con-
centration, 2.0
or DSL/AMX (various final concentration, 0.001–5000
PNPG (final concentration, 250
μg/mL), 70 μL PBS buffer (pH 7.4), 10 μL test compound
μ
M) and 10
μL
◦
μM) reacting for 30 min at 37 C. Be-
sides, the inhibition behavior of the selected inhibitor toward EcGUS
also was investigated by measuring the reaction rates with substrates
(0.2, 0.3, 0.5, and 1.0 mM) and inhibitors (Fig. 3) in various concen-
trations. Finally, the type of inhibition was determined and IC50 and
inhibition constant (Ki) values were calculated based on our previous
methods [16]. The calculated formulas about Ki were listed below
including the Eq. (1) for uncompetitive inhibition and Eq. (2) for mixed
inhibition.
1
ring overnight at room temperature. Spectroscopic data and H NMR,
13C NMR of fifteen compounds were provided in Supplementary
Materials.
(Vmax ꢀ v)/v = Km/[S] + [I]/K’
(1)
(2)
4.3. EcGUS preparation
i
EcGUS was prepared based on our previous study with a minor
change [18,35]. Firstly, Recombinant E. coli BL21 (DE3) was incubated
in a 200 mL LB liquid medium containing 1% kanamycin at 220 rpm and
(Vmax ꢀ v)/v = Km/[S] + (Km/Ki[S] + [I]/K’i)[I]
where [S] is the substrate concentration, [I] is the inhibitor concentra-
tion, Ki is the inhibition constant and Km is the substrate concentration at
half of the maximum velocity (Vmax) of reaction. (1)/K’i of the Eq. (1) is
◦
37 C until OD600 reached 0.5–0.6. Secondly, IPTG was added to 0.5
mM and the medium was further incubated for 16 h at 220 rpm and
Fig. 5. General synthetic procedure for test compounds 1–15.
5

T.-S. Zhou et al.
Bioorganic Chemistry 116 (2021) 105306
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previous study [18]. The X-ray crystal structure of EcGUS (PDB code
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Ethical statements
This research did not include any human subjects.
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
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Acknowledgments
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This work was financially supported by the National Key Research
and Development Program of China (2017YFE0103100), the programs
of the National Natural Science Foundation of China (No. 32072450, No.
81773628, and No. 81741165), the High-Level Talent Special Support
Plan of Zhejiang Province (No. 2019R52009), the National 111 Project
(No. D17012), the International Science and Technology Cooperation
Program in Guangdong (2020A0505100048) and the National Science
Fund for Distinguished Young Scholars of Guangdong Province
(2021B1515020107).
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