
==== Front
J Enzyme Inhib Med Chem
J Enzyme Inhib Med Chem
Journal of Enzyme Inhibition and Medicinal Chemistry
1475-6366
1475-6374
Taylor & Francis

39223707
10.1080/14756366.2024.2398561
2398561
Version of Record
Research Article
Research Article
Discovery of natural anthraquinones as potent inhibitors against pancreatic lipase: structure-activity relationships and inhibitory mechanism
Z.-Q. Chen et al.
Chen Zi-Qiang ab*
He Wen-Yao b*
Yang Si-Yuan c
Ma Hong-Hong d
Zhou Jing a
Li Hao b
Zhu Ya-Di de
Qian Xing-Kai ac
Zou Li-Wei b
a Translational Medicine Research Center, Guizhou Medical University, Guiyang, Guizhou, China
b Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China
c Department of Cardiac Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, China
d School of Basic Medicine, Guizhou Medical University, Guiyang, Guizhou, China
e Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases, Guizhou Medical University, Guiyang, Guizhou, China
* These authors contributed equally to this work.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/14756366.2024.2398561.

CONTACT Xing-Kai Qian qxkgood@hotmail.com Translational Medicine Research Center, Guizhou Medical University, Guiyang, Guizhou, China
Hao Li 651882314@qq.com Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Ya-Di Zhu 18337153105@163.com Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases, Guizhou Medical University, Guiyang, Guizhou, China
2 9 2024
2024
2 9 2024
39 1 239856117 5 2024
31 7 2024
17 8 2024
KnowledgeWorks Global Ltd.30 8 2024
published online in a building issue30 8 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

Obesity is acknowledged as a significant risk factor for various metabolic diseases, and the inhibition of human pancreatic lipase (hPL) can impede lipid digestion and absorption, thereby offering potential benefits for obesity treatment. Anthraquinones is a kind of natural and synthetic compounds with wide application. In this study, the inhibitory effects of 31 anthraquinones on hPL were evaluated. The data shows that AQ7, AQ26, and AQ27 demonstrated significant inhibitory activity against hPL, and exhibited selectivity towards other known serine hydrolases. Then the structure-activity relationship between anthraquinones and hPL was further analysed. AQ7 was found to be a mixed inhibition of hPL through inhibition kinetics, while AQ26 and AQ27 were effective non-competitive inhibition of hPL. Molecular docking data revealed that AQ7, AQ26, and AQ27 all could associate with the site of hPL. Developing hPL inhibitors for obesity prevention and treatment could be simplified with this novel and promising lead compound.

Keywords

Pancreatic lipase
inhibitor
structure-activity relationships
anthraquinones
National Natural Science Foundation of China 10.13039/501100001809 82304611 Basic Research Program of Guizhou Province ZK[2022]376 Key Laboratory of Basic Pharmacology of Ministry of Education (Zunyi Medical University) KY[2022]393 Science and Technology Foundation of Guizhou Provincial Health Commission gzwkj2022-088 This work was financially supported by the National Natural Science Foundation of China (82304611), Basic Research Program of Guizhou Province (ZK[2022]376), Key Laboratory of Basic Pharmacology of Ministry of Education (Zunyi Medical University) (KY[2022]393) and the Science and Technology Foundation of Guizhou Provincial Health Commission (gzwkj2022-088).
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pmcIntroduction

Obesity has been identified as a significant risk factor for numerous metabolic diseases, including stroke, diabetes, atherosclerosis, cardiovascular diseases, and some types of cancer1–3. Several studies indicate that blocking lipid intake has been validated as one of the most feasible strategies to reduce body weight. Human pancreatic lipase (triacylglycerol acyl hydrolase, hPL, E.C: 3.1.1.3) is the most extensively investigated member of the human lipase superfamily related to carboxyl esterase4,5. Pancreatic lipase is secreted from the acinar cells of the pancreas and has a strong preference for hydrolysing triacylglycerides over cholesterol esters, phospholipids, and galactolipids. Furthermore, besides playing a crucial role in the breakdown of triacylglycerol, research has additionally revealed its involvement in the hydrolysis of retinyl esters in vivo. The presence of colipase, a cofactor, is essential for the efficient digestion of dietary fat by pancreatic lipase6,7. As a result, targeting hPL has become a promising approach for treating obesity and hypertriglyceridemia8,9.

Pancreatic lipase inhibitors can block lipid digestion and absorption in the gastrointestinal system, which in turn bring beneficial effects in weight maintenance and obesity treatment10–13. In 1999, a potent inhibitor of mammalian pancreatic lipase called orlistat was introduced, which has been widely utilised as a primary anti-obesity agent in over 100 countries due to its strong inhibitory effect on the key serine hydrolase in the gastrointestinal tract14,15. However, despite its popularity, orlistat has been associated with significant side effects such as steatorrhoea, faecal spotting, diarrhoea, abdominal pain, anal fissures, and acute kidney injury. It has also been linked to an increased risk of osteoporosis, limiting its use in certain patient groups such as children, pregnant women, the elderly, and those with underlying health conditions16,17. Therefore, there is a need to discover and develop effective anti-obesity agents with better safety profiles and desirable pharmacokinetic properties.

Anthraquinones, also known as 9,10-dioxoanthracenes, are a significant class of natural and synthetic compounds with a diverse range of applications18. For centuries, anthraquinone-rich plants have been utilised in traditional Chinese medicine to treat various ailments19,20. Modern medical research has further revealed the potent pharmacological properties of anthraquinones, including their effectiveness as laxatives, anticancer agents, antibacterials, anti-inflammatories, antioxidants, and more21–24. As a result, compounds containing the anthraquinone ring structure are prevalent in traditional Chinese medicine and have garnered considerable attention for their therapeutic potential25. Although natural ingredients of flavonoids, saponins, alkaloids and terpenoids has an inhibitory effect on hPL7, but has not yet been reported finding Anthraquinones with hPL inhibition.

A recent study showed that some natural anthraquinone compounds has the effect of lowering lipid26–28. However, the anthraquinone compound responsible for the inhibition of pancreatic lipases and its mechanisms are yet to be elucidated. In traditional medicine, anthraquinones has been used primarily as a laxative and for obesity-related disease29. Therefore, the present study collected a group of anthraquinone compounds and evaluated their inhibitory effect on hPL. In addition, we further analysed the structure-activity relationship (SAR) of these compounds’ inhibition effect of hPL.

Experimental

Chemicals and reagents

Anthraquinones (purity >99%) were purchased from the Casmart Technology Co., Ltd. (Beijing, China). The molecular structures of all compounds are summarised in Figure 1. This study delved into the analysis of anthraquinones and their analogous compounds present in widely utilised raw materials currently available in the market. Beyond examining anthraquinones that encompass varying positions and quantities of hydroxyl groups, the study also focused on compounds featuring groups with diverse electron donating/accepting capabilities, thereby elucidating the intricate structure-activity relationship in terms of their inhibitory effects on pancreatic lipase activity. Anthraquinones were dissolved with dimethyl sulfoxide (DMSO, Sigma, USA) and ultrasound to form a 50 mM storage solution and store them in a −80 °C freezer. Further dilute the anthraquinone storage solution to the target concentration before measurement. The expression and purification methods of human pancreatic lipase, as well as its specific fluorescent probe 9H-1,3-Dichloro-7-oleoyl-9,9-dimethylacridine-2-one (DDAO-ol) and hydrolysis product 9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one (DDAO) synthesis methods, have been described in previous articles30. Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), dipeptidyl peptidase-IV (DPP-IV), prolyl endopeptidase (PREP), as well as fibroblast activation protein (FAP) were purchased from Adamas (Shanghai, China). N-alkylated D-luciferin methyl ester (NLMe) and glycyl-prolyl-N-butyl-4-amino-1,8-naphthalimide (GP-BAN) were synthesised by our group with a purity of >98%31,32. Fluorescent diacetate (FD) was purchased from Tedia (Shanghai, China). The substrate benzyloxycarbonyl-Gly-Pro-7-amido-4-methylcoumarin (Z-GP-AMC) for the PREP and FAP activity assays was purchased from Shanghai Hanhong Technology Co (Shanghai, China). ATChI, BTChI and DTNB were purchased from Bidepharm (Shanghai, China). The pure water used for testing was prepared by milli-Q (Millipore, USA). Luciferin detection reagent (LDR) was purchased from Promega (Madison, USA). All other reagents were the highest grade commercially available (Adamas, Shanghai, China).

Figure 1. Chemical structure of natural anthraquinones.

Enzyme inhibition assays

The measurement of hPL activity was based on the mechanism of hPL hydrolysing the fluorescent substrate DDAO-ol to produce DDAO. The inhibitory effects of 31 anthraquinones on hPL were evaluated by measuring the residual activity fraction. The assay was performed in a 96-well black microplate (Corning, USA) with a total incubation volume of 200 μL. The reaction was conducted in a mixed system containing 194 μL of buffer (25 mM Tris-HCl, 1 mM CaCl2, 150 mM NaCl, pH = 7.4), 2 μL of inhibitor (the final concentration of 1, 10, and 50 μM), and 2 μL of hPL (the final concentration of 0.025 mg/mL). The reaction mixture was pre-incubated at 37 °C for 10 min before the hydrolytic reaction was started by adding 2 μL DDAO-ol with the final concentration of 10 μM. A Solvent control was set up by replacing the enzyme solution with an equal volume of PBS solution to measure background fluorescence. A blank control was also set up by replacing the inhibitor solution with an equal volume of DMSO solution to measure 100% enzyme activity. A positive control was established using orlistat. The inhibition effect was expressed as the percentage of fluorescence intensity of the different inhibitor groups compared to the control group.

Inhibition kinetic analyses

For the inhibition kinetic analyses, the reaction mixture consisted of 25 mM Tris-HCl, 1 mM CaCl2, 150 mM NaCl, hPL (0.025 mg/ml) and different concentration inhibitors (DMSO as the control group), while the total volume is 200 μL (pH = 7.4). The 96-well black microplates were pre-incubated at 37 °C for 10 min, and then the 10 μM fluorescent substrate DDAO-ol was added for hydrolysis. Whereafter, kinetic analysis of the fluorescence of the hydrolysed product DDAO at excitation/emission wavelength = 600/660 nm (Gain = 500) was performed using SpectraMAX iD3® multimodal Molecular Devices (USA) with a time interval of 1 min and a total duration of 30 min. The relative activity of hPL was calculated by measuring the fluorescence intensity of the group containing the compound to be tested divided by the fluorescence intensity of the group containing only DMSO.

Selective analysis of inhibitors

All analyses were performed on the microplate and detect by SpectraMAX iD3®. The specific steps for different types of enzymes are as follows:

hCES1A: An incubation reaction with a total volume of 100 μL contains 1 μL of inhibitor or an equal volume of DMSO, 1 μL of human liver microsome (the final concentration 1 μg/mL), and 97 μL of PBS (pH = 6.5). The mixed solution was pre-incubated at 37 °C for 3 min. Then, 3 μM of the substrate NLMe was added to start the hydrolysis reaction for 10 min. The testing was performed on a white plate and the signal value was obtained through a full wavelength scan at 37 °C for 30 min with an interval of 1 min after adding LDR. The relative activity of hCES1A was calculated by maximum measuring the luminescence intensity from the group with the test compound divided by maximum measuring the luminescence intensity from the group with DMSO33.

hCES2A: For the hCES2A inhibition kinetic assay, the reaction mixture composed of 2 μL of human liver microsome (the final concentration of 2 μg/mL), 2 μL of inhibitor or an equal volume of DMSO, and 195 μL PBS (0.1 M, pH = 7.4) in a 96-well black microplate was pre-incubated at 37 °C for 10 min. The hydrolysis reaction was started by adding 1 μL fluorescent substrate FD with a final concentration of 5 μM. Then, the fluorescence of the hydrolysis product at excitation/emission wavelength = 480/525 nm for 30 min. The relative activity of hCES2A was calculated by measuring the fluorescence intensity from the group with the test compound divided by the fluorescence intensity from the group with only DMSO34.

AChE/BuChE: Make 0.25 U/mL of AChE/BuChE and 200 μM DTNB is pre-incubated with 2 μL of inhibitor or an equal volume of DMSO in 180 μL PBS (0.1 M, pH = 7.4) at 37 °C for 10 min. The reaction was started by adding 1 mM substrate ATChI/BTChI (dissolved in pure water). Then, the reaction was kinetically analysed for 30 min detection at 412 nm absorption. The relative activity of AChE/BuChE was calculated by measuring the optical density from the group with the test compound divided by the optical density from the group with DMSO35.

DPP-IV: For the DPP-IV inhibition assay, 200 μL of a reaction mixture composed of PBS (0.1 M, pH = 7.4), DPP-IV (0.1 μg/mL), and 2 μL of inhibitor (diluted in DMSO) or DMSO in a microplate was pre-incubated at 37 °C for 5 min. The reaction was started by adding 100 μM fluorescent substrate GP-BAN. Then, the fluorescence of the hydrolysis product BAN at excitation/emission wavelength = 430/535 nm was kinetically analysed for 20 min. The relative activity of DPP-IV was calculated by measuring the fluorescence intensity from the group with the test compound divided by the fluorescence intensity from the group with DMSO36.

PREP/FAP: Make a 10 μg/mL of enzyme sample is pre-incubated with 2 μL of inhibitor or an equal volume of DMSO in 194 μL PBS (0.1 M, pH = 7.4) at 37 °C for 10 min. Add 2 μL of Z-Gly-Pro-AMC (diluted in DMSO) to obtain a final concentration of 300 μM. Fluorescence is measured kinetically at excitation/emission wavelength = 380/465 nm for 30 min at 37 °C. The relative activity of PREP/FAP was calculated by measuring the fluorescence intensity from the group with the test compound divided by the fluorescence intensity from the group with DMSO37.

Molecular docking simulations

To study the binding mechanisms of compounds AQ7, AQ26 and AQ27 against hPL, molecular docking was performed with AutoDock Vina (Version 1.1.2) based on Lamarckian genetic algorithm. The hPL structures (PDB: 1ETH) were download from the website “https://www.rcsb.org/” as receptors while the compounds AQ7, AQ26 and AQ27 were used as ligand. Hydrogen atoms were added followed by assigning the Kollman charges. The grid box was enclosing the known binding sites reported previously, i.e. the Ser153 for the active site (Site I) and the surface between lipase and colipase for the interface site of hPL (Site II). The length, width and height of the grid box are set to 126*126*126 Å3 with a spacing of 0.375. The above steps indicate a favourable binding process with the lowest binding energy for subsequent calculations38,39.

Statistical analysis

All measurements are conducted in three parallel groups and the data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism version 9.5.0 for Windows (GraphPad Software, USA, www.graphpad.com).

Results and discussion

Screening of hPL inhibitors from anthraquinones

In this study, we collected and determined the inhibitory effects of 31 anthraquinone compounds against hPL (Figure 2). As shown in Figure 2(A), seventeen compounds including AQ2, AQ5, AQ6, AQ7, AQ9, AQ11, AQ12, AQ15, AQ16, AQ17, AQ18, AQ19, AQ22, AQ24, AQ26, AQ27 and AQ29 has been found a strong inhibition of hPL. It’s worth noting that some of these anthraquinone compounds displayed a better inhibitory effect than orlistat, a positive inhibitor of hPL. Hence, the inhibition of hPL by these compounds with different concentration gradients was further evaluated (Figure 2B). The results showed that these anthraquinone compounds can dose-dependent inhibit hPL, implying that these compounds may be the potential inhibitor on hPL.

Figure 2. Screening of hPL inhibitors from anthraquinones at a final concentration of 50 μM (A) and dose-dependent inhibition of hPL by anthraquinones (B).

Inhibitory effects of anthraquinones against hPL

To further quantify the inhibitory effects of these compounds against hPL, the dose-dependent inhibition curves were plotted using different inhibitor concentrations. As shown in Table 1 and Table S1, the catalytic activities of hPL were inhibited by each tested anthraquinones via a dose-dependent manner. The IC50 values of these compounds were all list in Table 1. It is worth noting that AQ7, AQ26, and AQ27 displayed a very strong inhibition of hPL. The IC50 values of these three anthraquinones were evaluated as 0.33 μM, 0.06 μM, and 0.10 μM, respectively. These results demonstrated that AQ7, AQ26, and AQ27 displayed strong to moderate inhibitory effects on hPL, which encouraged us to further investigate the inhibition mechanism of these three anthraquinones with strong inhibition on hPL.

Table 1. IC50 value of these anthraquinones against hPLa.

Compound	hPL residual activity (50 μM) (%)	hPL IC50 value (μM)	
AQ1	36.95	–	
AQ2	6.17	6.88 ± 1.35	
AQ3	42.24	–	
AQ4	25.61	–	
AQ5	6.75	2.94 ± 0.62	
AQ6	11.19	8.67 ± 1.45	
AQ7	4.89	0.33 ± 0.05	
AQ8	68.75	–	
AQ9	12.60	1.10 ± 0.25	
AQ10	57.39	–	
AQ11	8.10	10.81 ± 1.49	
AQ12	7.85	3.48 ± 0.63	
AQ13	52.11	–	
AQ14	60.31	–	
AQ15	3.90	2.20 ± 0.34	
AQ16	6.24	1.77 ± 0.25	
AQ17	23.59	12.14 ± 2.43	
AQ18	7.90	1.09 ± 0.10	
AQ19	7.76	2.26 ± 0.60	
AQ20	61.46	–	
AQ21	70.00	–	
AQ22	13.34	3.69 ± 0.70	
AQ23	103.35	–	
AQ24	8.34	31.56 ± 6.44	
AQ25	32.46	–	
AQ26	5.44	0.06 ± 0.01	
AQ27	5.43	0.10 ± 0.02	
AQ28	71.70	–	
AQ29	18.51	13.64 ± 1.59	
AQ30	94.95	–	
AQ31	43.89	–	
Orlistatb	–	1.70 ± 0.20 (nM)	
aAll experimental data are averages of at least three independent experiments.

bA positive inhibitor of hPL.

Inhibition mechanism analyses of identified inhibitors towards hPL

The strong inhibitory effects of AQ7, AQ26, and AQ27 on hPL prompted us to further investigate their inhibition types and constants (Ki) on hPL. Therefore, we performed inhibition kinetics of these three natural compounds against hPL-mediated DDAO hydrolysis. As shown in Figure 3, the Lineweaver-Burk plots clearly demonstrate that AQ7 functions as a mixed inhibition, while AQ26 and AQ27 function as non-competitive inhibition against hPL-mediated DDAO hydrolysis. The Ki values of AQ7, AQ26, and AQ27 were determined to be 8.49 μM, 0.10 μM, and 2.12 μM, respectively (Table 2, Figure S1). These results indicate that all three anthraquinones can effectively inhibit hPL-mediated DDAO hydrolysis with Ki values less than 10 μM. Notably, AQ26 showed the most potent inhibitory effect on hPL with a Ki value of 0.10 μM, suggesting that this natural compound may serve as a promising lead for developing new hPL inhibitors.

Figure 3. The Lineweaver-Burk plots of AQ7(A), AQ26(B) and AQ27(C) against hPL-mediated DDAO hydrolysis. All data were shown as mean ± SD.

Table 2. The Ki values and the inhibition types of AQ7, AQ26, and AQ27 against hPL-mediated DDAO hydrolysis.

Compound	Ki(µM)	Inhibition type	Goodness of fit (R2)	
AQ7	8.49	Mixed	0.99	
AQ26	0.10	Non-competitive	0.93	
AQ27	2.12	Non-competitive	0.98	

Specificity of AQ7, AQ26, and AQ27 towards hPL over other serine hydrolases

In order to further demonstrate the specificity of AQ7, AQ26, and AQ27 for hPL inhibition, the inhibitory effects of these three compounds were tested on a panel of well-known serine hydrolases, including human carboxylesterase 1 (hCES1A), human carboxylesterase 2 (hCES2A), acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), dipeptidyl peptidase IV (DPP-IV), fibroblast activation protein (FAP), and prolyl endopeptidase (PREP), using established assays. As shown in Table 3, compound AQ7 exhibited a lower inhibitory effect on hCES2A (IC50 = 12.50 μM) compared to hPL. Similarly, AQ26 and AQ27 showed lower inhibitory effects on PREP (IC50 = 10.66 and 3.57 μM, respectively) compared to hPL. However, all three compounds showed poor inhibitory effects on hCES1A, AChE, BuChE, DPP-IV, and FAP, with IC50 values greater than 50 μM. These results suggest that AQ7, AQ26, and AQ27 are effective inhibitors of hPL with some selectivity over other serine hydrolases.

Table 3. The inhibitory effects of AQ7, AQ26, and AQ27 towards eight serine hydrolases.

Compound	Target enzyme	Substrate	IC50 (µM)	
AQ7	hPL	DDAO-ol	0.33	
hCES1A	NLMe	>50	
hCES2A	FD	12.50	
AChE	ATChI/DTNB	>50	
BuChE	BTChI/DTNB	>50	
DPP-IV	GP-BAN	>50	
FAP	Z-GP-AMC	>50	
PREP	Z-GP-AMC	>50	
AQ26	hPL	DDAO-ol	0.06	
hCES1A	NLMe	>50	
hCES2A	FD	>50	
AChE	ATChI/DTNB	>50	
BuChE	BTChI/DTNB	>50	
DPP-IV	GP-BAN	>50	
FAP	Z-GP-AMC	>50	
PREP	Z-GP-AMC	10.66	
AQ27	hPL	DDAO-ol	0.10	
hCES1A	NLMe	>50	
hCES2A	FD	>50	
AChE	ATChI/DTNB	>50	
BuChE	BTChI/DTNB	>50	
DPP-IV	GP-BAN	>50	
FAP	Z-GP-AMC	>50	
PREP	Z-GP-AMC	3.57	
Data were shown as mean ± SD (n = 3).

Structure-activity relationship analysis

On the basis of the structure of anthraquinone, the changes of different positions and different functional groups showed different inhibitory effects. By analysing the structure-activity relationship (SAR) and summarising the rule, it could provide a reference for promoting the structural modification of the compound and enhancing the biological inhibitory effect. Compared with compound AQ1(anthraquinone), compound AQ2 showed a stronger inhibitory effect on hPL, suggesting that the inhibitory effect of compound AQ1 could be enhanced by changing the carbonyl group of anthraquinone to the C-1 and C-4 sites. According to compounds AQ3, AQ4, AQ5, AQ6, AQ16, AQ17, AQ18, AQ19, the substitution of hydroxyl, amino, chlorine and other functional groups at the α position (C-1/4/5/8) can enhance the inhibitory effect, among which the inhibitory effect of amino > hydroxyl > chlorine, and the inhibitory effect is positively correlated with the number of substitutions. compound AQ7 has two hydroxyl substitution in C-1 and C-2, which has a strong inhibitory effect on hPL, while the introduction of sulphonic acid group (AQ8) or dicarboxyaminomethyl group (AQ9) at the C-3 site leads to a slight decrease in its inhibitory effect. Compared with AQ5, the introduction of methyl groups at the C-3 site of compounds AQ10, AQ13 and AQ14 significantly reduced the inhibition of hPL, while the introduction of hydroxymethyl (AQ11) and carboxylic (AQ12) groups at C-3 slightly reduced the inhibition of hPL, suggesting that the introduction of hydrogen bond donor groups at the C-3 site may be detrimental to the inhibition of hPL. In compounds AQ20–25, the inhibition of hPL was not improved by introducing methyl, ethyl, carboxyl, hydroxyl, and bromine at the β position, while the inhibition of hPL was significantly enhanced by chlorine substitution. Dianthraquinones AQ15 and AQ26–27 showed strong inhibitory effects on hPL. In naphthoquinone, compound AQ28–31 generally had a weak inhibitory effect on hPL. Based on this, the results of the inhibiting effects of anthraquinones on hPL were summarised in the following SAR (Figure 4).

Figure 4. SAR summary of anthraquinone inhibiting hPL.

Docking simulation

Molecular docking simulations were performed to obtain insight into the interaction between hPL and inhibitors (AQ7, AQ26, and AQ27). Compound AQ7 and hPL were bound at positions that were investigated, as seen in Figure 5(A,B) and Figure S2A,B. With an energy of −9.8 kcal/mol, the primary interaction at Site I (Ser-153 for the active site) was hydrophobic with a distance of 2.34 Å. The catechol in AQ7 has a tendency to occupy the pocket and point in the direction of the solvent, which may have an impact on substrate entrance. With an energy of −7.0 kcal/mol, AQ7 can establish hydrogen bonds with Lys-42 at Site II, the lipase and colipase interface site. Comparably, AQ26 and AQ27 likewise have a tendency to occupy the pocket at Site II and point in the direction of the solvent. The glycoside included in AQ26/AQ27 has the ability to establish hydrogen bonds with colipase’s Arg-65 and lipase’s Arg-338, Asp-338, and Thr-330. On the other hand, as Figure 5(C,D) and Figure S2CD, demonstrate, dianthraquinone may establish π-alkyl contacts with both Try-370 of lipase and Leu-41 of colipase, with energies of −9.1 kcal/mol each. Colipase engages in interactions with the C-terminal domain of lipase and flap, a surface loop derived from the N-terminal domain. Thus, binding at Site II might influence flap movement, which would then influence substrate binding. Mixed inhibition from molecular docking suggests that AQ7 can influence hPL activity by binding to distinct conformations at Sites I and II. Due to their bigger molecular architectures, dianthraquinones like AQ26 and AQ27 might make it more difficult for them to enter the active cavity. Consequently, they display non-competitive inhibition and are limited to acting at Site II.

Figure 5. The stereo view of hPL docked with compound AQ7 at site I (A) and site II (B); The stereo view of hPL docked with compound AQ26 (C), AQ27 (D) at site II.

Conclusion

In summary, thirty-one anthraquinones were collected and their inhibitory effects against hPL were assayed. Seventeen anthraquinones displayed strong to moderate inhibition against hPL. The SAR summarised and discussed of anthraquinones was conducted based on the results of IC50 values against hPL. Dianthraquinones have shown strong inhibitory effects on hPL. The substitution of the hydroxyl group, amino group and chlorine group at α position and the substitution of the chlorine group at β position could enhance the inhibitory effect on hPL, and the substitution of the hydroxyl group at α and β position ortho-substituted had the strongest inhibitory effect. The anthraquinone AQ7 (a mixed inhibition) and AQ26, AQ27 (non-competitive inhibition), showed strong activity to inhibit hPL (IC50 values were 0.33 μM, 0.06 μM and 0.10 μM, respectively) and displayed some selectivity over other known serine hydrolase (hCES1A, hCES2A, AChE, BuChE, DPP-IV, FAP and PREP). In addition, inhibitory kinetics and molecular docking indicated that AQ7 can form hydrogen bond with Ser-153 and AQ7, AQ26 and AQ27 can form hydrogen bond with Lys-42, Arg-65, Arg-338, Asp-338 and Thr-330 against hPL. Collectively, anthraquinones are a good choice for designing and developing hPL inhibitors, as our findings suggest.

Supplementary Material

supplementary20240731.docx

Authors contributions

Zi-Qiang Chen and Wen-Yao He, performed most of the experiments. Hong-Hong Ma and Jing Zhou, prepared the test sample reagents, materials. Hao Li, constructed the screening method. Ya-Di Zhu and Si-Yuan Yang, performed the Molecular Docking. Xing-Kai Qian, Ya-Di Zhu and Li-Wei Zou, designed and analysed the experiments. Hao Li, Ya-Di Zhu, and Xing-Kai Qian, prepared the manuscript with contributions from all authors.

Disclosure statement

The authors declare that they have non-financial conflicts of interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
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