Identification of lignans as ATP citrate lyase (ACLY) inhibitors from the roots of Pouzolzia zeylanica

Abstract A new lignan, named pouzolignan P (1), together with 14 known ones (2 − 15) were isolated from the roots of Pouzolzia zeylanica (L.) Benn. Their structures were deduced based on the detailed spectroscopic analysis. All the isolates were evaluated for their inhibitory activities toward the ATP citrate lyase (ACLY). Among them, four lignans, isopouzolignan K (3), gnemontanins E (5), gnetuhainin I (6), and styraxlignolide D (15) showed excellent ACLY inhibitory effect with IC50 values of 9.06, 0.59, 2.63, and 7.62 μM, respectively. These compounds were further evaluated for their cholesterol-lowing effects on ox-LDL-induced high-cholesterol HepG2 cells. Compound 15 emerges as the most potent ACLY inhibitor, which significantly decreased the TC level in a dose-dependent manner. In addition, molecular docking simulations elucidated that 15 formed a strong hydrogen-bond interaction with Glu599 of ACLY, which was an important site responsible for the enzyme catalytic activity. Graphical Abstract


Introduction
ATP citrate lyase (ACLY) is a critical enzyme in cellular metabolism, linking glucose catabolism to lipogenesis by providing acetyl-CoA, an important precursor for fatty acid and cholesterol biosynthesis (Burke et al. 2019;Granchi 2022).Studies have shown that ACLY emerges as a promising therapeutic target for hypercholesterolemia and related cardiovascular diseases (Feng et al. 2020).In 2020, FDA approved bempedoic acid, the specific ACLY inhibitor, for the treatment of adults with heterozygous familial hypercholesterolemia or established atherosclerotic cardiovascular disease who require additional lowering of LDL-C, which provides further evidence for the therapeutic value of this target.The molecular mechanisms determining ACLY activity and function, in simple terms, is to form a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production (Bazilevsky et al. 2019).
The structural diversity of natural products makes them an important source of lead compounds for advanced drug discovery (Newman and Cragg 2020).Statins, represented by the first-line clinical lipid-lowering medicine mevastatin, were isolated from natural microorganisms (Endo et al. 1976).Therefore, in-depth studies on natural products with cholesterol-lowering effect is an important strategy to find new lead to attenuate hypercholesterolemia. Recently, several naturally occurring molecules have emerged to inhibit the activity of ACLY (Deng et al. 2019;Huang et al. 2021;Song et al. 2021;Wang et al. 2022;Jiang et al. 2023), providing viable strategies for the research and development of the drug discovery on ACLY-related diseases.
Pouzolzia zeylanica (L.) Benn.(Urticaceae) is widely distributed in Southern China and the tropical areas of Asia.The whole plant has been used as a traditional medicine treat furuncle, dysentery and enteritis (Chen et al. 2015).Previous phytochemistry studies have revealed that lignans and flavonoids are the major secondary metabolites of this plant species, and the lignans have been considered as the chemical markers for taxonomic identification of Pouzolzia species (Nhung et al. 2022).The bioactivity evaluation of the extracts was mainly focused on the antitumor, antioxidant, anti-inflammatory activities.Lignans constitute a group of natural phenolics found in plant species.They have been shown to have a wide range of biological activities, and the regulatory role of lignans in lipid metabolism has been widely reported (Ma et al. 2020).In our continuing effort to find novel interesting bioactive compounds from nature, phytochemical investigation of the roots of P. zeylanica and the evaluation of their biological activities based on lipid metabolism were undertaken.Herein, we report the isolation and identification of a new norlignan, named pouzolignan P (1), along with 14 known lignans (2-15) from the roots of P. zeylanica (Figure 1).The structures were determined by spectroscopic analysis, CD data analysis and electronic circular dichroism (ECD) calculations.The inhibitory activities of all the isolates toward the ACLY were evaluated, and the bioactive compounds were selected to evaluate their cholesterol-lowing effects on ox-LDL-induced high-cholesterol HepG2 cells.The binding mode of the most potent compound, styraxlignolide D (15), was also discussed within the active site of ACLY (PDB: 6UUZ).This is the first report of the cholesterol-lowing effects of this kind of lignans by inhibiting the activity of ACLY in vitro, which deserves further study.
Considering the flexible tetrahydrofuran ring exists with envelope and twist conformations, the relative conformations of 1 cannot be deduced by analysing the coupling constants of the protons on the ring.In the NoESY spectrum of 1, the NoE correlations among H-3 and H-2 and H-6 indicated their cis orientation, which was also supported by the NoE correlations among H-2′′/6′′ and H-4 and H-6′.The trans orientation of H-4 and H-5 can be concluded from the observation the NoESY cross-peaks of H-2′′′/6′′′ and H-4, H-2′′/6′′, and H-6′ (Supplementary material Figures S1 and S14).
Pouzolignan D (2), the homologue of 1, was obtained as a dextrorotation pale-yellow amorphous powder, which was first identified by Guo's group (Zhong et al. 2015).However, the absolute configurations of the compound were undetermined.In our study, using the same approach of 1, we performed the quantum-mechanical calculation of 2 for having (2S,3S,4S,5R).The dominant conformation of pouzolignan D (2) was similar to that of 1 (Supplementary material Figure S4).The ECD calculations for conformers 2a-2h (above 2% populations, with envelope type configuration) were done at the PCM/B3LYP/6-311+(d,p)//PCM/B3LYP/6-311+(d,p) levels.The ECD spectra were Boltzmann-averaged, and the resulted ECD spectrum agreed well with the experimental ECD spectrum (Supplementary material Figure S5), which suggested that the absolute configurations of 2 were identical to those of 1.
ACLY is a key enzyme in cellular metabolism, facilitating the de novo fatty acid synthesis.The expression changes are often related to hypercholesterolemia and even cardiovascular diseases.The inhibition of ACLY may be beneficial to attenuated hypercholesterolemia. Thus, we choose the active compounds (3, 5, 6, and 15) to evaluate their cholesterol-lowing effects on ox-LDL-induced high-cholesterol HepG2 cells.Co-incubation of HepG2 cells with ox-LDL (25 mg/mL) was treated with each of these active compounds (20 μM) for 48 h (1 μM atorvastatin as a positive control).The results showed that compounds 6 and 15 significantly reduced the total cholesterol (TC) in ox-LDL-induced HepG2 cells.Especially, the effect of styraxlignolide D (15) at 20 μM was almost equivalent to that of atorvastatin (1 μM) (Supplementary material Figure S6).Further experiments on the effects of 15 on the high-cholesterol HepG2 model were undertaken at the concentrations of 10, 20, and 40 μM.As shown in Figure 2, the compound significantly decreased the TC level in a dose-dependent manner, and the TC levels of the 40 μM-treated group were closed to those of NC group.In addition, the effects of 15 on fatty acid and sterol synthesis in HepG2 cells were also evaluated by measuring 1,2-[ 14 C]-acetate incorporation into lipids.As shown in Figure S7, styraxlignolide D (15) at concentration of 20 μM significantly downregulated the lipid synthesis in HepG2 cells.These results suggesting that 15 deserves further evaluation as a potential cholesterol-lowing agent.
Styraxlignolide D (15) was docked to the CoA domain of ACLY (PDB: 6UUZ) by conventional docking procedures by AutoDock 4.2 (Morris et al. 2009).The best-scored docking pose is shown in Figure S8 (Supplementary material), and its affinity value was −5.69 kcal/mol.The predicted binding mode showed that 6′′-oH in the glucose moiety of 15 formed a strong hydrogen-bond interaction with Glu599 via the hydroxy group.Studies have revealed that Glu599 is positioned to play an important catalytic role for stabilizing the phospho-citryl-CoA intermediate, resulting the significantly compromised activities of E599A and E599Q (Wei et al. 2020).In addition, the lactone ring of 15 formed hydrophobic interaction with Gln505 in the active site of ACLY, which also contributed to the binding affinity.

Conclusions
In our continuing effort to find novel bioactive compounds from nature, phytochemical investigation of the roots of Pouzolzia zeylanica (L.) Benn was undertaken.A new lignan, named pouzolignan P (1), together with 14 known ones (2 − 15) were isolated from the roots of Pouzolzia zeylanica (L.) Benn.Their chemical structures as well as absolute configurations were elucidated by extensive analysis of HR-ESI-MS, NMR, and CD spectra.All these compounds were evaluated for their inhibitory activities toward the ATP citrate lyase (ACLY).Among them, four lignans, isopouzolignan K (3), gnemontanins E (5), gnetuhainin I (6), and styraxlignolide D (15) showed excellent ACLY inhibitory effect with IC 50 values of 9.06, 0.59, 2.63, and 7.62 μM, respectively, and further selected to evaluate their cholesterol-lowing effects on ox-LDL-induced high-cholesterol HepG2 cells.Compound 15 emerges as the most potent ACLY inhibitor, which significantly decreased the TC level in a dose-dependent manner.Molecular docking simulations elucidated that 15 formed a strong hydrogen-bond interaction with Glu599 of ACLY, which was an important site responsible for the enzyme catalytic activity.This is the first report of the cholesterol-lowing effects of this kind of lignans by inhibiting the activity of ACLY in vitro, which deserves further study.

Figure 2 .
Figure 2. effects of styraxlignolide d (15) on ox-ldl-induced high-cholesterol hepG2 cells. 1 μM atorvastatin was used as a positive control.the data are presented as the percentage of Nc group and as the means ± standard error of the mean (sd).** p < 0.01 and *** p < 0.001 vs. model group, n = 3-4 for all groups.