A New Strategy for Rapidly Screening Natural Inhibitors Targeting the PCSK9/LDLR Interaction In Vitro

Li Li, Chen Shen, Ya-Xuan Huang, Ya-Nan Li, Xiu-Feng Liu, Xu-Ming Liu, Ji-Hua Liu, Li Li, Chen Shen, Ya-Xuan Huang, Ya-Nan Li, Xiu-Feng Liu, Xu-Ming Liu, Ji-Hua Liu

Abstract

The interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR) is a promising target for the treatment of hyperc-holesterolemia. In this study, a new method based on competitive affinity and tag detection was developed, which aimed to evaluate potent natural inhibitors preventing the interaction of PCSK9/LDLR directly. Herein, natural compounds with efficacy in the treatment of hypercholesterolemia were chosen to investigate their inhibitory activities on the PCSK9/LDLR interaction. Two of them, polydatin (1) and tetrahydroxydiphenylethylene-2-O-glucoside (2), were identified as potential inhibitors for the PCSK9/LDLR interaction and were proven to prevent PCSK9-mediated LDLR degradation in HepG2 cells. The results suggested that this strategy could be applied for evaluating potential bioactive compounds inhibiting the interaction of PCSK9/LDLR and this strategy could accelerate the discovery of new drug candidates for the treatment of PCSK9-mediated hypercholesterolemia.

Keywords: LDLR; PCSK9; hypercholesterolemia; interaction; natural products.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Strategy for evaluating the inhibition of natural products on the proprotein convertase subtilisin/kexin type 9 (PCSK9)/low-density lipoprotein receptor (LDLR) interaction. PCSK9 with a hexahistidine-tag was immobilized on Ni magnetic beads. The epidermal growth factor precursor homology domain A (EGF-A), a domain of the LDLR contributing to PCSK9 binding, was chosen for simulating the function of the LDLR with the addition of a glutathione S-transferase (GST)-tag. When the inhibitors were introduced, the PCSK9/EGF-A interaction was interrupted, and the ratios of the tags (GST/His) represented the inhibitory activities. The inhibition by the natural inhibitor was screened in this method and validated by cell assays.
Figure 2
Figure 2
Expression and purification of His-PCSK9 and GST-EGF-A. (A) Expression of His-PCSK9 (Lane M: prestained protein marker; Lane 1: cell lysate before induction with isopropylthio-β-d-galactopyranoside (IPTG); Lane 2: cell lysate after 24 h of expression); (B) Purification of His-PCSK9 (Lane M: pre-stained protein marker; Lane 1–6: elution by buffer with 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, and 250 mM imidazole, respectively); (C) Expression of GST-EGF-A (Lane M: pre-stained protein marker; Lane 1: cell lysate before induction with IPTG; Lane 2: cell lysate after 24 h of expression); (D) Purification of GST-EGF-A (Lane M: prestained protein marker; Lane 1–6: washing with 6-column volumes of buffer in turn; Lane 7–12: eluting with 6-column volumes of buffer containing glutathione in turn).
Figure 3
Figure 3
Establishment of the method for evaluating the PCSK9/LDLR interaction. The effects of the immobilized time of the PCSK9-MBs (A); the binding time between the ligands and the PCSK9-MBs (B) and the amounts of GST-EGF-A (C) on the binding assay were investigated; (D) The method established was verified by mixing GST-EGF-A (2.4 μg/μL PCSK9-MBs) and the PCSK9-MBs in presence of positive compound SBC-115076 with different concentrations (5, 15, and 50 nM), and the GST/His ratios were monitored by western blot. The control group was conducted without the addition of SBC-115076. The values are the mean ± SEM deviation of the three independent experiments. * p < 0.05; ** p < 0.01, compared with the control group.
Figure 4
Figure 4
The potential inhibitory effect of the natural compounds on the PCSK9/LDLR interaction. (A) Chemical structures of compounds 1–3, namely polydatin, tetrahydroxydiphenylethylene-2-O-glucoside and crocin I, respectively; (B–D) The potential inhibition of compounds 1–3 on the PCSK9/LDLR interaction were evaluated by the methods above. The GST/His ratios were monitored by western blot assay in the presence of compounds 1–3 at different concentrations (5, 15, and 50 nM), respectively. The values are the mean ± SEM deviation of the three independent experiments. * p < 0.05; ** p < 0.01, compared with the control group.
Figure 5
Figure 5
The activities of compounds 1–3 on preventing PCSK9-mediated LDLR degradation in the HepG2 cells. The LDLR levels in the HepG2 cells were monitored by western blot upon the treatment of PCSK9 in the presence of SBC-115076 (A); polydatin (B); tetrahydroxyl diphenylethylene-2-O-glucoside (C); and crocin I (D) at different concentrations (0, 0.5, 1.5, and 5.0 μM), respectively. The values are the mean ± SEM deviation of three independent experiments. #p < 0.05; ##p < 0.01; compared with the control group, * p < 0.05; ** p < 0.01, compared with the PCSK9 group.

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