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
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References
- Myler R.K., Ryan C., Dunlap R., Shaw R.E., Bashour T.T., Cumberland D.C., Mooney M.R. Dyslipoproteinemias in atherosclerosis, thrombosis and restenosis after coronary angioplasty. J. Invasive Cardiol. 1995;7:33–46.
- He X.X., Zhang R., Zuo P.Y., Liu Y.W., Zha X.N., Shan S.S., Liu C.Y. The efficacy advantage of evolocumab (AMG 145) dosed at 140 mg every 2 weeks versus 420 mg every 4 weeks in patients with hypercholesterolemia: Evidence from a meta-analysis. Eur. J. Intern. Med. 2017;38:52–60. doi: 10.1016/j.ejim.2016.10.009.
- Steinberg D., Witztum J.L. Inhibition of PCSK9: A powerful weapon for achieving ideal LDL cholesterol levels. Proc. Natl. Acad. Sci. USA. 2009;106:9546–9547. doi: 10.1073/pnas.0904560106.
- Li J., Tumanut C., Gavigan J.A., Huang W.J., Hampton E.N., Tumanut R., Suen K.F., Trauger J.W., Spraggon G., Lesley S.A., et al. Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity. Biochem. J. 2007;406:203–207. doi: 10.1042/BJ20070664.
- Lagace T.A., Curtis D.E., Garuti R., McNutt M.C., Park S.W., Prather H.B., Anderson N.N., Ho Y.K., Hammer R.E., Horton J.D. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J. Clin. Investig. 2006;116:2995–3005. doi: 10.1172/JCI29383.
- Abifadel M., Elbitar S., El Khoury P., Ghaleb Y., Chemaly M., Moussalli M.L., Rabes J.P., Varret M., Boileau C. Living the PCSK9 adventure: From the identification of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs. Curr. Atheroscler. Rep. 2014;16:439. doi: 10.1007/s11883-014-0439-8.
- Yadav K., Sharma M., Ferdinand K.C. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors: Present perspectives and future horizons. Nutr. Metab. Cardiovasc. Dis. NMCD. 2016;26:853–862. doi: 10.1016/j.numecd.2016.05.006.
- Ramanathan A., Gusarova V., Stahl N., Gurnett-Bander A., Kyratsous C.A. Alirocumab, a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes. PLoS ONE. 2016;11:e0154498. doi: 10.1371/journal.pone.0154498.
- Zhang Y., Eigenbrot C., Zhou L., Shia S., Li W., Quan C., Tom J., Moran P., Di Lello P., Skelton N.J., et al. Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor. J. Boil. Chem. 2014;289:942–955. doi: 10.1074/jbc.M113.514067.
- Wang Y., Ye J., Li J., Chen C., Huang J., Liu P., Huang H. Polydatin ameliorates lipid and glucose metabolism in type 2 diabetes mellitus by downregulating proprotein convertase subtilisin/kexin type 9 (PCSK9) Cardiovasc. Diabetol. 2016;15:19. doi: 10.1186/s12933-015-0325-x.
- Tai M.H., Chen P.K., Chen P.Y., Wu M.J., Ho C.T., Yen J.H. Curcumin enhances cell-surface LDLR level and promotes LDL uptake through downregulation of PCSK9 gene expression in HepG2 cells. Mol. Nutr. Food Res. 2014;58:2133–2145. doi: 10.1002/mnfr.201400366.
- Chen H.C., Chen P.Y., Wu M.J., Tai M.H., Yen J.H. Tanshinone IIA Modulates Low Density Lipoprotein Uptake via Down-Regulation of PCSK9 Gene Expression in HepG2 Cells. PLoS ONE. 2016;11:e0162414. doi: 10.1371/journal.pone.0162414.
- Nhoek P., Chae H.S., Masagalli J.N., Mailar K., Pel P., Kim Y.M., Choi W.J., Chin Y.W. Discovery of Flavonoids from Scutellaria baicalensis with Inhibitory Activity Against PCSK 9 Expression: Isolation, Synthesis and Their Biological Evaluation. Molecules. 2018;23:504. doi: 10.3390/molecules23020504.
- Zhang D.W., Lagace T.A., Garuti R., Zhao Z., McDonald M., Horton J.D., Cohen J.C., Hobbs H.H. Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation. J. Boil. Chem. 2007;282:18602–18612. doi: 10.1074/jbc.M702027200.
- Shan L., Pang L., Zhang R., Murgolo N.J., Lan H., Hedrick J.A. PCSK9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide. Biochem. Biophys. Res. Commun. 2008;375:69–73. doi: 10.1016/j.bbrc.2008.07.106.
- Zhang Y., Zhou L., Kong-Beltran M., Li W., Moran P., Wang J., Quan C., Tom J., Kolumam G., Elliott J.M., et al. Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain. J. Mol. Boil. 2012;422:685–696. doi: 10.1016/j.jmb.2012.06.018.
- Kwon H.J., Lagace T.A., McNutt M.C., Horton J.D., Deisenhofer J. Molecular basis for LDL receptor recognition by PCSK9. Proc. Natl. Acad. Sci. USA. 2008;105:1820–1825. doi: 10.1073/pnas.0712064105.
- Bottomley M.J., Cirillo A., Orsatti L., Ruggeri L., Fisher T.S., Santoro J.C., Cummings R.T., Cubbon R.M., Lo Surdo P., Calzetta A., et al. Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants. J. Boil. Chem. 2009;284:1313–1323. doi: 10.1074/jbc.M808363200.
- Gu H.M., Adijiang A., Mah M., Zhang D.W. Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding. J. Lipid Res. 2013;54:3345–3357. doi: 10.1194/jlr.M041129.
- Du J., Sun L.N., Xing W.W., Huang B.K., Jia M., Wu J.Z., Zhang H., Qin L.P. Lipid-lowering effects of polydatin from Polygonum cuspidatum in hyperlipidemic hamsters. Phytomed. Int. J. Phytother. Phytopharm. 2009;16:652–658. doi: 10.1016/j.phymed.2008.10.001.
- Lin P., Lu J.M., Wang Y.F., Gu W., Zhao R.H., Yu J. Prevention Mechanism of 2,3,5,4′-Tetrahydroxy-stilbene-2-O-beta-d-glucoside on Lipid Accumulation in Steatosis Hepatic L-02 Cell. Pharmacogn. Mag. 2017;13:245–253.
- Javandoost A., Afshari A., Nikbakht-Jam I., Khademi M., Eslami S., Nosrati M., Foroutan-Tanha M., Sahebkar A., Tavalaie S., Ghayour-Mobarhan M., et al. Effect of crocin, a carotenoid from saffron, on plasma cholesteryl ester transfer protein and lipid profile in subjects with metabolic syndrome: A double blind randomized clinical trial. ARYA Atheroscler. 2017;13:245–252.
- Benjannet S., Rhainds D., Essalmani R., Mayne J., Wickham L., Jin W., Asselin M.C., Hamelin J., Varret M., Allard D., et al. NARC-1/PCSK9 and its natural mutants: Zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol. J. Boil. Chem. 2004;279:48865–48875. doi: 10.1074/jbc.M409699200.
- Maxwell K.N., Breslow J.L. Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. Proc. Natl. Acad. Sci. USA. 2004;101:7100–7105. doi: 10.1073/pnas.0402133101.
- Park S.W., Moon Y.A., Horton J.D. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J. Boil. Chem. 2004;279:50630–50638. doi: 10.1074/jbc.M410077200.
- Yamamoto T., Lu C., Ryan R.O. A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor. J. Boil. Chem. 2011;286:5464–5470. doi: 10.1074/jbc.M110.199042.
- Miao Q., Shi X.P., Ye M.X., Zhang J., Miao S., Wang S.W., Li B., Jiang X.X., Zhang S., Hu N., et al. Polydatin attenuates hypoxic pulmonary hypertension and reverses remodeling through protein kinase C mechanisms. Int. J. Mol. Sci. 2012;13:7776–7787. doi: 10.3390/ijms13067776.
- Lin P., Lu J., Wang Y., Gu W., Yu J., Zhao R. Naturally Occurring Stilbenoid TSG Reverses Non-Alcoholic Fatty Liver Diseases via Gut-Liver Axis. PLoS ONE. 2015;10:e0140346. doi: 10.1371/journal.pone.0140346.
- Wang W., He Y., Lin P., Li Y., Sun R., Gu W., Yu J., Zhao R. In vitro effects of active components of Polygonum Multiflorum Radix on enzymes involved in the lipid metabolism. J. Ethnopharmacol. 2014;153:763–770. doi: 10.1016/j.jep.2014.03.042.
- Abdel-Meguid S., Abou-Gharbia M., Blass B., Childers W., Elshourbagy N. Anti-Proprotein Convertase Subtilisin Kexin Type 9 (Anti-PCSK9) Compounds and Methods of Using the Same in the Treatment and/or Prevention of Cardiovascular Diseases. 14,767,133. U.S. Patent. 2014 Mar 11;
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