七种蛋白酶抑制剂对多房棘球蚴DNA损伤诱导样1蛋白活性的影响

doi:10.11843/j.issn.0366-6964.2024.05.045

摘要/Abstract

摘要： 目前，多房棘球蚴病(alveolar echinococcosis, AE)尚无有效药物治疗手段，迫切需要开发新型治疗药物。前期研究表明，HIV蛋白酶抑制剂(HIV protease inhibitors，HIV PIs)具有潜在抗寄生虫功能。本文旨在研究HIV PIs对Echinococcus multilocularis (Emu)DNA损伤诱导样1蛋白(DNA damage inducible 1 protein，Ddi1)活性的影响。本研究通过构建真核表达重组载体pFastBac1-Emu Ddi1，在昆虫细胞系Sf9细胞中表达筛选出P1代和P2代，纯化出可溶性Ddi1重组蛋白，然后与目的蛋白的荧光底物检测纯化蛋白的活性，进一步检测沙奎那韦(saquinavir，SQV)、利托那韦(ritonavir，RTV)、安普那韦(amprenavir，APV)、阿扎那韦(atazanavir，ATV)、洛匹那韦(lopinavir，LPV)、福沙那韦(fosamprenavir，Fos)、达芦那韦(darunavir，DRV)等7种HIV PIs对Emu Ddi1重组蛋白活性的抑制能力。结果显示：细胞系内真核表达产物的酶活Km为1.422 μmol·L^-1，具有良好的亲和力和活性，最终筛到沙奎那韦对Ddi1蛋白二聚体活性位点的抑制率达67%，其IC₅₀为34，说明沙奎那韦对Emu Ddi1重组蛋白酶活性具有良好的抑制效果。以上结果提示：沙奎那韦抑制重组蛋白Ddi1的活性，可能成为Ddi1的靶向药物，以期为替代药物或开发联合用药提供基础。

关键词: 蛋白酶抑制剂, 多房棘球蚴, DNA损伤诱导样蛋白, 酶活性, 抑制率

Abstract: As no effective treatment for alveolar echinococcosis (AE) is currently available, the therapeutic drugs are needed urgently. Early stage studies have shown that the protease inhibitors of HIV could also be anticancer and anti-parasitic. The purpose of this paper is to study the effect of the inhibitors (HIV PIs) on the activity of DNA damage inducible 1 protein of Echinococcus multilocularis (EmuDdi1). The recombinant vector pFastBac1-EmuDdi1 was constructed and expressed in Sf9 cell, and the soluble protein was successfully purified in P2 generations. Then the enzyme activity of Ddi1 protein was detected with the specific fluorescent substrates, and the inhibition rate of seven HIV PIs including saquinavir (SQV), ritonavir (RTV), amprenavir (APV), atazanavir (ATV), lopinavir(LPV), fosamprenavir (Fos), tipranavir (TPV) and darunavir (DRV) on Ddi1 protein activity was further examined. The results showed that Emu Ddi1had high affinity and activity, and saquinavir showed the highest inhibition rate at 67% to inhibit protease activity of EmuDdi1 with a IC₅₀ at 34. These results suggested that saquinavir is effcetive to inhibit the activity of Ddi1, and could be used to develop a potential targeting drug for AE.

Key words: protease inhibitors, Echinococcus multilocularis, DNA damage inducible 1 protein, enzyme activity, inhibition rate

中图分类号:

S852.734

张生英, 刘仲藜, 郭爱疆, 王帅. 七种蛋白酶抑制剂对多房棘球蚴DNA损伤诱导样1蛋白活性的影响[J]. 畜牧兽医学报, 2024, 55(5): 2273-2280.

ZHANG Shengying, LIU Zhongli, GUO Aijiang, WANG Shuai. The Effect of Seven Protease Inhibitors on Activity of DNA Damage Inducible 1 Protein in Echinococcus multilocularis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2273-2280.

参考文献

[1] CASULLI A, BARTH T F E, TAMAROZZI F. Echinococcus multilocularis[J]. Trends Parasitol, 2019, 35(9):738-739.
[2] LIU Z L, GUO X L, GUO A J, et al. HIV protease inhibitor nelfinavir is a potent drug candidate against echinococcosis by targeting Ddi1-like protein[J]. eBioMedicine, 2022, 82:104177.
[3] KUMAR S, SUGUNA K. Crystal structure of the retroviral protease-like domain of a protozoal DNA damage-inducible 1 protein[J]. FEBS OpenBio, 2018, 8(9):1379-1394.
[4] WOOLSEY I D, MILLER A L. Echinococcus granulosus sensu lato and Echinococcus multilocularis: a review[J]. Res Vet Sci, 2021, 135:517-522.
[5] MÓTYÁN J A, MICZI M, TÖZSÉR J. Dimer interface organization is a main determinant of intermonomeric interactions and correlates with evolutionary relationships of retroviral and retroviral-like Ddi1 and Ddi2 proteases[J]. Int J Mol Sci, 2020, 21(4):1352.
[6] NOWICKA U, ZHANG D N, WALKER O, et al. DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin[J]. Structure, 2015, 23(3):542-557.
[7] GANTT S, CASPER C, AMBINDER R F. Insights into the broad cellular effects of nelfinavir and the HIV protease inhibitors supporting their role in cancer treatment and prevention[J]. Curr Opin Oncol, 2013, 25(5):495-502.
[8] ASAITHAMBI K, BISWAS I, SUGUNA K. Structural and functional insights into the DNA damage-inducible protein 1 (Ddi1) from protozoa[J]. Curr Res Struct Biol, 2022, 4:175-191.
[9] XIANG T, DU L Y, PHAM P, et al. Nelfinavir, an HIV protease inhibitor, induces apoptosis and cell cycle arrest in human cervical cancer cells via the ROS-dependent mitochondrial pathway[J]. Cancer Lett, 2015, 364(1):79-88.
[10] YIP M C J, BODNAR N O, RAPOPORT T A. Ddi1 is a ubiquitin-dependent protease[J]. Proc Natl Acad Sci U S A, 2020, 117(14):7776-7781.
[11] ELU N, OSINALDE N, BEASKOETXEA J, et al. Detailed dissection of UBE3A-mediated DDI1 ubiquitination[J]. Front Physiol, 2019, 10:534.
[12] PERTEGUER M J, GÓMEZ-PUERTAS P, CAÑAVATE C, et al. Ddi1-like protein from Leishmania major is an active aspartyl proteinase[J]. Cell Stress Chaperones, 2013, 18(2):171-181.
[13] WHITE R E, POWELL D J, BERRY C. HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites[J]. FASEB J, 2011, 25(5):1729-1736.
[14] DANAHER R J, WANG C M, ROLAND A T, et al. HIV protease inhibitors block oral epithelial cell DNA synthesis[J]. Arch Oral Biol, 2010, 55(2):95-100.
[15] MAXSON T, DEANE C D, MOLLOY E M, et al. HIV protease inhibitors block streptolysin S production[J]. ACS Chem Biol, 2015, 10(5):1217-1226.
[16] SCHWAKE C, BALDWIN M R, BACHOVCHIN W, et al. HIV protease inhibitors block parasite signal peptide peptidases and prevent growth of Babesia microti parasites in erythrocytes[J]. Biochem Biophys Res Commun, 2019, 517(1):125-131.
[17] HUDON S E, COFFINIER C, MICHAELIS S, et al. HIV-protease inhibitors block the enzymatic activity of purified Ste24p[J]. Biochem Biophys Res Commun, 2008, 374(2):365-368.
[18] REBELLO K M, ANDRADE-NETO V V, ZUMA A A, et al. Lopinavir, an HIV-1 peptidase inhibitor, induces alteration on the lipid metabolism of Leishmania amazonensis promastigotes[J]. Parasitology, 2018, 145(10):1304-1310.
[19] BACIGALUPO I, PALLADINO C, LEONE P, et al. Inhibition of MMP-9 expression by ritonavir or saquinavir is associated with inactivation of the AKT/Fra-1 pathway in cervical intraepithelial neoplasia cells[J]. Oncol Lett, 2017, 13(5):2903-2908.
[20] BARILLARI G, IOVANE A, BACIGALUPO I, et al. Ritonavir or saquinavir impairs the invasion of cervical intraepithelial neoplasia cells via a reduction of MMP expression and activity[J]. AIDS, 2012, 26(8):909-919.
[21] WU X D, LI Y Z, PENG K S, et al. HIV protease inhibitors in gut barrier dysfunction and liver injury[J]. Curr Opin Pharmacol, 2014, 19:61-66.
[22] FALKENSAMMER B, PILZ G, BEKTIĆ J, et al. Absent reduction by HIV protease inhibitors of Candida albicans adhesion to endothelial cells[J]. Mycoses, 2007, 50(3):172-177.
[23] ABOU-EL-NAGA I F, EL KERDANY E D, MADY R F, et al. The effect of lopinavir/ritonavir and lopinavir/ritonavir loaded PLGA nanoparticles on experimental toxoplasmosis[J]. Parasitol Int, 2017, 66(6):735-747.
[24] ABT D, BESSE A, SEDLARIKOVA L, et al. Improving the efficacy of proteasome inhibitors in the treatment of renal cell carcinoma by combination with the human immunodeficiency virus (HIV)-protease inhibitors lopinavir or nelfinavir[J]. BJU Int, 2018, 121(4):600-609.
[25] GRANATO Q M, SOUSA S I, ROSA T L S A, et al. Aspartic peptidase of Phialophora verrucosa as target of HIV peptidase inhibitors:blockage of its enzymatic activity and interference with fungal growth and macrophage interaction[J]. J Enzyme Inhib Med Chem, 2020, 35(1):629-638.
[26] BONDE C G, GAWAD J, BHOLE R P, et al. Effective drug candidates against global pandemic of Novel Corona Virus (nCoV-2019): A probability check through computational approach for public health emergency[J]. Russ J Bioorg Chem, 2023, 49(3):689-695.
[27] GOULIELMAKI E, KAFOROU S, VENUGOPAL K, et al. Distinct effects of HIV protease inhibitors and ERAD inhibitors on zygote to ookinete transition of the malaria parasite[J]. Mol Biochem Parasitol, 2018, 220:10-14.
[28] SANGENITO L S, D’AVILA-LEVY C M, BRANQUINHA M H, et al. Nelfinavir and lopinavir impair Trypanosoma cruzi trypomastigote infection in mammalian host cells and show anti-amastigote activity[J]. Int J Antimicrob Agents, 2016, 48(6):703-711.
[29] RICHMOND S R, CARPER M J, LEI X Y, et al. HIV-protease inhibitors suppress skeletal muscle fatty acid oxidation by reducing CD36 and CPT1 fatty acid transporters[J]. Biochim Biophys Acta, 2010, 1801(5):559-566.
[30] SZOKA L, KARNA E, ANDRULEWICZ-BOTULINSKA E, et al. The mechanism for differential effect of nelfinavir and indinavir on collagen metabolism in human skin fibroblasts[J]. Exp Dermatol, 2019, 28(7):845-853.
[31] YAO K, WANG Z, PENG C, et al. HIV protease inhibitor saquinavir inhibits toll-like receptor 4 activation by targeting receptor dimerization[J]. Immunopharmacol Immunotoxicol, 2023, 45(6):754-760.