HIV-1 非催化整合酶抑制剂的适宜分析方法研究

Study on suitable analysis method for HIV-1 non-catalytic integrase inhibitor.

机构信息

Department of Bio-Industrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Seoul, Korea.

Department of Biomedical Science and Engineering, College of Animal Bioscience and Technology, Konkuk University, Seoul, Korea.

出版信息

Virol J. 2021 Jan 13;18(1):17. doi: 10.1186/s12985-020-01476-x.

DOI:10.1186/s12985-020-01476-x

PMID:33436020

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7805210/

Abstract

BACKGROUND

Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs.

METHODS

In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed.

RESULTS

During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication.

CONCLUSION

Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

摘要

背景

整合酶（IN）是 HIV 复制所必需的一种关键蛋白，它在病毒感染的早期阶段催化将逆转录的病毒基因组插入宿主染色体。高效抗逆转录病毒疗法是一种 HIV/AIDS 治疗方法，它将三种或更多种抗病毒药物联合使用，这些药物通常是由抑制病毒逆转录酶和蛋白酶活性的化合物组成。早期的整合酶抑制剂（INIs）主要作为整合酶链转移抑制剂（INSTI），通过结合 IN 的催化核心结构域来破坏链转移。然而，IN 的突变可以导致对 INSTI 的耐药性。因此，已经开发了非催化整合酶抑制剂（NCINI）作为下一代 INIs。

方法

在本研究中，我们根据分析方法评估和比较了 INSTI 和 NCINI 的活性。使用 p24 ELISA 法结合 MT2 细胞和 TZM-bl 荧光素酶系统结合 TZM-bl 细胞比较抗病毒活性。将每种药物进行连续稀释并处理 MT2 和 TZM-b1 细胞，用 HIV-1 AD8 株感染并分别孵育 5 天和 2 天。此外，为了分析 INSTI 和 NCINI 的特性，进行了转移抑制试验和 3′-加工抑制试验。

结果

在使用 p24 ELISA 和 TZM-bl 荧光素酶系统筛选 INIs 时，我们发现 INSTI 和 NCINI 药物的结果不一致。用 T 嗜性 HIV-1 株感染 MT2 和 TZM-bl 细胞后，INSTI 和 NCINI 处理均在 MT2 细胞中显著减少 p24。然而，NCINI 在 TZM-bl 荧光素酶系统中没有抗病毒活性，表明这种广泛使用和方便的抗逆转录病毒检测方法不适用于筛选靶向 HIV-1 复制第二轮的 NCINI 化合物。