Abstract
BackgroundIntegrase (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.MethodsIn 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.ResultsDuring 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.ConclusionAccordingly, 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.
Highlights
Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-tran‐ scribed viral genome into the host chromosome during the early steps of viral infection
We were commissioned to evaluate the effectiveness of Non-catalytic integrase inhibitor (NCINI) candidates, but observed no activity using the TZM-bl system
BI 224436 was employed as the positive control in vitro to ascertain whether the TZM-bl system could be applied to assess the efficacy of NCINIs
Summary
Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-tran‐ scribed viral genome into the host chromosome during the early steps of viral infection. Active anti-retroviral therapy is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from com‐ pounds that inhibit the activities of viral reverse transcriptase and protease enzymes. IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain of IN. Antiretroviral drugs are divided into four classes: entry/fusion inhibitor, reverse transcription inhibitor (RTI), integrase inhibitor (INI), and protease inhibitor (PI) [5, 6]. Active antiretroviral therapy (HAART), a standard HIV/AIDS treatment method, is a cocktail therapy combining three or more antiretroviral. HAART effectively reduces the viral load and facilitates significant recovery of immune functions in HIV/AIDS patients, increasing the survival period by more than 7–10 years or even longer compared to single drug-treated patients [7,8,9]. Following the initial approval of zidovudine as a therapeutic NRTI by the US Food and Drug Administration (FDA) in 1987, various anti-HIV drugs have been developed as INI, starting with raltegravir (RAL) in 2007 followed by elvitegravir (EVG) and dolutegravir (DTG) [9, 11, 12]
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