Abstract
BackgroundCombination antiretroviral therapy efficiently suppresses HIV replication in infected patients, transforming HIV/AIDS into a chronic disease. Viral resistance does develop however, especially under suboptimal treatment conditions such as poor adherence. As a consequence, continued exploration of novel targets is paramount to identify novel antivirals that do not suffer from cross-resistance with existing drugs. One new promising class of targets are HIV protein–cofactor interactions. Transportin-SR2 (TRN-SR2) is a β-karyopherin that was recently identified as an HIV-1 cofactor. It has been implicated in nuclear import of the viral pre-integration complex and was confirmed as a direct binding partner of HIV-1 integrase (IN). Nevertheless, consensus on its mechanism of action is yet to be reached.ResultsHere we describe the development and use of an AlphaScreen-based high-throughput screening cascade for small molecule inhibitors of the HIV-1 IN–TRN-SR2 interaction. False positives and nonspecific protein–protein interaction inhibitors were eliminated through different counterscreens. We identified and confirmed 2 active compound series from an initial screen of 25,608 small molecules. These compounds significantly reduced nuclear import of fluorescently labeled HIV particles.ConclusionsAlphascreen-based high-throughput screening can allow the identification of compounds representing a novel class of HIV inhibitors. These results corroborate the role of the IN–TRN-SR2 interaction in nuclear import. These compounds represent the first in class small molecule inhibitors of HIV-1 nuclear import.
Highlights
Combination antiretroviral therapy efficiently suppresses HIV replication in infected patients, transforming HIV/AIDS into a chronic disease
To aid compound solubility at these concentrations, aside from the already present 0.1% (v/v) Tween-20 and 0.1% (w/v) bovine serum albumin (BSA), we increased the final concentration of dimethylsulfoxide (DMSO)
Our efforts evidenced that an Alphascreen-based high-throughput screening (HTS) can allow the identification of compounds representing a novel class of HIV inhibitors
Summary
Combination antiretroviral therapy efficiently suppresses HIV replication in infected patients, transforming HIV/AIDS into a chronic disease. Viral resistance does develop especially under suboptimal treatment conditions such as poor adherence. As a consequence, continued exploration of novel targets is paramount to identify novel antivirals that do not suffer from cross-resistance with existing drugs. Transportin-SR2 (TRN-SR2) is a β-karyopherin that was recently identified as an HIV-1 cofactor. It has been implicated in nuclear import of the viral pre-integration complex and was confirmed as a direct binding partner of HIV-1 integrase (IN). Viral resistance selection under suboptimal treatment conditions together with long-term adverse effects of chronic combination antiretroviral therapy (cART), highlight the need for novel antivirals with (1) a higher barrier towards resistance development, (2) pharmacokinetic properties. As a consequence of their common mechanism of action these inhibitors suffer a significant degree of cross-resistance. By binding the LEDGF/p75 binding site across the IN dimerization interface, LEDGINs block LEDGF/p75 binding and perturb IN multimerization, which in turn leads to inhibited catalytic activity (both 3′processing and strand transfer) and aberrant viral maturation [4,5,6,7, 11, 12]
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