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HIV/AIDS is still one of the leading causes of death worldwide. Current drugs that target the canonical steps of the HIV-1 life cycle are efficient in blocking viral replication but are unable to eradicate HIV-1 from infected patients. Moreover, drug resistance (DR) is often associated with the clinical use of these molecules, thus raising the need for novel drug candidates as well as novel putative drug targets. In this respect, pharmacological inhibition of the highly conserved and multifunctional nucleocapsid protein (NC) of HIV-1 is considered a promising alternative to current drugs, particularly to overcome DR. Here, using a multidisciplinary approach combining in silico screening, fluorescence-based molecular assays, and cellular antiviral assays, we identified nordihydroguaiaretic acid (6), as a novel natural product inhibitor of NC. By using NMR, mass spectrometry, fluorescence spectroscopy, and molecular modeling, 6 was found to act through a dual mechanism of action never highlighted before for NC inhibitors (NCIs). First, the molecule recognizes and binds NC noncovalently, which results in the inhibition of the nucleic acid chaperone properties of NC. In a second step, chemical oxidation of 6 induces a potent chemical inactivation of the protein. Overall, 6 inhibits NC and the replication of wild-type and drug-resistant HIV-1 strains in the low micromolar range with moderate cytotoxicity that makes it a profitable tool compound as well as a good starting point for the development of pharmacologically relevant NCIs.

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The described phenotypic assay can be adopted to evaluate the antiviral activity of licensed and investigational HIV-1 drugs targeting any of the three HIV-1 enzymes.

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Small molecule inhibitors of the HIV-1 nucleocapsid protein (NC) are considered as promising agents in the treatment of HIV/AIDS. In an effort to exploit the privileged 2-amino-4-phenylthiazole moiety in NC inhibition, here we conceived, synthesized, and tested in vitro 18 NC inhibitors (NCIs) bearing a double functionalization. In these NCIs, one part of the molecule is deputed to interact noncovalently with the NC hydrophobic pocket, while the second portion is designed to interact with the N-terminal domain of NC. This binding hypothesis was verified by molecular dynamics simulations, while the linkage between these two pharmacophores was found to enhance antiretroviral activity both on the wild-type virus and on HIV-1 strains with resistance to currently licensed drugs. The two most interesting compounds 6 and 13 showed no cytotoxicity, thus becoming valuable leads for further investigations.

Practical cell-based assays can accelerate anti-Zika (ZIKV) and anti-dengue (DENV) virus drug discovery. We developed an immunodetection assay (IA), using a pan-flaviviral monoclonal antibody recognizing a conserved envelope domain. The final protocol includes a direct virus yield reduction assay (YRA) carried out in the human Huh7 cell line, followed by transfer of the supernatant to a secondary Huh7 culture to characterize late antiviral effects. Sofosbuvir and ribavirin were used to validate the assay, while celgosivir was used to evaluate the ability to discriminate between early and late antiviral activity. In the direct YRA, at 100, 50, and 25 TCID50, sofosbuvir IC50 values were 5.0 ± 1.5, 2.7 ± 0.5, 2.5 ± 1.1 µM against ZIKV and 16.6 ± 2.8, 4.6 ± 1.4, 2.6 ± 2.2 µM against DENV; ribavirin IC50 values were 6.8 ± 4.0, 3.8 ± 0.6, 4.5 ± 1.4 µM against ZIKV and 17.3 ± 4.6, 7.6 ± 1.2, 4.1 ± 2.3 µM against DENV. Sofosbuvir and ribavirin IC50 values determined in the secondary YRA were reproducible and comparable with those obtained by direct YRA and plaque reduction assay (PRA). In agreement with the proposed mechanism of late action, celgosivir was active against DENV only in the secondary YRA (IC50 11.0 ± 1.0 µM) and in PRA (IC50 10.1 ± 1.1 µM). The assay format overcomes relevant limitations of the gold standard PRA, allowing concurrent analysis of candidate antiviral compounds against different viruses and providing preliminary information about early versus late antiviral activity.