Oxadiazols: a new class of rationally designed anti-human immunodeficiency virus compounds targeting the nuclear localization signal of the viral matrix protein

Abstract

Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

FIG. 1.

CADD of ITI-367. (A) Hypothetical structural pocket within the N-terminal NLS of HIV-1 MA protein. The amino acids shown (R22, K27, Y29, and H33) participate in the formation of the hypothetical binding pocket for ITI-367. (B) The chemical structure of ITI-367. (C) ITI-367 in its docked (hypothetical) conformation and orientation within the “tyrosine pocket” of HIV-1 MA. In this molecular model, the protein is rendered as a solid molecular surface (light gray/light blue) and ITI-367 is shown in space-filling representation (standard atom coloring: gray, carbon; blue, nitrogen; red, oxygen; green, fluorine).

FIG. 2.

ITI-367 inhibits HIV-1 replication. (A) HIV-1-producing OM10.1 cells (left panel) and HTLV-I-producing MT-2 cells (right panel) were gamma irradiated and cocultured with CEM cells without any drug or in the presence of 0.1% dimethyl sulfoxide (DMSO) or 10 μM ITI-367 (prepared from 10 mM stock in DMSO). Samples were collected for ELISA at the indicated time points after initiation of cocultures (p19 for HTLV-1 and p24 for HIV-1). Results for one representative experiment out of two performed are presented. (B) Primary monocyte-derived macrophages (left panel) or tonsil-derived T lymphocytes (right panel) were infected with R5 isolates ADA or M1, respectively, and cultured in the presence of the indicated concentrations of ITI-367. Analysis of virus replication, as measured by RT activity or p24 concentration in culture supernatant, is presented. Results are shown for one representative experiment out of three performed. (C) Dose-response analysis plotted from the results of three independent experiments.

FIG. 3.

Anti-HIV activity of combinations of ITI-367 and approved anti-HIV drugs. Cultures of tonsil-derived T lymphocytes were infected with a primary R5 HIV-1 isolate M1 and cultured in the presence of the indicated concentrations of ITI-367 in combination with various concentrations of the nonnucleoside reverse transcriptase inhibitor (NNRTI) Efavirenz (panel a), the nucleoside reverse transcriptase inhibitors (NRTIs) AZT (panel b) or d4T (panel c), or the protease inhibitors (PIs) Saquinavir (panel d) or Nelfinavir (panel e). Virus replication was analyzed by measuring p24 in the culture supernatant on day 7 after infection. The data are presented as means of the results for three independent wells.

FIG. 4.

Anti-HIV activity of ITI-367 in histocultures of human tonsils. Blocks of human tonsils were infected with X4 virus LAI or R5 strain SF-162 in the presence of the indicated concentrations of ITI-367. Virus replication was assayed on day 12 after infection by measuring p24 in culture medium. Results (mean ± standard deviation) are presented as percent inhibition of virus replication in drug-treated cultures. The results for one representative experiment out of three performed with tissue from different donors are shown.

FIG. 5.

HIV-1 carrying mutations in MA NLS-1 is resistant to ITI-367. (A) Monocyte-derived macrophages were infected with the wild-type NLHXADA virus (wt) or NLHXADAΔNLS-1 (ΔNLS-1) carrying substitutions of isoleucine residues for lysines in positions 26 and 27 of MA (48) and cultured for 11 days. Half of the medium was changed at days 4 and 8. Virus replication was assessed according to p24 levels in the culture supernatant and is presented as means ± the standard error of the means of the results for three independent wells. The zero time point sample was taken after the viral inoculum was washed away. (B) Triplicate cultures of macrophages were infected as for panel A, except that cells were cultivated in the presence or absence of the indicated concentrations of ITI-367. Analysis of viral replication was performed on day 8 after infection by measuring p24 in the culture medium. Inhibition of HIV-1 replication by ITI-367 was calculated for each test well by dividing the p24 value in that well by the mean p24 value of the corresponding untreated culture, expressing the result as a percent and subtracting from 100%. Results are presented as the means ± the standard errors of the means.

FIG. 6.

ITI-367 inhibits HIV-1 PIC binding to importin α. Cytoplasmic lysates of HIV-1-infected cells were incubated with importin α immobilized on Sepharose beads in the presence of the indicated concentrations of ITI-367. The amount of importin α-associated HIV-1 DNA was quantified in triplicate by real-time PCR. Results are presented as means ± the standard errors of the means of the numbers of HIV-1 DNA copies. Empty Sepharose beads retained less than 15 DNA copies. The graph shows results of one representative experiment out of two performed.

FIG. 7.

ITI-367 inhibits HIV-1 nuclear import. (A) HIV-1 DNA was analyzed 72 h after infection of macrophages with HIV-1 ADA in the presence or absence of ITI-367 (10 μM) or AZT (3 μM), using primers specific for the late-reverse transcription product or 2-LTR circle DNA. Results were normalized according to β-actin signals and are presented as means ± standard errors of the means of the reduction in DNA copy numbers from three independent experiments. (B) Monocyte-derived macrophages were infected with Alexa Fluor 488-labeled HIV-1 in the presence or absence of ITI-367 (10 μM) and were analyzed by fluorescent microscopy 72 h after infection. Nuclei were stained with TOPRO-3 iodide. Arrows point to HIV-infected cells. Two independent fields are shown for each treatment. The scale bar is 10 μm.