Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors with anti-HIV Activity

The interest in the synthesis of Se-containing compounds is growing with the discovery of derivatives exhibiting various biological activities. In this manuscript, we have identified a series of 2,2′-diselenobisbenzamides (DISeBAs) as novel HIV retroviral nucleocapsid protein 7 (NCp7) inhibitors. Because of its pleiotropic functions in the whole viral life cycle and its mutation intolerant nature, NCp7 represents a target of great interest which is not reached by any anti-HIV agent in clinical use. Using the diselenobisbenzoic scaffold, amino acid, and benzenesulfonamide derivatives were prepared and biologically profiled against different models of HIV infection. The incorporation of amino acids such as glycine and glutamate into DISeBAs <b>7</b> and <b>8</b> resulted in selective anti-HIV activity against both acutely and chronically infected cells as well as an interesting virucidal effect. DISeBAs demonstrated broad antiretroviral activity, encompassing HIV-1 drug-resistant strains including clinical isolates, as well as simian immunodeficiency virus (SIV). Time of addition experiments, along with the observed dose dependent inhibition of the Gag precursor proper processing, confirmed that their mechanism of action is based on NCp7 inhibition

Amidate Prodrugs of Deoxythreosyl Nucleoside Phosphonates as Dual Inhibitors of HIV and HBV Replication

The synthesis of four l-2′-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent antiviral activity displaying an EC₅₀ of 4.69 μM against HIV-1 and an EC₅₀ value of 0.5 μM against HBV, whereas completely lacking cytotoxicity. The synthesis of a number of phosphonomonoamidate and phosphonobisamidate prodrugs of PMDTA led to a boost in antiviral potency. The most potent congeners were a l-aspartic acid diisoamyl ester phenoxy prodrug and a l-phenylalanine propyl ester phosphonobisamidate prodrug that both display anti-HIV and anti-HBV activities in the low nanomolar range and selectivity indexes of more than 300

Amidate Prodrugs of Deoxythreosyl Nucleoside Phosphonates as Dual Inhibitors of HIV and HBV Replication

The synthesis of four l-2′-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent antiviral activity displaying an EC₅₀ of 4.69 μM against HIV-1 and an EC₅₀ value of 0.5 μM against HBV, whereas completely lacking cytotoxicity. The synthesis of a number of phosphonomonoamidate and phosphonobisamidate prodrugs of PMDTA led to a boost in antiviral potency. The most potent congeners were a l-aspartic acid diisoamyl ester phenoxy prodrug and a l-phenylalanine propyl ester phosphonobisamidate prodrug that both display anti-HIV and anti-HBV activities in the low nanomolar range and selectivity indexes of more than 300

Antiviral Activity of Flexibilane and Tigliane Diterpenoids from <i>Stillingia lineata</i>

In an effort to identify new potent and selective inhibitors of chikungunya virus and HIV-1 and HIV-2 virus replication, the endemic Mascarene species <i>Stillingia lineata</i> was investigated. LC/MS and bioassay-guided purification of the EtOAc leaf extract using a chikungunya virus-cell-based assay led to the isolation of six new (<b>4</b>–<b>9</b>) and three known (<b>1</b>–<b>3</b>) tonantzitlolones possessing the rare C₂₀-flexibilane skeleton, along with tonantzitloic acid (<b>10</b>), a new linear diterpenoid, and three new (<b>11</b>, <b>13</b>, and <b>15</b>) and two known (<b>12</b> and <b>14</b>) tigliane-type diterpenoids. The planar structures of the new compounds and their relative configurations were determined by spectroscopic analysis, and their absolute configurations were determined through comparison with literature data and from biogenetic considerations. These compounds were investigated for selective antiviral activity against chikungunya virus (CHIKV), Semliki Forest virus, Sindbis virus, and, for compounds <b>11</b>–<b>15</b>, the HIV-1 and HIV-2 viruses. Compounds <b>12</b>–<b>15</b> were found to be the most potent and are selective inhibitors of CHIKV, HIV-1, and HIV-2 replication. In particular, compound <b>14</b> inhibited CHIKV replication with an EC₅₀ value of 1.2 μM on CHIKV and a selectivity index of >240, while compound <b>15</b> inhibited HIV-1 and HIV-2 with EC₅₀ values of 0.043 and 0.018 μM, respectively. It was demonstrated further that potency and selectivity are sensitive to the substitution pattern on the tigliane skeleton. The cytotoxic activities of compounds <b>1</b>–<b>10</b> were evaluated against the HCT-116, MCF-7, and PC3 cancer cell lines

Inhibition of HIV infection of TZM-bl cell cultures.

<p>Monolayer TZM-bl cells were infected with HIV-1 in 0.2 ml DMEM medium in the presence of 10 μM or 25 μM test compound (in case of TE-10 also 250 μM) or an inhibitor control (nevirapin). Additionally, one well was mock infected (no virus, negative control). After 40 h, the cells were assayed for expression of luciferase activity. The value for the vehicle control was set to 100% in each individual experiment and the negative control was set to 0%. All other values were normalised against these reference values. The data are means of three independent experiments; the error bars represent the SEM. P-values are indicated if significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001).</p

Inhibition of syncytia formation in cocultures of HuT-78/HIV-1 and Sup-T1 cells.

<p>Inhibition of syncytia formation in cocultures of HuT-78/HIV-1 and Sup-T1 cells.</p

Time-of-drug-addition in HIV-1 infected MT-4 cell cultures.

<p>MT-4 cells were infected with HIV-1(III_B) for 1 h after which the cells were washed and distributed in a 96-well plate. Test compounds were added at different time points after the start of infection, indicated on the abscis of the graphs (Panels A & B). Whereas in Panel A, the focus lies on a wide array of time points in the infection cycle of HIV-1, Panel B focuses on the very early time points during the viral replication cycle. 31 h after the start of the experiments, cellular supernatants were harvested and HIV-1 replication was quantified using a p24 ELISA. AMD3100 was used at 625 nM, DS-8000 at 12.5 μM, AZT at 1.9 μM, ritonavir at 277 nM, T-20 at 4.5 μM and compound TE-2 at 50 μM. The graphs are representatives of 2–3 independent experiments.</p

Inhibition of the reduction of gp120 in the presence of the test compounds.

<p>An ELISA was performed with two set-ups for each compound, the reference compound (auranofin), a vehicle control (DMSO) and DTT. In the first set-up (TrxR1 + Trx1)_preincub., TrxR1 was shortly (15 min) pre-incubated with Trx1 before adding the compounds. In the second set-up (TrxR1 + compounds)_preincub, TrxR1 was shortly (15 min) pre-incubated with the compounds before adding Trx1. The final reaction mixture contained 1 μM Trx1 + 100 nM TrxR1 + 240 μM NADPH + 100 μM compound or auranofin. DTT was used as a reduction control. ELISA plates were coated with recombinant human gp120. The reaction mixtures were added to the wells to allow reduction of the coated gp120, incubated for 15 min at room temperature and exposed to streptavidine-ALP and p-nitrophenyl phosphate. The value for the DTT was set to 100% in each individual experiment and all other values were normalised against this reference value. The data are means of three independent experiments; the error bars represent the SEM. P-values were calculated for each reaction in relation to its control and are indicated if significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001).</p

Anti-HIV and cytostatic activities of organotellurium compounds in CEM and HeLa cell cultures.

<p>Anti-HIV and cytostatic activities of organotellurium compounds in CEM and HeLa cell cultures.</p

Inhibition of HIV-1 protease.

<p>(A) Inhibition of HIV-1 protease activity: The HIV-1 protease (100 ng/well) and the compounds (25 μM concentration) or an inhibitor control (pepstatin A) were added to a flat-bottom 96-well plate. The plate was incubated for 15 minutes at 37°C and the provided protease substrate was added to all wells. The fluorescence was measured at Ex/Em = 490 nm/520 nm. The value for the vehicle control was set to 100% in each individual experiment and all other values were normalised to this reference value. The data are means of three independent experiments; the error bars represent the SEM. P-values are indicated if significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001).(B) Inhibition of HIV-1 protease activity in viral particles: Persistently infected HuT-78/HIV-1 cells were incubated in the presence or absence of compound (3.7 μM AZT, 2.8 μM ritonavir or 1μM/20μM TE-10). After 43 h, virus particles in the cellular supernatants were harvested, lysed and subjected to Western blotting. P24 and its precursor p55 were detected using an anti-p24 antibody. Negative control: no compound added during incubation. M: MagicMark XP Western Protein Standard.</p