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The inhibition of HIV-1 infection by aristolactam derivatives was tested in TZM-bl cells, which contain long terminal repeat (LTR)-driven firefly luciferase and lacZ (bgalactosidase), and express human CD4, CXCR4, and CCR5, in order to facilitate HIV-1 infection, as described previously (Platt et al. 1998). bl-DTR (TZM-bl-derived dual Tat reporter) cells were generated from TZM-bl cells by transforming two doxycycline-inducible lentiviral expression cassettes encoding flag-tagged tat and Renilaluciferase genes; these cells were then used to determine Tat-mediated HIV-1 transcriptional activity, as described previously (Shin et al. 2017). TZM-bl and bl-DTR cells were cultured in Dulbecco's modified Eagle's medium supplemented with 1% penicillin–streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (all obtained from Gibco-BRL, Gaithersburg, MD, USA). The bl-DTR cells were additionally supplemented with 1 μg/mL puromycin and 200 μg/mL zeocin. Peripheral blood mononuclear cells (PBMCs) were purchased from AllCells (Alameda, CA, USA) and cultured, as described previously (Yoon et al. 2015). HIV-1 clones pNL4-3 and AD8, as well as TZM-bl and A3.01 cells, were obtained from the National Institute of Health's AIDS Research and Reference Reagent Program (NIH, Bethesda, MD, USA). Organic chemical compounds were provided by the Korea Research Institute of Chemical Technology (KRICT) (Choi et al. 2009). The chemicals were renamed as follows: (1-(2-(dimethylamino)ethyl)-9-methoxybenzo[6, 7]oxepino[4, 3, 2-cd]isoindol-2(1H)-one (ID 262860): 1, 2, 8, 9-tetramethoxy-5-(2- (pyrrolidin-1-yl)ethyl)dibenzo[cd, f]indole-4(5H)-one < Compound 1 > : 1, 2, 8, 9- tetramethoxy-5-(2-(piperidin-1-yl)ethyl)- dibenzo[cd, f]indole-4(5H)-one < Compound 2 > : 5-(2-(diethylamino)ethyl)-tetramethoxydibenzo[cd, f]indole-4(5H)-one < Compound 3 > : 5-(2-(diethylamino)ethyl)-1, 2-dimethoxydibenzo[cd, f]indole-4(5H)-one < Compound 4 > : 1, 2, 9-trimethoxy-5-(2-(piperidin-1-yl)ethyl)dibenzo[cd, f]indole-4(5H) -one < Compound 5 > : 1, 2-dimethoxy-5-(2-piperidin-1- yl)ethyl)dibenzo[cd, f]indole-4(5H)-one < Compound 6 > : 2-amino-5-(2-piperidin-1-yl)ethyl)dibenzo[cd, f]indole-4(5H)- one < Compound 7 > : 8-fluoro-1, 2-dimethoxy-5-(2-piperidin-1-yl)ethyl) dibenzo[cd, f]indole-4(5H)-one < Compound 8 > : 8-cloro-1, 2-dimethoxy-5-(2-piperidin-1-yl)ethyl) dibenzo- [cd, f]indole-4(5H)-one < Compound 9 > .
Seliciclib, azidothymidine (AZT), adefovir (ADV), raltegravir (RAL), and elvitegravir (ELV) were purchased from Sigma Aldrich (St. Louis, MO, USA).
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To determine the inhibitory effect of the compounds on HIV-1 infection, TZM-bl cells were used, as described previously (Shin et al. 2020), with some minor modifications. In brief, 2 × 104 cells were cultured in 96-well plates for 24 h and then treated with compounds at a final concentration of 3 μmol/L (single dose assay; Fig. 1 and Table 1) or with 1:2 serially diluted compounds at concentrations ranging from 0 to 25 μmol/L (dose-dependent assay; Table 2). At 1 h after treatment, the cells were infected with the HIV-1NL4-3 virus at a multiplicity of infection (MOI) of 1. After 48 h, the inhibitory effect of the compounds was determined using a Bright Glo luciferase assay kit (Promega). The infectivity data are presented as a percentage relative to the DMSO control (vehicle).
Figure 1. Comparison of the anti-HIV-1 effect of aristolactam derivatives. TZM-bl cells (2 × 104 cells) were treated with 3 μmol/L of each Compound 1 h prior to infection with the HIV-1NL4-3 strain at an MOI of 1. After 48 h, viral infectivity was determined with a firefly luciferase assay kit. Cell viability was assessed using an MTT-based cell viability reagent. The tested compounds are as follows: A Chemical structure of aristolactam derivatives and seliciclib; B dibenzo[cd, f]indol-4(5H)-one (Compound 1), similar compounds containing benzo[6, 7]oxepino[4, 3, 2-cd]isoindol-2(1H)-one (ID 262860), and purine (seliciclib); C 50N substitution of 5-(2-(pyrrolidinyl)ethyl) (Compound 1), 5-(2-(piperidinyl)ethyl) (Compound 2), 5-(2-(diethylamino)ethyl) (Compound 3), and H-substitution at R6-R7 of 5-(2-(diethylamino)ethyl) (Compound 4) on dibenzo[cd, f]indol- 4(5H)-one; D methoxy-deleted forms on 5-(2-(piperidinyl)ethyl) (Compound 2). The graphical data are presented as a value relative to the vehicle (DMSO)-treated controls, as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 compared with cells treated with the vehicle.
Compound R1 R3 R4 R6 R7 HIV-1 infectivity (%) Cell survival rate (%) 1 -CH2CH2N pyrrolidine OMe OMe OMe OMe 3.62±0.27 88.07±1.88 2 -CH2CH2N piperidine OMe OMe OMe OMe 16.61±7.37 107.46±9.21 3 -CH2CH2N-(CH2CH3)2 OMe OMe OMe OMe 47.78±4.35 86.89±1.97 4 -CH2CH2N-(CH2CH3)2 OMe OMe H H 42.71±3.95 103.37±5.57 5 -CH2CH2N piperidine OMe OMe OMe H 0.98±0.27 106.63±11.69 6 -CH2CH2N piperidine OMe OMe H H 8.32±0.51 95.84±2.26 7 -CH2CH2N piperidine NH2 H H H 18.69±0.00 74.18±1.17 8 -CH2CH2N piperidine OMe OMe H F 0.48±024 85.50±7.76 9 -CH2CH2N piperidine OMe OMe H Cl 1.65±0.25 93.67±2.12 Seliciclib 45.01±1.99 103.57±4.30 ID 262860 29.35±1.33 46.34±0.93 The inhibitory effects on HIV-1 infection and cell viability were determined in TZM-bl cells infected with HIV-1NL4-3 at an MOI of 1. Table 1. Structure and inhibitory effect of aristolactam derivatives 1–9 on HIV-1 infection.
Compound IC50 (μmol/L)a CC50 (μmol/L)b SIc 1 0.69 ± 0.09 6.88 ± 0.31 9.94 2 1.03 ± 0.38 16.91 ± 3.22 16.45 3 3.73 ± 1.00 17.15 ± 0.34 4.59 4 3.07 ± 0.22 6.98 ± 0.09 2.27 5 1.07 ± 0.06 4.51 ± 0.96 4.23 6 1.06 ± 0.03 4.97 ± 0.62 4.70 7 2.00 ± 0.61 3.62 ± 0.15 1.80 8 0.55 ± 0.01 3.72 ± 0.01 6.74 9 0.44 ± 0.01 3.64 ± 0.08 8.31 Seliciclib 2.29 ± 0.40 25.49 ± 0.17 11.11 The inhibitory effects on HIV-1 infection and cell viability were determined in TZM-bl cells infected with HIV-1NL4-3 at an MOI of 1.
aIC50: half-maximal inhibitory concentration.
b CC50: concentration that reduces cell viability by 50%.
cSI: selectivity index, i.e. the ratio of IC50 to CC50.Table 2. Concentration–responses of aristolactam derivatives on cytotoxicity and anti-HIV activity.
The inhibitory effects of the compounds on viral replication were determined, as described previously (Shin et al. 2020). In brief, 5 × 104 cells/well of A3.01 cells, PBMCs and MOLT4-R5 cultured without activation were infected with HIV-1NL4-3 or HIV-1AD8 at an MOI of 0.1 in 96-well plates for 4 h. After infection, the compounds were added to the infected cells at a final concentration of 3 μmol/L. After 72 h of treatment, the inhibitory effect of the compounds on viral replication was determined by measuring the amount of p24, a HIV capsid protein, using an HIV-1 p24 ELISA kit. Cell viability was determined using the 3-2, 5-diphenyltetrazolium bromide (MTT)-based PrestoBlue Cell Viability Reagent (Invitrogen) according to the manufacturer's instructions.
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To determine the inhibitory effect of the compounds on Tat-mediated transcription, a concentration–response assay was performed, as described previously (Shin et al. 2020). In brief, 1 × 104 bl-DTR cells cultured in a 100 μL medium were treated with serial dilutions of the compounds (0–25 μmol/L), after which the expression of Tat and renilla luciferase was induced by adding 50 μL doxycycline to achieve a final concentration of 50 ng/mL. After 24 h of treatment, the activities of Tat-induced firefly luciferase and doxycycline-induced renilla luciferase were determined using a dual luciferase assay kit (Dual Glo, Promega), as described previously. The data are presented as a percentage relative to the DMSO control (vehicle) in the presence of doxycycline. The experiment was performed in triplicate.
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Reverse transcriptase activity was determined, as described previously, with certain modifications (Clouser et al. 2010). In brief, after treatment with DNase I, 2 × 105 TZM-bl cells were treated with the indicated compounds for 1 h prior to infection with HIV-1NL4-3 (at a MOI ratio of 1). Sixteen hours after infection, cytosolic DNA from the cells was isolated and the levels of reverse transcription (RT) products were determined by quantitative PCR (qPCR). The primers for RT products were 5′- GGTCCAAAATGCGAACCCAG-3′ (forward) and 5′- TCTTGCTTTATGGCCGGGTC-3′ (reverse). To determine the relative levels of the RT products, rRNA from the lysed cells were analyzed with one-step quantitative RTPCR using the following primer sets for 18S rRNA: 5′- GTAACCCGTTGAACCCCATT-3′ (forward) and 5′- CCATCCAATCGGTAGTAGGG-3′ (reverse). The relative level of each RT product was analyzed using the delta/ delta CT method, as described previously (Shin et al. 2020).
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To determine how the compounds inhibited HIV-1 infection, the integrase activity was assessed using an XpressBio HIV-1 integrase assay kit, according to the manufacturer's protocols.
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All data are expressed as the mean ± SD (n = 3). The data were compared using a Student's t test, and *P < 0.05, ** P < 0.01 was considered to be statistically significant. All statistical analyses were performed using Prism software (v.5.0; Graph Pad Software, San Diego, CA, USA).
Cells, Virus, and Reagents
Inhibition of HIV-1 Infection
Inhibition of Tat-mediated Transcription
Reverse Transcriptase Assay
Integrase Assay
Statistical Analysis
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Initially, a random screen of an organic compound library identified an aristolactam derivative (Compound 1) that inhibited HIV-1 infection. To explore whether the dibenzoindole moiety in the core of the aristolactam derivative is critical for the inhibition of HIV-1 infection, the antiviral effects of benzo[6, 7]oxepino[4, 3, 2-cd]isoindol-2(1H)-one (ID 262860), a compound that is structurally similar to dibenzo[cd, f]indol-4(5H)-one (Compound 1), and seliciclib, which bears a purine moiety that is structurally similar to an indole moiety (Fig. 1A), were compared at a final concentration of 3 μmol/L. As shown in Fig. 1B, the compound bearing a benzo-oxepino-isoindol moiety (ID 262860) had a moderate inhibitory effect on HIV-1 infection and was cytotoxic. Seliciclib, a known inhibitor of HIV-1 infection, had a moderate inhibitory effect on HIV-1 infection and was not cytotoxic. Dibenzo[cd, f]indol-4(5H)- one (Compound 1) exhibited the greatest inhibitory effect on HIV-1 infection and was slightly cytotoxic at the concentration tested (Fig. 1B and Table 1). To investigate whether variation of the R1 side chain on dibenzo[cd, f]indol-4(5H)-one influences the anti-HIV-1 effect, 5-(2- (pyrrolidiny)lethyl) of Compound 1 was substituted to 5-(2-(piperidinyl)ethyl) (Compound 2) and 5-(2-(diethylamino)ethyl) (Compound 3). Although the 5-(2-(piperidinyl)ethyl) (Compound 2) substitution inhibited HIV-1 infection slightly less than the Compound 1 containing pyrrolidine, Compound 2 exhibited no cytotoxicity at the same concentration. However, 5-(2-(diethylamino)ethyl) (Compound 3) had a low inhibitory effect on HIV-1 infection and cell viability (Fig. 1C and Table 1). Since 5-(2-(piperidinyl)ethyl) (Compound 2) exhibited no cytotoxicity and had an efficient antiviral effect, we explored an extended set of derivatives substituted at the R3-R4-R6-R7 position on Compound 2 in order to find agents that could have a potent inhibitory effect on HIV-1 infection while lowering toxicity. As shown in Fig. 1D and Table 1, the substitution of R7-H (Compound 5) greatly improved the inhibitory effect of the compound on HIV-1 infection without increasing cytotoxicity. However, the additional deletion of the methoxy moiety (Compound 6 and 7) slightly reduced the inhibitory effect of the compound and increased cytotoxicity, whereas substitution with R7-F (Compound 8) or R7-Cl (Compound 9) greatly improved the inhibitory effect on HIV-1 infection and decreased cell viability (Fig. 1D and Table 1). The deletion effect of these methoxy was not detected in a compound bearing the 5-(2- (diethylamino)ethyl) moiety (Compound 4) (Fig. 1C). These results show that the aristolactam derivatives containing a dibenzo-indole core inhibited HIV-1 infection, and provided proof-of-principal that R1-substitution of (piperidinyl)ethyl and (pyrrolidinyl)ethyl moieties connected to the lactam ring increased anti-HIV-1 activity, and that substitutions at R3-R4-R6-R7 on Compound 2 influenced antiviral activity and cell viability. The inhibitory effect of the aristolactam derivatives on HIV-1 infection and cell viability (tested at a concentration of 3 μmol/L) are summarized in Table 1.
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To accurately evaluate the potency of the aristolactam derivatives, TZM-bl cells infected with HIV-1NL4-3 were treated with 1:2 serially diluted compounds at a starting concentration of 25 μmol/L. The cytotoxicity of the compounds was also evaluated. As a control, seliciclib had a half-maximal inhibitory concentration (IC50) value of 2.29 μmol/L and a selectivity index (SI, the ratio of IC50 to CC50: a concentration which reduces cell viability by 50%) of 11.11; this value was similar to that reported in a previous study (Shin et al. 2020). As shown in Table 2, 5-(2- pyrrolidinyl)ethyl) (Compound 1) exhibited a strong inhibitory effect, with an IC50 value of 0.69 μmol/L. The IC50 values of 5-((2-piperidinyl)ethyl) (Compound 2) and 5-(2- diethylamino)ethyl) substituents (Compound 3) were 1.03 μmol/L and 3.73 μmol/L, respectively. The CC50 values of Compound 1, Compound 2, and Compound 3 were 6.88 μmol/L, 16.91 μmol/L, and 17.15 μmol/L, respectively, and the selectivity indices were 9.94, 16.45, and 4.59, respectively. From these results, the 5-(2-piperidinyl)ethyl) substitution at R1 (Compound 2) showed the highest SI with low cytotoxicity. The effect of substituting side chains at R3-R4-R6-R7 on 5-(2-(piperidinyl)ethyl)- dibenzo[cd, f]indol-4(5H)-one (Compound 2) was evaluated; two derivatives substituted to R7-H (Compound 5) and R6-R7-H (Compound 6) had IC50 values of 1.07 μmol/L and 1.06 μmol/L, respectively, which were similar to the IC50 value of tetra-methoxy (Compound 2); however, they exhibited severe cytotoxicity, as indicated by CC50 values of 4.51 μmol/L and 4.97 μmol/L, respectively, and low SIs (4.23 and 4.70, respectively). The compound with H-substitution at positions R4-R6-R7 (Compound 7) showed the highest cytotoxicity, as indicated by the lowest SI (1.80). Substitution of R7-F (Compound 8) and R7-Cl (Compound 9) increased the antiviral effect (IC50 values of 0.55 μmol/L and 0.44 μmol/L, respectively), but decreased cell viability (CC50 values of 3.72 μmol/L and 3.64 μmol/L, respectively), and resulted in low SIs of 6.74 and 8.31, respectively. An H-substituent (Compound 4) at R6-R7 on 5-(2-(diethylamino)ethyl) (Compound 3) increased cytotoxicity (CC50 value of 6.98 μmol/L), but its antiviral effect was not higher than that of Compound 3. Consequently, among the 50N-substituents (R1), 5-(2-(piperidinyl)ethyl) (Compound 2) exhibited good inhibition of HIV-1 infection and the lowest cytotoxicity. H-substitution at positions R6-R7 on Compound 2 increased cytotoxicity, and substitution to F and Cl at position R7 increased the anti-HIV-1 effect, but this was accompanied by high cytotoxicity (Table 2). These data indicate that aristolactam derivatives potently inhibited HIV-1 infection, while cytotoxicity increased greatly at concentrations of over 6.25 μmol/L (data not shown).
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To determine the inhibitory effect of the aristolactam derivatives on the overall HIV-1 life cycle, HIV-1 replication assays were performed in A3.01 T-cell lines (Fig. 2A) and PBMCs (Fig. 2B) infected with CXCR4 tropic HIV-1NL4-3 in the presence or absence of these compounds. 5-(2-pyrrolidinyl)ethyl (Compound 1) and derivatives of 5-(2-piperidinyl)ethyl (Compounds 2 and 5–9) almost completely abrogated viral replication at a concentration of 3 μmol/L, with a cell survival rate of over 50%. 5-(2-(diethylamino)ethyl) (Compound 3) and its R6- R7-H substituted form (Compound 4) showed less inhibition of viral replication, which accorded with its reduced effect on viral infection. As a representative compound, Compound 1 suppressed replication of the CCR5 tropic HIV-1AD8 strain in the MOLT4-R5 cell line and in PBMCs; these effects were similar to the inhibitory effect of this compound on replication of the CXCR4 tropic HIV-1NL4-3 strain at the same concentration (Fig. 2C).
Figure 2. Inhibitory effect on viral replication. A3.01 cells (A) and PBMCs (B) (both 5 × 104) cultured without activation were infected with HIV-1NL4-3 at an MOI of 0.1 for 4 h and then treated with compounds (3 μmol/L) for 72 h. The inhibitory effect of the compounds on viral replication was determined by measuring the amount of p24 using an HIV-1 p24 ELISA kit. Cell viability was determined as described in Materials and Methods. C Cells (5 × 104 of MOLT4-R5 and PBMCs) infected with HIV-1AD8 at an MOI of 0.1 for 4 h were treated with (Compound 1) (3 μmol/L) for 72 h, and the p24 levels and cell viability were determined as shown above. Data (A–C) are presented as a value relative to the vehicle (DMSO)-treated controls, as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 compared with the cells treated with the vehicle.
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To further define the mode of action associated with the inhibitory effects of the compounds on HIV-1 infection, a HIV-1 reverse transcriptase (RTase) assay and an integrase assay were performed. Initial studies used Compound 1 as a representative compound, since it was efficacious in inhibiting HIV-1 infection. RTase inhibitors azidothymidine (AZT) and integrase inhibitors (elvitegravir; ELG and raltegravir; RAL) inhibited the activity of RTase and integrase, respectively, but Compound 1 did not inhibit the activity of either enzyme (Fig. 3A, 3B).
Figure 3. Determination of the anti-HIV-1 mode of action of the aristolactam derivatives. A Each compound was administered to TZM-bl cells for 1 h prior to infection with HIV-1NL4-3 at a final concentration of 10 μmol/L. After 16 h of infection, the cells were harvested and the levels of the RT product were determined as described in Materials and Methods. B An INTase assay was performed with 10 μmol/L of the indicated compounds according to the manufacturer's protocol. C bl-DTR cells (1 × 104) were treated with 3 μmol/L of the indicated compounds and then cultured in the presence of doxycycline (50 ng/mL). After 24 h of treatment, firefly luciferase and renilla luciferase activity was determined using the Dual-Glo-Luciferase assay system. The data (A–C) are presented as a value relative level to the vehicle (DMSO)-treated controls, as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 compared with the cells treated with vehicle. AZT; azidothymidine, ADV; adefovir, ELG; elvitegravir, RAL: raltegravir, T20: a fusion inhibitor.
To examine whether the aristolactam derivatives can inhibit HIV-1 transcription induced by the viral transcriptional factor Tat, a dual reporter system was employed. As this system compares the Tat-induced activity of firefly luciferase (F-Luc) or β-galactosidase (β-gal) with renilla luciferase (R-Luc) activity indicating normal cellular transcriptional activity, it can be used to assess the inhibitory effect of compounds on Tat-induced HIV-1 transcription. Compound 1 inhibited Tat-induced HIV-1 transcription without inhibiting R-Luc activity (Fig. 3C). Thus, a concentration–response assay of each of the aristolactam derivatives was conducted to determine their potency at inhibiting Tat-mediated viral transcription. All derivatives impaired HIV-1 transcriptional activity as measured by decreases in both F-Luc and β-gal activity at below 6.25 μmol/L, with no decrease in R-Luc activity (Fig. 4). Notably, 5-(2-pyrrolidinyl)ethyl (Compound 1) at a concentration of approximately 3 μmol/L completely inhibited HIV-1 viral transcription, with an IC50 value of 1.1 μmol/L (Table 3). R1 substitution of Compound 1 to 5-(2-piperidinyl)ethyl (Compound 2) and 5-(2-diethylamino)ethyl (Compound 3) resulted in IC50 values of 2.85 μmol/L and 2.58 μmol/L, respectively. Among the derivatives of Compound 2, an R7-H derivative (Compound 5) exhibited the highest inhibitory effect, with an IC50 value of 1.36 μmol/L, and the IC50 values of the other derivatives (Compound 6–9) were between * 1.78–3.38 μmol/L (Fig. 4 and Table 3). The inhibitory effect of each of the derivatives on HIV-1 transcription was generally similar to their inhibitory effect on HIV-1 inhibition (Fig. 4 and Table 2). These data revealed that aristolactam derivatives bearing a dibenzo[cd, f]-indol backbone exerted an inhibitory effect on Tat-induced viral transcription, which accounts for their inhibitory effects on HIV-1 infection. The increase in R-Luc activity in combination with a decrease in F-Luc activity might have been due to an excess of common machinery for transcription caused by the suppression of F-Luc expression (Shin et al. 2020).
Figure 4. Concentration–response of the aristolactam derivatives. blDTR cells (1 × 104) were cultured in 100 μL medium for 24 h and then two-fold serial dilutions of each compound were added prior to addition of doxycycline (final concentration, 50 ng/mL). After 24 h of treatment, the activity of firefly luciferase (F-Luc, closed circle) and renilla luciferase (R-Luc, open circle) was determined using the DualGlo-Luciferase assay system. Beta-galactosidase activity (triangle) was measured using the β-galactosidase enzyme assay system in parallel with the luciferase assay. Seliciclib was used as the experimental control. The data are presented as the mean ± SD (n = 3).
Compound IC50 (μmol/L)a 1 1.11 ± 0.02 2 2.85 ± 0.17 3 2.58 ± 0.05 4 3.40 ± 0.02 5 1.36 ± 0.01 6 2.47 ± 0.26 7 1.78 ± 0.15 8 2.25 ± 0.24 9 3.38 ± 0.05 Seliciclib 2.25 ± 0.14 aThe compounds were assessed in bl-DTR cells using a concentration–response test. Table 3. Concentration–responses of aristolactam derivatives on the inhibition of Tat-induced HIV-1 transcription.