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Antimicrob Agents Chemother. Sep 2008; 52(9): 3169–3179.
Published online Jul 14, 2008. doi:  10.1128/AAC.00274-08
PMCID: PMC2533482
Heterocyclic Compounds That Inhibit Rev-RRE Function and Human Immunodeficiency Virus Type 1 Replication[down-pointing small open triangle]
Deidra Shuck-Lee,1 Fei Fei Chen,1 Ryan Willard,1 Sharmila Raman,1 Roger Ptak,2 Marie-Louise Hammarskjold,1 and David Rekosh1*
Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908,1 Southern Research Institute, Department of Infectious Disease Research, Frederick, Maryland 217012
*Corresponding author. Mailing address: Department of Microbiology, Jordan Hall 7087, P.O. Box 800734, University of Virginia, Charlottesville, VA 22908. Phone: (434) 982-1599. Fax: (434) 982-1590. E-mail: dr4u/at/
Received February 27, 2008; Revised May 4, 2008; Accepted July 7, 2008.
A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 μM concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.
Most of the current drugs in use for the treatment of AIDS work by targeting the enzymatic activities of the human immunodeficiency virus (HIV) reverse transcriptase or protease, although entry (7) and integrase (13) inhibitors are starting to be used, and presently there is also promising development of other novel targets (51, 59). However, because of the emergence of drug-resistant virus that commonly occurs as the result of treatment, there remains a great need to continue the search for alternative therapies that target other essential viral activities.
The Rev protein is absolutely essential for HIV replication (for a review see reference 49). Proviral clones lacking a functional rev gene have no replicative ability, even in established tissue culture cell lines or peripheral blood mononuclear cells (PBMCs). In the absence of Rev, genomic RNA and several other HIV mRNAs cannot exit the nucleus (22, 30, 42). Thus, viral structural proteins are not made and the infectious cycle cannot continue. It is thus clear that modalities inhibiting the function of Rev could form the basis for therapy against HIV infection and AIDS.
Although the Rev/RRE (Rev response element) export pathway is still not fully understood, several important steps have been identified (see reference 49). The pathway starts with the import of Rev into the nucleus (34, 58). Rev then binds specifically to RNA containing the RRE (17, 28, 30, 42, 53) and multimerizes on the RRE in a process believed to involve protein-protein as well as protein-RNA interactions (12, 14, 16, 32, 36, 43, 67). The Rev-RRE complex is then recognized by Crm1 (exportin 1; official gene symbol, XPO1) and RAN-GTP (1), which initiates the export process and eventually targets the complex to the nuclear pore, where it interacts with nucleoporins (1, 4, 25, 70). This results in translocation of the complex to the cytoplasmic side. Many details in the pathway have yet to be elucidated, and other cellular proteins (e.g., RIP, EIF5A, actin, and RNA helicases) may also play specific, albeit yet unclear, roles (3, 35, 54, 65, 66). It is also not clear what happens once the complex reaches the cytoplasm, but some studies suggest that Rev also promotes translation (15, 48).
From the standpoint of therapeutic development, it is important that the interactions that mediate some of the steps of Rev function are completely viral in nature. Rev-RRE binding and multimerization can readily be demonstrated in vitro with purified viral components (12, 14, 16, 32, 36, 43, 67). Cellular factors are not necessary for these interactions in vitro, although they are likely to have influences in the cell. Thus, targeting of the virus-specific interactions by a therapeutic agent could potentially lead to specific inhibition of viral replication, with no or only minimal side effects on cellular functions. Proof of principle that viral replication can be inhibited by interfering with various steps in the Rev pathway has already been given through the use of different classes of Rev mutants (2), as mutants in each of the Rev functional domains that abolish viral replication have been described (for a review see reference 49).
Some small-molecule compounds that inhibit HIV type 1 (HIV-1) replication and Rev-RRE function have been identified (33, 46, 64, 69), but none has progressed very far in preclinical development, in part because of cellular toxicity issues. The aminoglycoside antibiotic neomycin, previously known to inhibit structures in rRNAs, has been shown to inhibit Rev function by binding the RRE and blocking Rev-RRE interaction (29, 39, 68). Leptomycin B has been shown to target Crm1 (25), preventing the transport of Rev-RRE mRNA through the nuclear pore complex (21, 63). Various peptides are also under investigation for Rev inhibition (18, 20, 57). RRE decoys (40, 56) and a transdominant-negative version of the Rev protein (41, 45, 62) have also been described as potential anti-Rev therapeutic candidates.
In this study, we performed a cell-based drug screening assay that targeted the postintegration phase of the HIV life cycle. From this screen, two small molecules were identified that inhibit HIV replication through inhibition of HIV Rev function. The molecules represent good candidates for further anti-HIV drug development and provide proof of principle that targeting Rev function with small-molecule inhibitors is worthy of further pursuit.
Compound library.
Compounds used for screening were obtained in a library of approximately 40,000 compounds from Specs (Delft, The Netherlands) by Message Pharmaceuticals (Malvern, PA).
Primary screening assay.
The 5BD.1 cells used in this assay have been previously described (55). They were made by calcium phosphate transfection of CMT3 COS cells with pCMVGagPol-RRE, pCMVrev, or pCMVenv. They express pseudovirions in a Rev-dependent fashion. For screening, the cells were suspended in Iscove's modified Dulbecco's medium (IMDM), supplemented with 10% fetal calf serum, 0.2 mg/ml hygromycin B, 1.5 mg/ml G418, and 0.50 μg/ml gentamicin sulfate, and added to each well of 384-well plates at a concentration of 4,500 cells per 40 μl per well. Compound was then added to 10 μM for 16 h. The medium was removed and discarded, and fresh medium (40 μl) with 10 μM compound was applied. The cells were incubated for an additional 8 h, at which time 25 μl of supernatant was collected and used in a p24 enzyme-linked immunosorbent assay (ELISA) (61). Compounds that inhibited p24 production by at least 50% were selected as primary hits.
Dose-response assay.
5BD.1 cells suspended in medium (IMDM supplemented with 10% fetal calf serum, 0.2 mg/ml hygromycin B, 1.5 mg/ml G418, and 0.50 μg/ml gentamicin sulfate) were added to each well of 96-well plates at a concentration of 20,000 cells per 135 μl per well. The plates were then incubated in the presence of various concentrations (30, 10, 3, 1, 0.3, 0.1, or 0 μM for controls) of the 192 primary selected compounds for 16 h. After 16 h the medium was removed and discarded and 135 μl of fresh medium with the appropriate concentration of compound was added. The cells were incubated for an additional 24 h, and supernatant was collected and used in a p24 ELISA.
Toxicity assays.
The toxicity of the compounds in 5BD.1 and MT-4 cells and PBMCs was evaluated by use of the Promega nonradioactive cell proliferation assay. Cells were incubated with compound at the concentration and the length of time indicated in the text or figure legend, and the assay was performed according to the manufacturer's directions. The assay uses a tetrazolium salt [MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and the electron coupling reagent phenazine methosulfate. MTS is reduced by dehydrogenases in cells into formazan that can be measured by absorbance at 492 nm. The production of formazan is proportional to the number of living cells. Toxicity is calculated based on formazan production, in comparison to cells that did not receive compound.
Dual-luciferase Rev assay.
Inhibition of the HIV-1 Rev function was directly assayed using a HeLa cell line that had been specifically engineered to monitor Rev function (R. Ptak, unpublished data). To make this line, the pDM128 Rev reporter plasmid, which has been previously described by Hope et al. (37), was modified by replacing the chloramphenicol acetyltransferase coding sequence with that of Renilla luciferase and substituting the Tet-Off promoter for the cytomegalovirus (CMV) immediate-early promoter. This vector was then used to transfect a line of HeLa cells, which had been previously engineered to express both firefly luciferase and HIV-1 Rev from a bicistronic mRNA that was also under the control of the Tet-Off promoter. Thus, in the presence of doxycycline none of the transfected genes are expressed. However, in the absence of doxycycline, the resultant cell line expresses both luciferases and Rev, with Renilla luciferase expression being dependent upon Rev function and firefly luciferase expression being independent of Rev function. Firefly luciferase can therefore be used to assay for nonspecific or toxic compounds and also as a surrogate marker for Rev expression levels. Compounds that inhibit the Rev function specifically, without inhibiting Rev expression, would be expected to reduce the expression of Renilla luciferase and have no effect on the expression of firefly luciferase. Assays were performed in a 96-well format by plating cells (2 × 104/well) in the presence of test compound (triplicate wells). Luciferase expression levels were determined after 24 h of incubation with the use of dual-luciferase assay reagents (Promega) following the manufacturer's instructions.
Transient-transfection assay.
pCMVGagPol constructs containing either RRE and pCMV Rev or Mason-Pfizer monkey virus constitutive transport element (CTE) were transfected into 293T cells using a standard calcium phosphate protocol as previously described (38). Various concentrations of the compounds (0, 1.875, 3.75, 7.5, and 15 μM) were tested by suspension in the transfection culture medium (IMDM supplemented with 10% fetal calf serum and 50 μg/ml gentamicin sulfate). Supernatants were harvested 72 h posttransfection and evaluated for p24 by ELISA.
U1 cell latency reactivation assay.
U1 cells (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, courtesy of Thomas Folks) (24) were suspended in medium (RPMI supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin), and 100 μl was added to wells of 96-well plates at a concentration of 50,000 cells per ml. Cells were induced with 5 ng/ml tumor necrosis factor alpha (TNF-α), and compounds were simultaneously added at various concentrations (compound 103833: 0, 0.12, 0.4, 1.2, 3.8, and 11.9 μM; compound 104366: 0, 0.25, 0.8, 2.5, 7.9, and 25.1 μM). Cultures were incubated for 3 days, and the supernatants were harvested for reverse transcriptase analysis (8). Toxicity was determined by MTS dye reduction of the cells as described above. In some cases, cells were also harvested and analyzed for protein expression using monoclonal antibodies directed against either p24 or Nef.
In vitro electrophoretic mobility (gel shift) assay.
Plasmids containing a 234-bp RRE were linearized and transcribed in vitro using [32P]UTP and T7 polymerase into an RNA probe that was gel purified using a Riboprobe transcription kit (Promega). The RNA was then folded by suspension in a renaturation buffer (500 mM NaCl, pH 7.8, 100 mM HEPES-KOH, 20 mM MgCl2) at 80°C for 3 min, followed by slow cooling to 4°C. To perform the gel shift assay, 0.5 ng of the RRE was incubated with 38 ng of bacterially synthesized Rev protein in binding buffer (200 mM KCl, pH 7.8, 20 mM HEPES-KOH, 4 mM MgCl2, 1 mM EDTA, 2 mM dithiothreitol, 20% glycerol) on ice for 10 min in the presence of various concentrations of compounds. The complexes were then run on a native 4% polyacrylamide gel. The gel was analyzed using a phosphorimager.
HIV-1 replication assay in PBMCs.
HIV-1 93BR021 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, courtesy of the WHO-UNAIDS Network for HIV Isolation and Characterization. Fresh human PBMCs, seronegative for HIV and hepatitis B virus, were isolated from blood of screened donors (Biological Specialty Corporation, Colmar, PA) using lymphocyte separation medium (Cellgro by Mediatech, Inc.; density, 1.078 ± 0.002 g/ml) following the manufacturer's instructions. Cells were stimulated by incubation in 4 μg/ml phytohemagglutinin (PHA; Sigma) for 48 to 72 h. Mitogenic stimulation was maintained by the addition of 20 U/ml recombinant human interleukin-2 (R&D Systems, Inc.) to the culture medium. PHA-stimulated PBMCs from at least two donors were pooled, diluted in fresh medium, and added to 96-well plates at 5 × 104 cells/well. Cells were infected (final multiplicity of infection, ≈0.1) in the presence of nine different concentrations of test compounds (triplicate wells/concentration) and incubated for 7 days. To determine the level of virus inhibition, cell-free supernatant samples were collected for analysis of reverse transcriptase activity (8). Following removal of supernatant samples, compound cytotoxicity was measured by the MTS toxicity assay as described above.
HIV replication assay in SupT1 cells.
SupT1 cells were placed in six-well plates at the concentration of 5 million cells per well in 2 ml of RPMI 1640 medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin sulfate. Two hundred nanograms of hexadimethrine bromide (Polybrene) was added to each well, and the plate was incubated for 30 min at 37°C. Fifty nanograms of p24 equivalents of HIV-1 (NL4-3) was then added to the corresponding well, and the plate was incubated for an additional 4 h at 37°C. Upon completion of the incubation, the 2-ml well contents were transferred to corresponding 15-ml conical tubes and centrifuged at 3,000 rpm for 5 min. The supernatant was discarded, and the infected cells were suspended in 5 ml of fresh RPMI 1640 medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin sulfate and the appropriate amount of compound. The resultant cultures were placed in 25-cm2 flasks, which were split every 3 days by removing 3 ml of each culture and adding back 3 ml of fresh medium with the appropriate level of compound to each culture flask. Virus yield into the supernatant of each culture was measured by p24 ELISA.
Measurement of p24 by ELISA.
p24 levels in viral culture or transfection supernatants were measured using a p24 ELISA following a published procedure (61).
Calculation of EC50 and TC50.
We have expressed our inhibition data using the concept of 50% effective concentration of inhibition (EC50), rather than 50% inhibitory concentration, since our experiments use whole cells in which the actual intracellular concentration of compound is not known. Similarly 50% toxicity concentration (TC50) is also an effective concentration. The EC50 and TC50 were calculated using a formular fit analysis for the initial screening and dose-response experiments. For all other experiments the values were calculated by interpolation between the two data points surrounding the value to be calculated.
Cell-based screening assay for postintegration steps in the HIV life cycle.
We previously have described a cell line that constitutively expresses high levels of the HIV-1 structural proteins in a Rev-dependent fashion (55). This cell line, 5BD.1, produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. We reasoned that this cell line could be used to screen for compounds that inhibit postintegration steps in the HIV life cycle, because addition of a compound to the cells that inhibits a critical postintegration HIV function should cause a decrease in particle production. This could easily be quantified by measuring p24 levels in the medium.
The assay using these cells was performed at Message Pharmaceuticals (Malvern, PA) in a 384-well microtiter plate format. (Message Pharmaceuticals suspended operations in early 2004.) Forty thousand compounds in 1% dimethyl sulfoxide (DMSO) from a library supplied by Specs B.V. (Delft, The Netherlands) were screened at a final concentration of 10 μM (0.001% DMSO). To promote the identification of compounds that might be inhibitors of Rev function, we carried out a two-step compound addition procedure, where compound was added for 16 h, washed off with the medium, and readded. After an additional 8 h of incubation, medium was collected and assayed for p24. Since Rev acts at an upstream step in the gene expression-particle assembly pathway, this two-step drug addition procedure, which discards the p24 produced during the first 16 h of drug addition, is needed to allow Rev inhibitors to score in the assay. Figure Figure11 shows the p24 values obtained for a representative 5,120 of the 40,000 compounds, expressed as a percentage of the control value. Compounds that inhibited p24 production by more than 50% were retained for further study. In the representative batch shown, 13 compounds gave p24 values that were less than 50% of the control. In total, 192 compounds, from the 40,000 screened, caused greater than 50% inhibition of p24.
FIG. 1.
FIG. 1.
Primary screening assay in 5BD.1 cells. 5BD.1 cells constitutively producing HIV-like particles in a Rev-dependent manner were used to test the initial 40,000 heterocyclic compounds. p24 production was measured by ELISA to compare viral particle production (more ...)
Each of the 192 compounds was further screened for specificity and toxicity in six-point dose-response assays, using the same 5BD.1 cell line. To perform the assays, compounds were added to the cells that were plated into 96-well plates at 20,000 cells per well. The plates were incubated in the presence of various concentrations of the 192 compounds for 16 h. After 16 h the medium was removed and discarded. One hundred thirty-five microliters of fresh medium with the appropriate concentration of compound was applied, and the cells were incubated for an additional 24 h. Supernatants were collected and used in a p24 ELISA. To measure toxicity, the cells were collected after 40 h and subjected to the Promega CellTiter One Solution cell proliferation assay (Promega, Madison, WI). This assay measures the bioreduction of MTS, which is a measure of cells that have metabolically active dehydrogenases.
At all concentrations tested, the compounds showed little toxicity in the 5BD.1 cell line. To further examine the toxicity of the compounds in a cell line that is infectible by HIV, the compounds were tested in the MT-4 T-cell line (52) after 5 days of incubation. In total, 11 compounds emerged as candidate molecules. The structure of each of these 11 compounds is shown in Fig. Fig.2,2, and the EC50s and TC50s from dose-response assays are shown in Table Table1.1. (The code used for naming each compound was generated by Message Pharmaceuticals.)
FIG. 2.
FIG. 2.
Structures of compounds selected for further study. The structures of each of the compounds that gave EC50s lower than 30 μM in the 5BD.1 cell assay, with at least a 1-log difference in toxicity, are shown.
EC50s and TC50s of compounds selected from the efficacy and toxicity dose-response assaysa
Figure 3A and B show the efficacy and toxicity dose-response assays for two of the compounds, 3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (103833) and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile (104366). Both compounds showed a good linear response up to concentrations of 30 μM, with little toxicity. Figure Figure3C3C shows the toxicity of the compounds in the MT-4 T-cell line after 5 days of incubation. In this case, both compounds showed little toxicity up to 10 μM, after which toxicity was readily apparent.
FIG. 3.
FIG. 3.
Efficacy and toxicity dose-response assays for 103833 and 104366 in 5BD.1 cells. All assays were performed in triplicate, and the error bars represent standard deviations. (A) Six concentrations of 103833 were tested in 5BD.1 cells for inhibition of p24 (more ...)
Activity of the compounds in a cell-based assay for Rev function.
Since our laboratory has a longstanding interest in HIV Rev function, we next tested the compounds in an assay that was designed to measure inhibition of Rev activity. The assay uses a derivative of a HeLa cell line that contains a stably integrated Rev reporter construct. The construct is a variation of a well-established reporter plasmid that expresses the chloramphenicol acetyltransferase gene in a Rev-dependent context from the CMV immediate-early promoter, in that it has Renilla luciferase substituted for the chloramphenicol acetyltransferase gene (37). The cell line also contains a second reporter, firefly luciferase, which serves as a specificity control. The firefly luciferase is expressed from a stably integrated bicistronic expression construct that also expresses the HIV-1IIIB rev gene. To perform the assay, compound is added to the medium at various concentrations, and doxycycline is removed to induce both luciferases and Rev expression. The two luciferases are measured after 24 h of incubation at 37°C. If a drug candidate is a specific inhibitor of Rev function, it would be expected to decrease the amount of Renilla luciferase produced but not the amount of firefly luciferase.
Table Table22 shows the EC50s and TC50s for the inhibition of the two luciferases for all 11 compounds tested. Figures 4A and B show the dose-response curves for 103833 and 104366, which generated EC50s of 1.07 μM and 2.24 μM, respectively, for inhibition of the Renilla luciferase, with at least a 10-fold-higher value for the inhibition of the firefly luciferase (TC50) by each compound. Although some inhibition of firefly luciferase was observed for 103833 at concentrations near the Renilla luciferase EC50, an unexplained plateau value was reached, and a good differential was observed at higher concentrations. As a control leptomycin B was also tested (Fig. (Fig.4C).4C). This is a known inhibitor of HIV Rev function, since it is known to bind to and inhibit the Rev cofactor Crm1 (25). At a concentration of 10 nM, a clear differential between the two luciferase activities was observed, confirming the specificity of the assay, although the compound is quite toxic to cells at higher concentrations.
Specificities of compounds in the Rev functional assaya
FIG. 4.
FIG. 4.
Dose-response assays for 103833 and 104366 comparing their effects on Rev-dependent and Rev-independent luciferases. HeLa cells containing stably integrated expression plasmids for Renilla and firefly luciferases were treated with the indicated concentrations (more ...)
The data in Table Table22 and Fig. Fig.44 show that compounds 89246, 103833, and 104366 scored best as potential specific inhibitors of Rev function. However, due to supply limitations of compound 89246, only compounds 103833 and 104366 were chosen for further study.
103833 and 104366 show specific inhibition of the Rev-RRE pathway versus the CTE pathway.
HIV-1 uses the Rev-RRE pathway to mediate export of unspliced and incompletely spliced mRNA. Other retroviruses use different mechanisms to accomplish the same task (31). The prototypical example of a simpler retrovirus that utilizes a pathway that is distinct from the Rev pathway is Mason-Pfizer monkey virus. This virus uses a cis-acting RNA element called the CTE that interacts with the cellular protein Tap to mediate export (6, 27). The Tap export pathway works independently of the Rev cofactor Crm1 and is not sensitive to inhibition by leptomycin B (47, 63). In order to determine if compounds 103833 and 104366 specifically affected the Rev-RRE pathway but not the CTE-mediated pathway, we compared HIV-1 particle production mediated by the two pathways for the ability to be inhibited by the compounds, using transient transfection. To do this experiment, 293T cells were either cotransfected with pCMVGagPol-RRE and pCMVRev or transfected with pCMVGagPol-CTE. The former transfection produces HIV pseudovirions using the Rev-dependent pathway, while the latter produces particles using the Rev-independent CTE pathway. Different concentrations of each of the two compounds were added to each culture at the start of the transfection, and p24 was measured in the medium after 72 h.
Figure Figure55 shows the dose-response curves for each transfection. p24 production mediated by the Rev-RRE pathway was sensitive to each of the compounds in a dose-dependent fashion, while p24 production mediated by the CTE pathway was largely insensitive. Importantly, the EC50s for the inhibition in this assay were in the same low micromolar range as in the other assays described above.
FIG. 5.
FIG. 5.
Dose-response assays for 103833 and 104366 with a Rev/RRE-dependent construct and a CTE-dependent construct. 293T cells were transfected with either pCMVGagPol-RRE and pCMV Rev ([filled square]) or pCMVGagPol-CTE (○). Compounds were added at the indicated (more ...)
Activities of the compounds in assays that measure first-round viral replication.
To further examine the specificity of the compounds for the Rev-RRE pathway, we next performed a dose-response assay using U1 cells, which are promonocytic histiocytic lymphoma cells that contain an integrated HIV-1 provirus. They can be induced by TNF-α to start viral replication (9, 11, 23, 24). After induction, the cells progress through the entire postintegration phase of the virus replication cycle. To perform the dose-response assay, cells were induced with TNF-α, test compound was added at various concentrations, and virus production was monitored by measuring reverse transcriptase released into the medium after 72 h. Temacrazine, a known inhibitor of HIV transcription (60), was used as a control to demonstrate how a compound that inhibited p24 expression would score in this assay. Figure Figure66 shows the results of this assay. Both compounds effectively inhibited HIV particle production in a dose-dependent fashion. Again the inhibition was in approximately the same concentration range in which inhibition was observed in the other assays, and the compounds showed little toxicity in the range of their EC50s as measured in the MTS assay that was performed at 72 h.
FIG. 6.
FIG. 6.
Dose-response assay for 103833 and 104366 in U1 cells. U1 cells were induced with TNF-α and tested for viral replication in the presence of the compounds at the indicated concentrations. Virus release from cells was measured as reverse transcriptase (more ...)
We reasoned that analysis of the viral proteins produced in the cells released from latency in the presence of the compounds should allow us to determine if the compounds were specific for functional inhibition of Rev. This is because Rev function is required for export only of the unspliced and incompletely spliced HIV mRNA species and is not required by the mRNAs that are fully spliced. Thus, inhibition of Rev would be expected to have little or no effect on Nef protein production, since this protein is made from a fully spliced mRNA. However, Pr55gag and p24 synthesis should be inhibited, because these proteins are produced from unspliced RNA whose transport is completely Rev dependent.
To perform the analysis, U1 cells were treated with TNF-α and 5 μM of 104366 or 10 μM of 103833 was added. After 24 h of incubation, cells were harvested and analyzed by Western blotting for p24, Pr55gag, and Nef levels, using p24 and Nef-specific monoclonal antibodies. The blot was then quantified using an infrared imaging system (Odyssey; LiCor Inc.), and the results are shown in Fig. Fig.7A.7A. Analysis of the data demonstrates that both p24 and Pr55gag were significantly diminished in expression by treatment with both compounds, while Nef protein levels remained largely unchanged.
FIG. 7.
FIG. 7.
Western blot analysis of cells treated with 103833 or 104366. (A) U1 cells were treated with TNF-α (lanes I) or not treated with TNF-α (lanes U) and 5 μM of 104366 or 10 μM of 103833. After 24 h cells were harvested and (more ...)
We also examined the issue of compound specificity for the Rev pathway, using a proviral clone transfected into 293T cells where we were able to examine the protein products produced in the presence of compound during first-round replication. In this experiment, 293T cells were transfected with the pNL4-3 proviral clone and no compound, 8 μM of 104366, or 10 μM of 103833 was added. After 24 h of incubation, cells were harvested and analyzed by Western blotting for p24 and Nef levels, using p24 and Nef-specific monoclonal antibodies, respectively, as was done for the U1 cells. Figure Figure7B7B shows that essentially the same results were obtained. Again, in the presence of either 103833 or 104366, p24 levels were significantly diminished while Nef levels remained unaffected. Taken together, these results strongly support our hypothesis that the compounds specifically inhibit Rev function.
Lack of activity of the compounds in a Rev-RRE binding assay.
The total data presented so far strongly suggest that both 103833 and 104366 work by interfering with Rev function. An early step in the RNA export pathway that is mediated by Rev is Rev-RRE binding. This step can be studied in vitro with bacterially synthesized purified Rev protein and a radiolabeled in vitro-transcribed RRE RNA probe, using an electrophoretic mobility shift assay (EMSA). To assess whether the compounds were capable of inhibiting this interaction, we first determined Rev-RRE binding conditions that lead to a significant shift of the radiolabeled probe, being sure to stay in a linear range with respect to the response of the probe to Rev concentration (data not shown). The EMSA was then performed by incubating Rev, the radiolabeled probe, and compound together on ice for 10 min. Neomycin was included as a control since it is a known inhibitor of the Rev-RRE interaction (29, 39, 68). The complexes that formed were then analyzed by polyacrylamide gel electrophoresis. The gel was scanned using a phosphorimager. Figure Figure88 shows the results of the experiment. The data show that the neomycin control completely prevented the Rev-RRE complexes from forming, but neither 103833 nor 104366 had any effect on complex formation, even at concentrations of up to 100 μM. Thus, under the conditions tested, neither compound appears to affect Rev-RRE binding.
FIG. 8.
FIG. 8.
Dose-response assay for in vitro Rev-RRE binding. An EMSA was performed by incubating radiolabeled RRE probe and Rev protein expressed and purified from bacteria plus or minus the test compounds at the indicated concentrations on ice for 10 min. The resulting (more ...)
Lastly we examined the effect of 103833 and 104366 on HIV-1 replication in two different assays and with two different viral strains. Figure Figure99 shows a growth assay that was performed in PBMCs. For this assay the primary HIV-1 Brazilian isolate 93BR021 (CCR5-tropic, group M, subtype B) was used to infect PHA-stimulated PBMCs, and the virus produced in the presence of different drug concentrations was measured by assaying the amount of reverse transcriptase that was secreted into the medium after a 7-day period (Fig. 9A and B). The inhibition by the reverse transcriptase inhibitor zidovudine (AZT) is shown as a control (Fig. (Fig.9C)9C) to demonstrate how a bona fide inhibitor of replication would score in this assay. The toxicity of the compounds was also measured at 7 days using the MTS assay. The data show clearly that both compounds inhibited replication of the Brazilian virus with EC50s that were similar to the values in the other assays.
FIG. 9.
FIG. 9.
Dose-response assay for viral replication in PBMCs. PHA-stimulated PBMCs were infected with the primary HIV-1 Brazilian isolate 93BR021 at a multiplicity of infection of 0.1. The indicated concentrations of 103833, 104366, or AZT were added at the time (more ...)
In Fig. Fig.10,10, the growth of the laboratory isolate NL4-3 was assayed over a 30-day period, using a standard replication assay (19) in which virus was passaged, in SupT1 cells, in the presence of 0.8 μM and 8 μM drug for 104366 (Fig. 10A) and 1 μM and 10 μM drug for 103833 (Fig. 10B) or in the absence of drug. Growth of NL4-3 was also greatly inhibited at both the concentrations that were tested.
FIG. 10.
FIG. 10.
Virus growth inhibition assay. SupT1 cells (5 × 106) were infected with HIV-1 NL4-3 virus (50 ng of p24) in the presence of compound, and the cultures were passaged for 30 to 31 days by removing three-fifths of the culture on the indicated days (more ...)
The essential function of the Rev-RRE pathway makes it an attractive target for anti-HIV therapies. However, at this point, no therapies are clinically available, despite studies which have clearly shown that inhibitors of the pathway can be effective inhibitors of HIV replication (reviewed in reference 49).
Previous screening assays for compounds that inhibit Rev function have, for the most part, involved in vitro assays that interfere with the high-affinity binding of Rev and the RRE (50, 64, 68). However, while compounds discovered in this fashion often work well in vitro, many have shown considerable toxicity in cells or fail to specifically inhibit HIV replication.
In contrast, our previous efforts in this area used a cell-based screen that involved transient transfection with expression vectors producing Rev and Rev-dependent HIV GagPol (10). p24 secretion into the medium was used as an end point to measure possible inhibition of Rev function. This screen identified a series of related compounds that inhibited HIV replication. However, the compounds did not inhibit Rev-RRE binding in vitro, and their mechanism of action was not studied further. Adapting this transient assay to more extensive screens proved cumbersome, and so a cell line that stably expresses HIV GagPol in a Rev-dependent context was created to allow higher throughput.
The present study utilized this stable cell line (5BD.1), which was made with the same plasmids used in the transient-transfection assay. Approximately 40,000 compounds were screened over a 4-week period. Eleven compounds were identified as primary hits, and in a secondary assay that more directly measured the inhibition of cellular Rev function, three emerged as Rev inhibitors. Thus, the data validate the use of the 5BD.1 cell line as a method for initially identifying inhibitors of Rev function. However, since the end point of the screening assay with the 5BD.1 cells was p24 release into the medium, inhibition at any of the other specific steps leading up to viral assembly and release would also score. It seems likely that some of these steps were the targets for some of the primary hits that did not score in the subsequent Rev assay. While we have not examined this issue in any detail, we believe that our results clearly support the utility of using the 5BD.1 cell line for further screening of compounds targeting many of the postintegration events in the HIV life cycle other than Rev function.
Since the compounds were identified using a broad-based cellular screening assay in which many specific steps (some of which are unknown) contributed to the readout of Rev function, further experimentation was needed to determine exactly how the compounds act. There is a dearth of knowledge about the specific steps in the Rev-RRE pathway, but it is generally accepted that the first step involves Rev-RRE recognition. Compounds 103833 and 104366 were tested in a Rev-RRE EMSA in vitro at concentrations up to 100 μM, which is at least 10-fold above their EC50s, where they failed to show any inhibitory effect. While the conditions used in vitro may not directly reflect the in vivo situation, this result suggests that the compounds do not directly interfere with the primary Rev-RRE interaction. However, there are limitations to this interpretation, since the in vitro assay measures the binding of a bacterially made Rev protein to the viral RRE in the absence of all other components. Additionally, the bacterial protein is clearly lacking posttranslational modifications that might be present in the eukaryotic cells. The role that these factors might play in the in vivo binding step is largely not known, although it has been suggested that phosphorylation of Rev increases its RRE binding affinity (26).
Since the compounds failed to inhibit Rev-RRE binding, some other step in the Rev pathway is likely to be inhibited, but our data do not allow us to distinguish if the compounds act directly on Rev or if they work by inhibiting a cellular factor that Rev requires. Inactivation of the function of such a factor could indirectly debilitate Rev, causing it to function less well in the presence of compound. Since our data do show that expression of protein from spliced mRNA or RNA whose export is driven by a CTE is largely unaffected by the compounds, it seems likely that, if a cellular factor is involved, its inhibition can be tolerated by the cell.
It is intriguing that both 103833 and 104366 share a great deal of structural similarity to bona fide kinase inhibitors, and there are some data to suggest that Rev function may be modulated by site-specific phosphorylation (26). Compound 103833 (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide) belongs to a class of compounds, thienopyridines, that have been described before as potent inhibitors of a variety of cellular kinases (44). Compound 104366 [4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile] shares some similarities with the anilino-3-quinolinecarbonitrile inhibitors of epidermal growth factor receptor and Src family kinases (5). It will thus be interesting to examine the phosphorylation patterns on Rev in the presence or absence of compound. However, even without a clear definition of the mechanism of action of the present compounds, the present study has demonstrated that small-molecule targeting of Rev function can be a viable way to inhibit HIV replication.
We thank Gordon Powers of Message Pharmaceuticals for performing the initial drug screening assay, Thomas J. Hope of Northwestern University for the generous gift of the pDM128 plasmid, Yeou-cherng Bor of the University of Virginia for a critical scientific reading of the manuscript, and Eileen Trainum for proofreading the manuscript.
This work was supported by National Institutes of Health grants CA097095 and AI054335 to M.-L.H. and AI054213 and AI068591 to D.R. Salary support for M.L.H. and D.R. was provided by the Charles H. Ross Jr. and Myles H. Thaler Endowments at the University of Virginia.
[down-pointing small open triangle]Published ahead of print on 14 July 2008.
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