- Open Access
A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions
- Thomas Vercruysse†1,
- Beata Basta†2,
- Wim Dehaen3,
- Nicolas Humbert2,
- Jan Balzarini1,
- François Debaene4, 5,
- Sarah Sanglier-Cianférani4, 5,
- Christophe Pannecouque1,
- Yves Mély2Email author and
- Dirk Daelemans1Email author
© Vercruysse et al.; licensee BioMed Central Ltd. 2012
Received: 31 August 2012
Accepted: 24 October 2012
Published: 12 November 2012
Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides.
Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed.
This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.
Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS (Acquired Immune Deficiency Syndrome), still represents a serious global public health problem. Although established anti-HIV treatments are relatively effective, they are sometimes poorly tolerated, highlighting the need for further refinement of the existing antiviral drugs and the development of novel anti-HIV strategies. In this respect, the use of topical virucides would be an interesting therapeutic strategy to prevent HIV transmission. Several topical agents for preventing HIV transmission have been described, including i) agents that inactivate HIV such as detergents (e.g. Nonoxynol-9 or SAVVY®) or pH modifiers (e.g. BufferGel; ReProtect ), ii) agents that target viral replication (e.g. the reverse transcriptase inhibitors UC-781, TMC-120, tenofovir) and iii) agents that target viral entry (e.g. PRO 2000, cellulose sulfate). With the exception of tenofovir , most microbicide candidate drugs that have been subject of large clinical trials, have proved to be non-effective or even toxic upon long-term exposure of the vaginal environment to these products.
The HIV-1 nucleocapsid NCp7 is essential for and plays multiple roles in virus replication . NCp7 contains two CCHC zinc-finger motifs and binds the viral genomic RNA in the interior of the virion. The binding of NCp7 to nucleic acids results in their condensation and protection from nuclease degradation [4–6]. Therefore, this nucleoprotein complex protects the genomic RNA ensuring viral stability. A number of classes of compounds targeting the retroviral NCp7 have been described, including, 3-nitrosobenzamide (NOBA) , 2,2′-dithiobisbenzamides (DIBA) , cyclic 2,2′-dithiobisbenzamides (e.g. SRR-SB3) , 1,2-dithiane-4,5-diol-1,1-dioxide , azadicarbonamide (ADA) [11, 12], pyridinioalkanoyl thiolesters (PATEs) , bis-thiadiazolbenzene-1,2-diamines  and S-acyl-2-mercaptobenzamide thioesters (SAMTs) . The latter class of compounds was recently considered for testing as topical microbicide for the prevention of HIV transmission . These SAMT compounds were able to efficiently prevent vaginal transmission of SHIV upon exposure of nonhuman primates . Here, we have identified a low-molecular-weight molecule, 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine, WDO-217, that inhibits HIV replication. Mechanism-of-action studies reveal WDO-217 as a potent NCp7 zinc ejector that directly inactivates HIV-1 and HIV-2 virions and inhibits the transmission of DC-SIGN captured virus to CD4+ lymphocytes. WDO-217 qualifies as a potential microbicide lead compound for further (pre)clinical studies.
Results and discussion
Inhibition of HIV and SIV in cell culture
Antiretroviral activity and cytotoxicity of WDO-217 in the MT-4/MTT-assay
7.9 ± 3.3
2.3 ± 0.3
5.3 ± 1.5
72 ± 11
0.007 ± 0.001
0.005 ± 0.0008
0.016 ± 0.007
Anti-HIV-1 and -HIV-2 activity and cytostatic properties of WDO-217 in human T-lymphocyte (CEM) cells
8.3 ± 1.8
15 ± 6.7
94 ± 15
0.058 ± 0.030
0.055 ± 0.020
Antiretroviral activity and cytotoxicity of WDO-217 in the anti-HIV
1.04 ± 0.2
0.9 ± 0.05
1.2 ± 0.1
1.7 ± 0.9
1.5 ± 0.6
1.3 ± 0.4
1.04 ± 0.3
75 ± 11
Inactivation of HIV-1, including clinical isolates, and HIV-2 virions by 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine
Inactivation of isolated HIV particles by WDO-217
1 × 106
5.4 × 106
AZT (3.7 μM)
1 × 106
5.4 × 106
Triton X-100 (0.5%)
WDO-217 (0.2 μM)
5.4 × 106
1.6 × 106
WDO-217 (1 μM)
1.6 × 106
1 × 106
WDO-217 (5 μM)
1 × 106
2.1 × 106
WDO-217 (25 μM)
WDO-217 (125 μM)
WDO-217 (625 μM)
Inactivation of clinical isolates from different HIV-1 subtypes by WDO-217
WDO-217 (125 μM)
Effect of 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine on the capture of HIV-1 by Raji/DC-SIGN cells and on subsequent virus transmission to CD4+T cells
Inhibition of HIV-1 NL4.3 capture and transmission* by Raji/DC-SIGN cells, quantified by p24-ELISA
HIV-capture procedure A
Transmission procedure B
Transmission procedure C
2.2 ± 1
8.3 ± 0.4
0.014 ± 0.01
0.002 ± 0.001
Next, we evaluated whether WDO-217 could prevent the transmission of captured HIV-1 from DC-SIGN-expressing cells to CD4+ T cells. Therefore, the DC-SIGN+ cells that efficiently captured drug-treated virus were co-cultured with uninfected C8166 cells. In these co-cultures, WDO-217 dose-dependently inhibited syncytium formation at an IC50 of 2.2 μM, whereas abundant syncytium formation occurred within 24 to 48 h post co-cultivation when the captured virus had not been pre-exposed to the drug (Table 6, procedure B). When virus was first given the opportunity to be captured by Raji/DC-SIGN cells in the absence of compound and then the Raji/DC-SIGN cells were co-cultured with C8166 cells in the presence of various concentrations of compound, WDO-217 still prevented the transmission of DC-SIGN-captured virus with an IC50 value of 8.3 μM. This suggests that WDO-217 can inactivate virus particles even when they are bound to DC-SIGN (Table 6, procedure C). All together, these results demonstrate that 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine does not inhibit the viral capture by DC-SIGN but is able to inactivate captured virus and efficiently prevents the transmission of HIV-1 from DC-SIGN-expressing cells to CD4+ T-lymphocytes, underlining its potential use as a microbicide agent.
Treatment of HIV-1 with WDO-217 decreases the virion-associated viral RNA content
Zinc ejection from the retroviral zinc fingers
Inhibition of NCp7(11-55)-induced destabilization of cTAR
Effect of WDO-217 on the interaction of NCp7(11-55) with SL3 or PBS
Next, we monitored the changes in the emission spectrum of 3HC-NCp7(11-55) that was preincubated with WDO-217 for 30 minutes, before addition to SL3 (Figure 7C). The incubation of 3HC-NCp7(11-55) with WDO-217 for 30 minutes leads to a decrease in the overall intensity of the 3HC probe as well as its N*/T* ratio (from 1.0 to 0.86) in respect with the free 3HC-NCp7(11-55). This is likely the result of a stronger interaction of the 3HC probe with the peptide backbone when it is in the zinc-free form. Addition of SL3 to the zinc-depleted 3HC-NC(11-55) induced a further decrease of the fluorescence emission resulting in a spectrum similar to that obtained when WDO-217 was added to the preformed 3HC-NC(11-55)/SL3 complex (Figure 7B). This result confirms that WDO-217 is able to eject zinc from the NC/ODN complex.
WDO-217 does not cause accumulation of unprocessed Gag polyprotein
The retroviral zinc fingers of the HIV-1 NCp7 are strictly conserved and functionally obligatory in the viral replication and virion stability. In contrast to the zinc fingers, the N-terminal domain is less conserved, but the positions of the basic residues are mainly conserved; also the basic linker between the two zinc fingers is highly conserved [36, 37]. Early studies have demonstrated that disruption of the zinc fingers by removal of the zinc led to a loss of viral replication . Different classes of zinc ejectors have been reported to efficiently eject zinc from the NCp7 zinc fingers. More specifically, the N-substituted S-acyl-2-mercaptobenzamides (SAMTs) are suggested as candidate pluripotent, HIV-specific, virucidal microbicides . Such nucleocapsid inhibitors were directly virucidal by preventing the initiation of reverse transcription and modifying the CCHC amino acid domain conformation within Gag [11, 39]. Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a very potent ejector of zinc ions from the HIV-1 NCp7 zinc fingers, inactivating HIV-1 and HIV-2 virions and relieving the protection of the viral RNA by the retroviral nucleocapsid protein. The exact detailed mechanism by which the RNA is degraded is currently under study. Interestingly, WDO-217 is able to dissociate zinc ions from NCp7 even when it is bound to nucleic acids. In agreement with the comparable affinities of the native and zinc-depleted NCp7 forms for oligonucleotides [29, 40, 41], ejection of zinc from NCp7 by WDO-217 should not dissociate the protein from the oligonucleotide. However, a change in the binding mode is expected since native NCp7 was shown to strongly interact with the bases of its nucleic acid targets through van der Waals, H-bonding and stacking interactions [32, 33, 42–44], while the zinc-depleted forms of NCp7 are thought to interact mainly through electrostatic interactions with the phosphate groups of the DNA or RNA backbone [45, 46]. The differences in the binding modes of the native and zinc-depleted forms have been clearly evidenced by the fact that only the former can modify the dynamics of the bases and structurally rearrange the oligonucleotides [32, 45, 46]. In contrast to the DIBA compounds  WDO-217 is found not to form a covalent complex with NCp7. This could be of interest because it would in principle allow using a lower concentration of compound as one molecule can react with more than one NCp7 as compared to compounds that react covalently. A disadvantage of a non-covalent binder could be that zinc ejection may be reversible if it is not followed by oxidation of the cysteines in the zinc fingers. As a consequence, the concentration of WDO-217 should be high enough to maintain an efficient depletion of zinc, so that oxidation of cysteines will occur. Although its antiviral selectivity is somewhat moderate, WDO-217 represents the first lead compound among zinc-ejecting compounds with a unique mode of action. Due to its moderate therapeutic index this lead compound would be more appropriate as microbicide then as a systemic therapeutic agent. Future structure activity relationship studies and molecular modeling studies, however, will likely enable improvement on activity, selectivity and pharmacological properties of the compound. Although WDO-217, as such, is a simple molecule there is room for further modifications and subsequent SAR studies. Modifications are possible introducing substituents at different positions on the phenyl ring. It is certainly worthwhile to further investigate the importance of the relative positions and nature of the substituents on the 1,2,4-thiadiazol ring. Furthermore it would be interesting switching to other heterocyclic rings, the most obvious being a series of 4,5-disubstituted -2-aminothiazole derivatives.
Interestingly, WDO-217 treatment does not inhibit the direct gp120-mediated capture of virus on DC-SIGN suggesting that it is able to inactivate HIV but with a preservation of the conformational and functional integrity of the surface envelope proteins. This preservation of surface proteins has earlier been demonstrated with the prototypical NCp7 zinc ejector AT-2 . Moreover, WDO-217 inactivates virus particles even when they are bound to DC-SIGN and prevents the transmission of DC-SIGN-captured HIV to CD4+ cells. This makes WDO-217 suitable, not only as a valuable component in topical microbicides, but also for the inactivation of virions to be used as vaccine antigens . Indeed, HIV vaccine strategies using DCs are currently being investigated. DCs detect viruses in peripheral tissues and, following activation and virus capture/uptake, migrate to lymph nodes to trigger adaptive immune responses . However, HIV is able to modulate these DCs to facilitate infection and transmission to T-cells causing their disregulation . This justifies the need to develop strategies that prevent this modulation and disregulation. In this respect, WDO-217 appears of high interest, since it does not prevent the capture by DC SIGN but prevents subsequent transmission and infection.
This new compound is useful as a lead for the design of a future new generation NCp7 inhibitors that could be applied as an agent in microbicide formulations and vaccine strategies.
Cells and viruses
MT-4, Jurkat A72, CEM, HuT-78 and Raji/DC-SIGN cells were grown and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.1% sodium bicarbonate and 20 μg gentamicin per ml. The HIV-1(IIIB) strain was provided by R.C. Gallo and M. Popovic (at that time at the NIH, Bethesda, MD, USA). HIV-2(ROD) was obtained from L. Montagnier (at that time at the Pasteur Institute, Paris, France) and SIV(Mac251) from C. Bruck. Raji/DC-SIGN were kindly provided by L. Burleigh (Paris, France).
In vitroantiviral assays
Evaluation of the antiviral activity of the compounds against HIV-1 strain IIIB in MT-4 cells was performed using the MTT assay as previously described [18, 19]. Stock solutions (10 x final concentration) of test compounds were added in 25 μl volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated HIV- and mock-infected cell samples were included as controls. HIV-1(IIIB) stock (50 μl) at 100-300 CCID50 (50% cell culture infectious doses) or culture medium was added to either the infected or mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effects of test compound on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 minutes at 220 g, and the supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/ml and 50 μl volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically using the MTT assay. The MTT assay is based on the reduction of yellow coloured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros Organics) by mitochondrial dehydrogenase activity in metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the median absorbance value of three wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The concentration achieving 50% protection against the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC50).
The antiviral activity of the compounds against HIV was evaluated in Jurkat cells stably transformed to express the LTR-GFP (A72 cells) [20, 50]. In 96-well plates, 3 × 104 A72 cells were infected with HIV in the presence of various concentrations of test compound. Three days post infection, cells were harvested and fixed in 3% paraformaldehyde. GFP-expression was quantified on a single cell basis by flow cytometry [51, 52]. Toxicity of the compounds was tested using an MTT-based method.
HIV-1 core antigen (p24 Ag) in the supernatant was analyzed by the p24 Ag enzyme-linked immunosorbent assay (Perkin Elmer).
Aliquots of a HIV stock (IIIB or ROD) were incubated with various concentrations of compound in a final volume of 100 μl RPMI-1640 culture medium with 10% FCS for 1 hour at 37°C. For the clinical isolates of different clades, stock was incubated with 125 μM WDO-217 for 1 hour at 37°C. Subsequently, the samples were diluted 4000 times with complete medium so that the residual concentration of compound present was far below its IC50. The drug-treated and diluted virus suspension was then used to infect susceptible MT-4 T-cells to quantify the viral infectivity by titration and CCID50 calculation . The different clinical isolates were titrated on freshly isolated PBMCs from a healthy donor. Control experiments with AZT indicated that this procedure effectively diluted the compound to concentrations well below its effective antiviral concentration.
Inhibitory effect on virus production from HuT-78/IIIBpersistently infected cells, virion analysis and western blot
Chronically-infected HIV-1 IIIB HuT-78 cells (HuT-78/IIIB) were washed four times with PBS to remove all free virions before treatment and 2 × 105 cells were resuspended in 1 ml compound-containing medium for 43 hours at 37°C. Then, virions were prepared from clarified supernatants (10 min at 300 g) by centrifugation at 36670 g for 2 hours at 4°C. Protein from lysed virions were separated by SDS-PAGE on a NuPage® Novex 4-12% Bis-Tris gel (Invitrogen) and transferred on a hydrophobic polyvinylidene difluoride (PVDF) membrane (Amersham Hybond™-P). The blot was blocked overnight at 4°C by 5% dry milk powder in Western blot wash solution (WBWS; PBS + 0.5% Tween 20), washed three times for 5 minutes with WBWS and incubated with a mouse anti-HIV-1 p24 antibody (1:5000) from Abcam. The blot was then washed three times for 5 minutes with WBWS and incubated for 1 h with a goat anti-mouse IgG-HRP secondary antibody (1:2500) from Santa Cruz Biotechnology. The blot was washed three times for 5 minutes with WBWS and after 5 minutes of incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) it was developed.
Effect of WDO-217 on the exposure of HIV-1 to Raji/DC-SIGN cells
In a first set of experiments (procedure A), HIV-1 (NL4.3) was exposed to WDO-217 at 105 μM (in 0.5 ml culture medium) for 60 minutes at 37°C. Then, 0.5 ml exponentially growing Raji/DC-SIGN cells (106 cells) was added, and the suspension further incubated at 37°C for 60 minutes. Subsequently, 39 ml medium was added and the cell suspension was centrifuged at 300 g for 10 minutes. The obtained pellet was washed again with 40 ml medium and after centrifugation, the cell pellet (containing DC-SIGN-bound virus) was analyzed for p24 antigen content by ELISA. In a second set of experiments (procedure B), Raji/DC-SIGN cells treated as in procedure A were co-cultured in the presence of an equal amount of C8166 cells (106 cells) (total volume of 1 ml). Replication in C8166 was measured after ~ 20 hr of incubation. In a third set of experiments (procedure C), Raji/DC-SIGN cells were given the opportunity to capture HIV-1/NL4.3 by mixing Raji/DC-SIGN cells with virus (106 cells/ml). After one hour of incubation at 37°C, C8166 cells (106 cells/ml) were added in the presence of WDO-217 at different concentrations and giant cell formation in the cultures was examined microscopically after approximately 24 hr. In the above-described experiments, the mannose-binding entry inhibitor HHA was included as a reference compound.
Total mRNA from virus stock was extracted using the QIamp viral RNA kit (Qiagen) followed by DNA digestion using RNase-free DNase I (Invitrogen). DNase I treated mRNA was used to generate cDNA along with Thermoscript reverse transcriptase (Invitrogen) and oligo(dT)20. qRT-PCR for genomic unspliced HIV mRNA was performed according to a protocol described earlier , using 0.2 mM primers TCAGCCCAGAAGTAATACCCATGT and TGCTATGTCAGTTCCCCTTGGTTCTCT, and 0.2 mM FAM-BHQ1 fluorescent probe ATTAACAGAAGGAGCCACCCCACAAGA. Control reactions omitted reverse transcriptase, and the number of cDNA copies was determined using a HIV-1NL4.3 molecular clone DNA standard.
Zinc ejection and inhibition of NC(11-55)-induced destabilization of cTAR monitored by fluorescence techniques
The NC(11-55) peptide was synthesized by solid phase peptide synthesis on a 433A synthesizer (ABI, Foster City, CA), as previously described . The lyophilized peptide was dissolved in water, and its concentration was determined using an extinction coefficient of 5,700 M–1 x cm–1 at 280 nm. Next, 2.5 molar equivalents of ZnSO4 were added to the peptide and pH was raised to its final value, by adding buffer. The increase of pH was done only after zinc addition to avoid oxidization of the zinc-free peptide. Zinc ejection was monitored through the changes in the intrinsic fluorescence of the Trp37 residue of NC(11-55) [23, 24], after addition of a 10-fold excess of WDO-217 (10 μM) to 1 μM NC(11-55).
To monitor the inhibition by WDO-217 of the NC(11-55)-induced destabilization of cTAR, we used doubly labelled cTAR, synthesized at a 0.2 μmol scale by IBA GmbH Nucleic Acids Product Supply (Göttingen, Germany). The 5′ terminus of cTAR was labeled with 6-carboxyrhodamine (Rh6G) via an amino-linker with a six carbon spacer arm. The 3′ terminus of cTAR was labeled with 4-(4′-dimethylaminophenylazo)benzoic acid (Dabcyl) using a special solid support with the dye already attached. The doubly labeled cTAR was purified by reverse-phase HPLC and polyacrylamide gel electrophoresis. An extinction coefficient at 260 nm of 521,910 M–1 x cm–1 was used for cTAR. All experiments were performed at 20°C in 25 mM Tris–HCl, pH 7.5, 30 mM NaCl, and 0.2 mM MgCl2. The effect of WDO-217 on the NC(11-55)-induced destabilization of cTAR was observed after addition of 10 μM WDO-217 to 0.1 μM Rh6G-cTAR-Dabcyl preincubated with 1 μM NC(11-55).
where Im is the measured fluorescence of the protein, Ip is the fluorescence intensity of the protein in the absence of inner filter, dp is the absorbance of the protein, ds is the absorbance of WDO-217 at the excitation wavelength, and dr is the absorbance of WDO-217 at the emission wavelengths.
Zinc ejection monitored by supramolecular mass spectrometry
Before mass spectrometry (MS) analysis, NC(11-55) was dissolved and buffer exchanged with 50 mM ammonium acetate pH 7.0 using 4 cycles of microcentrifuge size exclusion filtering (Vivaspin 500 5kD, Sartorius Stedim biotech, Aubagne, France) and peptide concentration was measured by a Bradford assay.
ESI-MS measurements were performed in the positive ion mode on an electrospray time-of-flight mass spectrometer (LCT, Waters, Manchester, UK) equipped with an automated chip-based nanoESI source (Triversa Nanomate, Advion Biosciences, Ithaca, NY). Calibration of the instrument was performed using multiply charged ions of a 2 μM horse heart myoglobine solution. For analysis in denaturing conditions, samples were diluted to 2 μM in a 1/1 water/acetonitrile mixture (v/v) acidified with 1% formic acid and standard interface parameters were used to obtain best mass accuracy. In these conditions, noncovalent interactions are disrupted, allowing the measurement of the molecular weight of the monomer with a good accuracy (better than 0.01%).
Analyses under non-denaturing conditions were carried out after careful optimization of instrumental settings to avoid dissociation of noncovalent bonds and obtain sensible detection of protein/zinc complexation states. The accelerating voltage (Vc) was fixed to 20 V, and the pressure in the first pumping stage of the instrument (Pi) to 6 mbar. Zinc ejection measurements were performed after 30 minutes incubation at room temperature of a 20 μM solution of NC(11-55) with either 40 μM or 100 μM WDO-217. Data analysis was performed with the MassLynx 3.5 software (Waters, Manchester, UK). Peak intensities were used to estimate the ratios of the different ions detected.
We thank L. Bral, C. Heens, K. Erven K. Uyttersprot and L. Ingels for excellent technical assistance; a number of reagents were obtained through the NIH AIDS Reagent Program.
The work was supported by grants from the KU Leuven (GOA 10/14 and PF 10/18), the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO)” number 18.104.22.168 and the Agence Nationale de la Recherche sur le Sida (ANRS). BB is supported by an ANRS fellowship.
- Mayer KH, Peipert J, Fleming T, Fullem A, Moench T, Cu-Uvin S, Bentley M, Chesney M, Rosenberg Z: Safety and tolerability of BufferGel, a novel vaginal microbicide, in women in the United States. Clin Infect Dis. 2001, 32: 476-482. 10.1086/318496.View ArticlePubMedGoogle Scholar
- Andrei G, Lisco A, Vanpouille C, Introini A, Balestra E, van den Oord J, Cihlar T, Perno CF, Snoeck R, Margolis L, Balzarini J: Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication. Cell Host Microbe. 2011, 10: 379-389. 10.1016/j.chom.2011.08.015.PubMed CentralView ArticlePubMedGoogle Scholar
- Thomas JA, Gorelick RJ: Nucleocapsid protein function in early infection processes. Virus Res. 2008, 134: 39-63. 10.1016/j.virusres.2007.12.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Krishnamoorthy G, Roques B, Darlix JL, Mely Y: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection. Nucleic Acids Res. 2003, 31: 5425-5432. 10.1093/nar/gkg738.PubMed CentralView ArticlePubMedGoogle Scholar
- Lapadat-Tapolsky M, De Rocquigny H, Van Gent D, Roques B, Plasterk R, Darlix JL: Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle. Nucleic Acids Res. 1993, 21: 831-839. 10.1093/nar/21.4.831.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanchou V, Gabus C, Rogemond V, Darlix JL: Formation of stable and functional HIV-1 nucleoprotein complexes in vitro. J Mol Biol. 1995, 252: 563-571. 10.1006/jmbi.1995.0520.View ArticlePubMedGoogle Scholar
- Rice WG, Schaeffer CA, Graham L, Bu M, McDougal JS, Orloff SL, Villinger F, Young M, Oroszlan S, Fesen MR, et al: The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes. Proc Natl Acad Sci U S A. 1993, 90: 9721-9724. 10.1073/pnas.90.20.9721.PubMed CentralView ArticlePubMedGoogle Scholar
- Rice WG, Supko JG, Malspeis L, Buckheit RW, Clanton D, Bu M, Graham L, Schaeffer CA, Turpin JA, Domagala J, et al: Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS. Science. 1995, 270: 1194-1197. 10.1126/science.270.5239.1194.View ArticlePubMedGoogle Scholar
- Witvrouw M, Balzarini J, Pannecouque C, Jhaumeer-Laulloo S, Este JA, Schols D, Cherepanov P, Schmit JC, Debyser Z, Vandamme AM, et al: SRR-SB3, a disulfide-containing macrolide that inhibits a late stage of the replicative cycle of human immunodeficiency virus. Antimicrob Agents Chemother. 1997, 41: 262-268.PubMed CentralPubMedGoogle Scholar
- Rice WG, Baker DC, Schaeffer CA, Graham L, Bu M, Terpening S, Clanton D, Schultz R, Bader JP, Buckheit RW, et al: Inhibition of multiple phases of human immunodeficiency virus type 1 replication by a dithiane compound that attacks the conserved zinc fingers of retroviral nucleocapsid proteins. Antimicrob Agents Chemother. 1997, 41: 419-426.PubMed CentralPubMedGoogle Scholar
- Rice WG, Turpin JA, Huang M, Clanton D, Buckheit RW, Covell DG, Wallqvist A, McDonnell NB, DeGuzman RN, Summers MF, et al: Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein. Nat Med. 1997, 3: 341-345. 10.1038/nm0397-341.View ArticlePubMedGoogle Scholar
- Goebel FD, Hemmer R, Schmit JC, Bogner JR, de Clercq E, Witvrouw M, Pannecouque C, Valeyev R, Vandevelde M, Margery H, Tassignon JP: Phase I/II dose escalation and randomized withdrawal study with add-on azodicarbonamide in patients failing on current antiretroviral therapy. AIDS. 2001, 15: 33-45. 10.1097/00002030-200101050-00007.View ArticlePubMedGoogle Scholar
- Turpin JA, Song Y, Inman JK, Huang M, Wallqvist A, Maynard A, Covell DG, Rice WG, Appella E: Synthesis and biological properties of novel pyridinioalkanoyl thiolesters (PATE) as anti-HIV-1 agents that target the viral nucleocapsid protein zinc fingers. J Med Chem. 1999, 42: 67-86. 10.1021/jm9802517.View ArticlePubMedGoogle Scholar
- Pannecouque C, Szafarowicz B, Volkova N, Bakulev V, Dehaen W, Mely Y, Daelemans D: Inhibition of HIV-1 replication by a bis-thiadiazolbenzene-1,2-diamine that chelates zinc ions from retroviral nucleocapsid zinc fingers. Antimicrob Agents Chemother. 2010, 54: 1461-1468. 10.1128/AAC.01671-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Jenkins LM, Byrd JC, Hara T, Srivastava P, Mazur SJ, Stahl SJ, Inman JK, Appella E, Omichinski JG, Legault P: Studies on the mechanism of inactivation of the HIV-1 nucleocapsid protein NCp7 with 2-mercaptobenzamide thioesters. J Med Chem. 2005, 48: 2847-2858. 10.1021/jm0492195.View ArticlePubMedGoogle Scholar
- Turpin JA, Schito ML, Jenkins LM, Inman JK, Appella E: Topical microbicides: a promising approach for controlling the AIDS pandemic via retroviral zinc finger inhibitors. Adv Pharmacol. 2008, 56: 229-256.View ArticlePubMedGoogle Scholar
- Wallace GS, Cheng-Mayer C, Schito ML, Fletcher P, Miller Jenkins LM, Hayashi R, Neurath AR, Appella E, Shattock RJ: Human immunodeficiency virus type 1 nucleocapsid inhibitors impede trans infection in cellular and explant models and protect nonhuman primates from infection. J Virol. 2009, 83: 9175-9182. 10.1128/JVI.00820-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Pannecouque C, Daelemans D, De Clercq E: Tetrazolium-based colorimetric assay for the detection of HIV replication inhibitors: revisited 20 years later. Nat Protoc. 2008, 3: 427-434. 10.1038/nprot.2007.517.View ArticlePubMedGoogle Scholar
- Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P, Desmyter J, De Clercq E: Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods. 1988, 20: 309-321. 10.1016/0166-0934(88)90134-6.View ArticlePubMedGoogle Scholar
- Jordan A, Defechereux P, Verdin E: The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J. 2001, 20: 1726-1738. 10.1093/emboj/20.7.1726.PubMed CentralView ArticlePubMedGoogle Scholar
- Yarchoan R, Klecker RW, Weinhold KJ, Markham PD, Lyerly HK, Durack DT, Gelmann E, Lehrman SN, Blum RM, Barry DW, et al: Administration of 3′-azido-3′-deoxythymidine, an inhibitor of HTLV-III/LAV replication, to patients with AIDS or AIDS-related complex. Lancet. 1986, 1: 575-580.View ArticlePubMedGoogle Scholar
- Bertaux C, Daelemans D, Meertens L, Cormier EG, Reinus JF, Peumans WJ, Van Damme EJ, Igarashi Y, Oki T, Schols D, et al: Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions. Virology. 2007, 366: 40-50. 10.1016/j.virol.2007.04.008.View ArticlePubMedGoogle Scholar
- Bombarda E, Grell E, Roques BP, Mely Y: Molecular mechanism of the Zn2 + -induced folding of the distal CCHC finger motif of the HIV-1 nucleocapsid protein. Biophys J. 2007, 93: 208-217. 10.1529/biophysj.106.101378.PubMed CentralView ArticlePubMedGoogle Scholar
- Mely Y, De Rocquigny H, Morellet N, Roques BP, Gerad D: Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy. Biochemistry. 1996, 35: 5175-5182. 10.1021/bi952587d.View ArticlePubMedGoogle Scholar
- Loo JA, Holler TP, Sanchez J, Gogliotti R, Maloney L, Reily MD: Biophysical characterization of zinc ejection from HIV nucleocapsid protein by anti-HIV 2,2-dithiobis[benzamides] and benzisothiazolones. J Med Chem. 1996, 39: 4313-4320. 10.1021/jm960253w.View ArticlePubMedGoogle Scholar
- Azoulay J, Clamme JP, Darlix JL, Roques BP, Mely Y: Destabilization of the HIV-1 complementary sequence of TAR by the nucleocapsid protein through activation of conformational fluctuations. J Mol Biol. 2003, 326: 691-700. 10.1016/S0022-2836(02)01430-4.View ArticlePubMedGoogle Scholar
- Beltz H, Azoulay J, Bernacchi S, Clamme JP, Ficheux D, Roques B, Darlix JL, Mely Y: Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7. J Mol Biol. 2003, 328: 95-108. 10.1016/S0022-2836(03)00244-4.View ArticlePubMedGoogle Scholar
- Bernacchi S, Stoylov S, Piemont E, Ficheux D, Roques BP, Darlix JL, Mely Y: HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence. J Mol Biol. 2002, 317: 385-399. 10.1006/jmbi.2002.5429.View ArticlePubMedGoogle Scholar
- Beltz H, Clauss C, Piemont E, Ficheux D, Gorelick RJ, Roques B, Gabus C, Darlix JL, de Rocquigny H, Mely Y: Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis. J Mol Biol. 2005, 348: 1113-1126. 10.1016/j.jmb.2005.02.042.View ArticlePubMedGoogle Scholar
- Bernacchi S, Mely Y: Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure. Nucleic Acids Res. 2001, 29: E62-10.1093/nar/29.13.e62.PubMed CentralView ArticlePubMedGoogle Scholar
- Shvadchak VV, Klymchenko AS, de Rocquigny H, Mely Y: Sensing peptide-oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein. Nucleic Acids Res. 2009, 37: e25-10.1093/nar/gkn1083.PubMed CentralView ArticlePubMedGoogle Scholar
- Bourbigot S, Ramalanjaona N, Boudier C, Salgado GF, Roques BP, Mely Y, Bouaziz S, Morellet N: How the HIV-1 nucleocapsid protein binds and destabilises the (-)primer binding site during reverse transcription. J Mol Biol. 2008, 383: 1112-1128. 10.1016/j.jmb.2008.08.046.View ArticlePubMedGoogle Scholar
- De Guzman RN, Wu ZR, Stalling CC, Pappalardo L, Borer PN, Summers MF: Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science. 1998, 279: 384-388. 10.1126/science.279.5349.384.View ArticlePubMedGoogle Scholar
- Mahmood N, Jhaumeer-Lauloo S, Sampson J, Houghton PJ: Anti-HIV activity and mechanism of action of macrocyclic diamide SRR-SB3. J Pharm Pharmacol. 1998, 50: 1339-1342.View ArticlePubMedGoogle Scholar
- Miller Jenkins LM, Ott DE, Hayashi R, Coren LV, Wang D, Xu Q, Schito ML, Inman JK, Appella DH, Appella E: Small-molecule inactivation of HIV-1 NCp7 by repetitive intracellular acyl transfer. Nat Chem Biol. 2010, 6: 887-889. 10.1038/nchembio.456.View ArticlePubMedGoogle Scholar
- Darlix JL, Godet J, Ivanyi-Nagy R, Fosse P, Mauffret O, Mely Y: Flexible Nature and Specific Functions of the HIV-1 Nucleocapsid Protein. J Mol Biol. 2011, 410: 565-581. 10.1016/j.jmb.2011.03.037.View ArticlePubMedGoogle Scholar
- Godet J, Boudier C, Humbert N, Ivanyi-Nagy R, Darlix JL, Mely Y: Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1. Virus Res. 2012, In pressGoogle Scholar
- Rice WG, Schaeffer CA, Harten B, Villinger F, South TL, Summers MF, Henderson LE, Bess JW, Arthur LO, McDougal JS, et al: Inhibition of HIV-1 infectivity by zinc-ejecting aromatic C-nitroso compounds. Nature. 1993, 361: 473-475. 10.1038/361473a0.View ArticlePubMedGoogle Scholar
- Schito ML, Soloff AC, Slovitz D, Trichel A, Inman JK, Appella E, Turpin JA, Barratt-Boyes SM: Preclinical evaluation of a zinc finger inhibitor targeting lentivirus nucleocapsid protein in SIV-infected monkeys. Curr HIV Res. 2006, 4: 379-386. 10.2174/157016206777709492.View ArticlePubMedGoogle Scholar
- Urbaneja MA, Kane BP, Johnson DG, Gorelick RJ, Henderson LE, Casas-Finet JR: Binding properties of the human immunodeficiency virus type 1 nucleocapsid protein p7 to a model RNA: elucidation of the structural determinants for function. J Mol Biol. 1999, 287: 59-75. 10.1006/jmbi.1998.2521.View ArticlePubMedGoogle Scholar
- Vuilleumier C, Bombarda E, Morellet N, Gerard D, Roques BP, Mely Y: Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study. Biochemistry. 1999, 38: 16816-16825. 10.1021/bi991145p.View ArticlePubMedGoogle Scholar
- Amarasinghe GK, De Guzman RN, Turner RB, Chancellor KJ, Wu ZR, Summers MF: NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition. J Mol Biol. 2000, 301: 491-511. 10.1006/jmbi.2000.3979.View ArticlePubMedGoogle Scholar
- Bazzi A, Zargarian L, Chaminade F, Boudier C, De Rocquigny H, Rene B, Mely Y, Fosse P, Mauffret O: Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC. Nucleic Acids Res. 2011, 39: 3903-3916. 10.1093/nar/gkq1290.PubMed CentralView ArticlePubMedGoogle Scholar
- Morellet N, Demene H, Teilleux V, Huynh-Dinh T, de Rocquigny H, Fournie-Zaluski MC, Roques BP: Structure of the complex between the HIV-1 nucleocapsid protein NCp7 and the single-stranded pentanucleotide d(ACGCC). J Mol Biol. 1998, 283: 419-434. 10.1006/jmbi.1998.2098.View ArticlePubMedGoogle Scholar
- Avilov SV, Piemont E, Shvadchak V, de Rocquigny H, Mely Y: Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine. Nucleic Acids Res. 2008, 36: 885-896.PubMed CentralView ArticlePubMedGoogle Scholar
- Godet J, Ramalanjaona N, Sharma KK, Richert L, de Rocquigny H, Darlix JL, Duportail G, Mely Y: Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer. Nucleic Acids Res. 2011, 39: 6633-6645. 10.1093/nar/gkr274.PubMed CentralView ArticlePubMedGoogle Scholar
- Rossio JL, Esser MT, Suryanarayana K, Schneider DK, Bess JW, Vasquez GM, Wiltrout TA, Chertova E, Grimes MK, Sattentau Q, et al: Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins. J Virol. 1998, 72: 7992-8001.PubMed CentralPubMedGoogle Scholar
- Altfeld M, Fadda L, Frleta D, Bhardwaj N: DCs and NK cells: critical effectors in the immune response to HIV-1. Nat Rev Immunol. 2011, 11: 176-186. 10.1038/nri2935.PubMed CentralView ArticlePubMedGoogle Scholar
- van der Vlist M, van der Aar AM, Gringhuis SI, Geijtenbeek TB: Innate signaling in HIV-1 infection of dendritic cells. Curr Opin HIV AIDS. 2011, 6: 348-352. 10.1097/COH.0b013e328349a2d1.View ArticlePubMedGoogle Scholar
- Jordan A, Bisgrove D, Verdin E: HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J. 2003, 22: 1868-1877. 10.1093/emboj/cdg188.PubMed CentralView ArticlePubMedGoogle Scholar
- Daelemans D, De Clercq E, Vandamme AM: A quantitative GFP-based bioassay for the detection of HIV-1 Tat transactivation inhibitors. J Virol Methods. 2001, 96: 183-188. 10.1016/S0166-0934(01)00330-5.View ArticlePubMedGoogle Scholar
- Daelemans D, Pannecouque C, Pavlakis GN, Tabarrini O, De Clercq E: A novel and efficient approach to discriminate between pre- and post-transcription HIV inhibitors. Mol Pharmacol. 2005, 67: 1574-1580. 10.1124/mol.104.010249.View ArticlePubMedGoogle Scholar
- Daelemans D, Pauwels R, De Clercq E, Pannecouque C: A time-of-drug addition approach to target identification of antiviral compounds. Nat Protoc. 2011, 6: 925-933. 10.1038/nprot.2011.330.View ArticlePubMedGoogle Scholar
- Vercruysse T, Pardon E, Vanstreels E, Steyaert J, Daelemans D: An intrabody based on a llama single-domain antibody targeting the N-terminal alpha-helical multimerization domain of HIV-1 rev prevents viral production. J Biol Chem. 2010, 285: 21768-21780. 10.1074/jbc.M110.112490.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.