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Antimicrobial Agents and Chemotherapy, May 2004, p. 1895-1899, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1895-1899.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Tat-trans-Activation-Responsive Region Interaction by an Antiviral Quinolone Derivative

Sara Richter,1 Cristina Parolin,2 Barbara Gatto,1 Claudia Del Vecchio,2 Egidio Brocca-Cofano,3 Arnaldo Fravolini,4 Giorgio Palù,2* and Manlio Palumbo1

Department of Pharmaceutical Sciences,1 Department of Histology, Microbiology and Medical Biotechnologies, Section of Microbiology and Virology, University of Padua, 35131 Padua,2 Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrara, 44100 Ferrara,3 Department of Pharmaceutical Chemistry and Technology, University of Perugia, 06123 Perugia, Italy4

Received 4 September 2003/ Returned for modification 6 November 2003/ Accepted 8 January 2004


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ABSTRACT
 
WM5, a 6-aminoquinolone derivative, binds with high affinity to the bulge of the trans-activation-responsive region (TAR), whereas it displays low binding affinity for the loop and stem regions of TAR and for random RNA and DNA sequences. Furthermore, WM5 disrupts the natural protein-nucleic acid complex with a 50% inhibitory concentration in the low micromolar range in both in vitro and in vivo assays.


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TEXT
 
Although combination of antiretroviral drugs (highly active antiretroviral therapy) has changed the outcome of human immunodeficiency virus type 1 (HIV-1) infection, leading to a dramatic reduction in AIDS-related morbidity and mortality (27), AIDS therapies still face many constraints, including some inadequate therapeutic responses and frequent intolerable drug toxicity. In addition, a high rate of genetic variation of the HIV-1 genome, combined with natural selection under therapy, gives rise to the development and outgrowth of virus variants resistant to one or more of the administered agents (15). One way to circumvent this problem would be the identification of new targets for drug therapy characterized by being essential for viral replication and therefore less prone to mutational changes.

Tat is one of the six HIV-1 regulatory proteins essential for viral replication; hence, inhibition of Tat function provides an attractive target for antiviral therapy. Tat is an RNA-binding protein that requires specific interactions with an RNA structure called the trans-activation-responsive region (TAR) to enhance the processivity of RNA polymerase II elongation complexes that initiate at the HIV long terminal repeat (LTR) region. TAR RNA is a 59-base stem-loop structure located at the 5' ends of all nascent HIV-1 transcripts (4); it contains a six-nucleotide loop and a three-nucleotide pyrimidine bulge that separate two helical stem regions (3).

Quinolones represent an important class of broad-spectrum antibacterials whose main structural feature is a 1,4 dihydro-4-oxo-quinolinyl moiety bearing an essential carbonyl group at position 3. Quinolone derivatives have been shown to inhibit HIV-1 replication in acutely and chronically infected cells (1, 2, 14, 22, 24-26, 36). Recently, our group developed a new class of 6-substituted quinolones and tested their antibacterial and anti-HIV-1 activities (6, 30). A 6-amino quinolone bearing a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at the C-7 position (WM5) was shown to inhibit HIV-1 replication (6). Recently, it was suggested to have a possible involvement in the trans-activation process, because WM5 is able to bind with high affinity to wild-type TAR RNA (28).

To define the specificity of WM5 (Fig. 1A) binding to TAR (Dharmacon), we constructed two mutant TAR structures in which all-annealed RNA segments replaced either the loop (loopless TAR) or both the loop and bulge regions (double-stranded TAR [dsTAR]), maintaining the overall wild-type TAR nucleotide sequence (Fig. 1B). We also used tRNA from calf thymus (Sigma) as a control for random RNA sequences containing bulges and hairpins; single-stranded DNA (ssDNA; Gentium) and dsDNA (Sigma) were used as controls for deoxyribonucleic sequences.



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  		FIG. 1. Structures of the molecules used in this study. (A) Chemical structure of WM5 and ciprofloxacin. The CC50s for WM5 and ciprofloxacin were >50 µM, as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method (28). (B) Secondary structures of wild-type and mutant TAR RNAs. Wild-type TAR spans the minimal sequences that are required for Tat responsiveness in vivo (21) and for in vitro binding of Tat-derived peptides (8). Loopless TAR is formed by two RNA strands that display complementary bases in the loop portion (the three nucleotides in the loop mutated from the wild-type sequence are shown in boldface; dsTAR is constituted by two all-cRNA strands (the three inserted nucleotides complementary to the three-nucleotide bulge and the three mutated nucleotides in the loop are shown in bold). (C) Schematic representation of the HIV-1 Tat protein various domains. The Tat peptide used in this study is highlighted. Its sequence spans from amino acids 38 to 72 and corresponds to the basic and part of the core domain; the minimal region required for TAR binding is shown in bold.

Based on the fluorescence emission displayed by the quinolone, we used this technique to evaluate RNA-binding properties, as previously described (28). Under these conditions, the apparent affinity of WM5 to this nucleic acid, expressed as the concentration of TAR needed to form a complex with 50% of the drug (C50), corresponded to 0.2 µM. When the aminoquinolone was titrated with loopless TAR, the affinity for the RNA was essentially similar, with a minor decrease in the apparent binding affinity. Finally, no variations in the emission spectra were observed upon titration of WM5 with either dsTAR, tRNA, ssDNA, or dsDNA, suggesting lack of interaction. The titration curves for the three TAR structures are shown in Fig. 2A.



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  		FIG. 2. (A) Binding of WM5 to wild-type TAR (wt), loopless TAR (ll), and dsTAR (ds). Shown is the fraction of bound quinolone ({nu}) versus the RNA concentration, as inferred by fluorometric titrations. (B) WM5 inhibition of Tat-TAR interaction. An electrophoretic mobility shift assay shows drug inhibition of Tat(38-72)-TAR complex formation. The amounts of TAR RNA and Tat peptide were fixed to 0.2 and 0.1 µM, respectively, in all lanes to get 40% of complex formation on total RNA; the amounts of WM5 were 0, 1, 2, 5, 10, and 50 µM in lanes 2 to 7, respectively. Lane C is a control lane for Tat-TAR complex in the absence of inhibitor; lane R is a control lane for TAR alone. The same experiment was performed with the antibacterial ciprofloxacin (CPF) as a negative control. Band identities (free TAR and Tat-TAR complex) are shown to the left of the gel.

These data provide evidence that the bulge region of wild-type TAR RNA is a specific target for the tested aminoquinolone. Since the trinucleotide bulge is essential for high-affinity and specific binding to the Tat protein (10, 11), while the loop region is required for in vitro trans-activation but is not involved in Tat binding (3, 8, 10, 12, 31), we investigated if WM5-specific binding to TAR could disrupt the interaction between Tat and the RNA.

It has been shown by a number of groups that Tat-derived peptides, which contain the basic arginine-rich region of Tat, are able to form in vitro complexes with TAR RNA (5, 7-9, 35). To achieve specific RNA binding by a Tat fragment, we hence synthesized a Tat peptide (amino acids 38 to 72) that contains the RNA-binding domain and 11 amino acids from the core domain of the wild-type sequence of Tat protein (Fig. 1C). The synthesis was performed as described previously (33): calculated mass for Tat(38-72) C175H292N64O51 = 4,108.6; found 4,108.6 (M + H).

Tat(38-72)-TAR complex formation was assessed by electrophoresis mobility shift assay. Under these conditions, the affinity of Tat peptide for TAR at 50% of binding is 55 nM, which is in accordance with Kd values previously measured for this system (17, 18, 33; data not shown). We hence measured the ability of WM5 to disrupt Tat(38-72)-TAR interaction by titrating a constant amount of Tat-TAR complex (30% of complex on total RNA present in solution) with increasing amounts of the drug. We used ciprofloxacin (Fig. 1A) as a negative control quinolone. The results are shown in Fig. 2B. WM5 was able to inhibit Tat-TAR complex formation with an apparent Ki of 3.5 µM, while ciprofloxacin did not produce any inhibitory effect on the peptide-nucleic acid interaction. Furthermore, the fluorescence-based experiments excluded a WM5-Tat interaction (data not shown).

Next, the ability of WM5 to inhibit Tat-dependent trans-activation within cells was investigated. To this end, we utilized a HeLa cell line (HL3T1) containing a stably integrated chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HIV-1 LTR. In these cells, expression of Tat in trans is able to trans-activate the HIV-1 LTR leading to CAT expression. HL3T1 cells were transfected with an effector plasmid expressing Tat (pRPneo-c-TAT/S) or its control (pRPneo-c). After transfection, the cells were incubated with various concentrations of the compounds for 48 h and assayed for the CAT activity. As shown in Fig. 3 A and B, WM5 significantly inhibited Tat-mediated trans-activation from HIV-1 LTR in a dose-dependent manner without affecting cell viability (concentration of compound required to reduce HL3T1 cell viability by 50% [CC50] >50 µM). Compared to control untreated cells, between 77 and 73% inhibition of Tat-dependent trans-activation was obtained in the presence of 10 and 5 µM WM5, respectively. To determine the specificity of the observed inhibition by WM5, the effect of transcription regulated by the human cytomegalovirus (HCMV) promoter was examined. To this end, 293T cells (an adenovirus 5-transformed human embryonic kidney 293 cell line constitutively expressing the simian virus 40 large-T antigen) were transfected with the pCDNA3.1 CAT plasmid carrying the CAT gene under the control of the human cytomegalovirus promoter and assayed as described above. As shown in Fig. 3C and D, WM5 was unable to inhibit transcription in this system even at the highest concentration.



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  		FIG. 3. Effect of WM5 on Tat-mediated trans-activation in vivo. Cultures were transfected with the pRPneo-c-TAT/S plasmid or pRPneo-c control plasmid (HL3T1 cells [A and B]) or with the pCDNA3.1 CAT plasmid (293T cells [C and D]) and incubated in the absence or presence of WM5 at the concentrations shown. Forty-eight hours later, cells were harvested and analyzed for CAT activity for a given amount of lysate. In panels A and C, the results are reported as percent conversion (conv.) of [14C]chloramphenicol to acetylated forms above the spots. A representative experiment is shown. neg., negative. (B and D) CAT activities were normalized to that observed at a 0 µM drug concentration (assigned a value of 100%). The values reported were derived from at least two independent experiments. Means and standard deviations are shown.

Since the trinucleotide bulge is essential for high-affinity and specific binding of the Tat protein, a synthetic compound that interacts with the bulge would compete for the formation of a stable Tat-TAR complex. Different compounds have been reported to disrupt Tat-TAR interaction—in particular, backbone-modified Tat analogues, including various peptoid-based structures (19, 20, 23, 29), a ß-peptide (13), a D-amino acid-containing peptide (16), an oligocarbamate (34), and an oligourea (32). The above compounds were designed with the aim of mimicking the natural substrate, Tat; however, they usually carry the burden of high molecular weights, cumbersome synthesis, nonfavorable pharmacokinetic properties, and elevated degradation rates. On the contrary, small organic molecules, such as quinolones, are extremely versatile: around 10,000 quinolones were described in the literature, and their relatively simple synthetic routes and well known biochemical properties render these molecules ideal pharmacophoric structures. The encouraging characteristics of WM5 should be amenable to refinement in future studies with the aim of deriving modified quinolone analogues that exhibit more effective anti-HIV activity while remaining nontoxic for the cells.


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ACKNOWLEDGMENTS
 
This work was supported by AIDS grants from the Istituto Superiore di Sanità (OAG/F19, Rome-AIDS Projects no. 40D.64 and no. 30D.60), the Fondazione Cassa di Risparmio di Padova e Rovigo, Regione Veneto, MIUR, FIRB, CNR Target Project on Biotechnology, and AIRC.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, via A. Gabelli 63, 35121 Padua, Italy. Phone: 39-049-827-2350. Fax: 39-049-827-2355. E-mail: giorgio.palu{at}unipd.it. Back


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Antimicrobial Agents and Chemotherapy, May 2004, p. 1895-1899, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1895-1899.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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