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LettersJuly 2, 2014

Functional and Structural Characterization of 2-Amino-4-phenylthiazole Inhibitors of the HIV-1 Nucleocapsid Protein with Antiviral Activity
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ACS Chemical Biology

Cite this: ACS Chem. Biol. 2014, 9, 9, 1950–1955

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https://doi.org/10.1021/cb500316h

Published July 2, 2014

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Abstract

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The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.

Subjects

During the past 25 years, the arsenal of drugs to combat HIV-1 infection has increased continuously, even though most anti-HIV-1 drugs experienced clinical failure due to antiretroviral drug resistance resulting from mutations in HIV-1 protein targets as a consequence of the error-prone nature of HIV-1 reverse transcriptase (RT) and its lack of a proofreading function. The most common and realistic strategy adopted so far to overcome this issue is the design of more potent and selective inhibitors of entry/fusion, RT, Integrase (IN), and Protease (PR), but these compounds often lack full activity against drug-resistant virus strains selected by the same drug classes. (1) Therefore, a medical need in this field is still felt, and additional conserved drug targets need to be explored to achieve virus suppression in drug-experienced patients or patients infected with drug-resistant viruses. In this context, the HIV-1 nucleocapsid protein (NC), which is highly conserved throughout different HIV-1 subtypes and exerts essential functions in virus replication, is a promising anti-HIV-1 target. (2, 3) NC is a 55-amino-acid small basic protein with two zinc fingers that assists RT during reverse transcription by chaperoning the annealing of the cellular primer tRNA to the primer binding site (PBS) and the two obligatory DNA strand transfers necessary for the synthesis of a complete functional double-stranded vDNA with two long terminal repeats. (4) NC is also thought to protect the nascent vDNA against nucleases (5) and to assist IN for the integration of the viral DNA into the host genome. (6) As a domain of the Gag structural polyprotein precursor, NC selects the genomic RNA and promotes its dimerization and packaging during virus assembly. (7-9) Finally, NC binding to RNA is critical for Gag processing by PR and for the subsequent formation of the condensed ribonucleoprotein architecture within the virion. (10) NC can hardly mutate, so that NC mutants are generally nonfunctional and result in noninfectious viruses. (11, 12) Therefore, specific inhibition of NC is thought to generate a sustained antiretroviral activity.

Inhibition of NC has been achieved by different strategies leading to antiretroviral effects in vitro and in vivo, (13) although generally through unspecific mechanisms and thus causing significant cytotoxicity. Indeed, there is a critical deficiency of small molecule NC inhibitors (NCIs) endowed with a clear and safe mechanism of action as well as with antiviral activity in vitro, which may be developed as effective and safe antiretroviral agents.

Targeting protein/nucleic acids interaction by small molecules is a highly promising strategy in anti-HIV therapy, even if generally considered as a tough challenge due to several difficulties, most often related to the intrinsic structural disorder and highly basic nature of nucleic acids-binding proteins. In continuation of our interest in discovering specific NCIs competing with nucleic acids for the binding to NC and exhibiting antiviral activity, we established a multidisciplinary strategy based on three pioneer works in the field. Indeed, Shvadchak et al. have developed a high-throughput screening assay based on targeting the NC chaperone activities, which led to the identification of five molecular fragments that efficiently inhibit NC but do not show antiviral activity. (14) Recently, Goudreau and co-workers have provided the first solution structure of NC in complex with an NCI that mimics the guanosine found in many NC/RNA complexes. (15) Moreover, as NC is a highly flexible protein, it was essential to use molecular dynamics (MD) simulations to study the conformational behavior of NC in complex with nucleic acids and to develop a new strategy based on virtual screening to design small molecule NCIs showing antiretroviral activity. (16, 17)

On the basis of a combination of molecular simulations, biophysical studies, NMR analysis, and antiviral assays, here we elucidate the NC inhibitory activity and structural requirements for NC binding of AN3, a new NCI targeting efficiently NC nucleic acid chaperone activity and endowed with antiviral activity in cells. Notably, AN3 was designed starting from NCI fragments identified by Shvadchak, (14) whose complexes with NC have been already investigated by molecular docking simulations and GRID-based analysis. (16) Starting from docking complexes, here we performed preliminary MD simulations of 25 ns each, showing that fragment A10 had the highest conformational stability within the hydrophobic pocket of NC located at the top of the folded zinc fingers (Supporting Information, Figure S1), which play a key role in NC chaperoning activities. (18-20) For this reason, A10 was selected for further studies aimed at corroborating the validity of the 2-amino-4-phenylthiazole scaffold for NC inhibition as well as to develop NCI with antiviral activity in cells.

In agreement with our previous observations, (16) MD analysis showed that the aminothiazole moiety nicely fits the hydrophobic pocket of NC and may behave as a guanine-mimetic. The catechol phenyl ring is stacked with the Trp37 side chain, whereas hydroxyl groups are projected toward the solvent but do not bind to basic residues that have been shown to play a key role in binding to nucleic acids. (21) Moreover, catechol species are particularly sensitive to oxidation, giving quinone or semiquinone radicals, which may interfere with the functional characterization of the mechanism of action through binding to proteins or nucleic acids. Indeed, a number of catechols of polyhydroxylated compounds have been previously shown to inhibit NC functions by an unclear mechanism, but without showing antiviral activity in vitro. (22) Therefore, our strategy focused on the structure-guided replacement of the catechol moiety in A10 with functional groups that may eventually improve membrane permeability. To this end, compounds sharing the common 2-amino-4-phenylthiazole substructure and bearing substitutions on the phenyl ring were downloaded from the ZINC database (about 950 molecules) and submitted to the virtual screening protocol already described. (16) In parallel, these molecules were also docked toward the NMR structure of NC in complex with an NCI. (15) Compounds AN1, AN2, and AN3 (Figure 1A and Supporting Information, Table S1, for basic properties) were prioritized by virtual screening and further studied by means of 0.3 μs of MD for checking their conformational stability (Supporting Information). Then, these molecules were purchased and tested in vitro.

We first monitored the effect of AN compounds on the NC(11-55)-induced destabilization of the secondary structure of cTAR DNA, which is exquisitely dependent on the hydrophobic plateau at the top of the properly folded zinc finger motifs, (19) by using the double-labeled Rh6G-5′-cTAR-3′-Dabcyl derivative. In the absence of NC, cTAR is mainly in a nonfluorescent closed form, where the Rh6G and Dabcyl labels at the 5′ and 3′ termini of the cTAR stem are close together. Addition of a 10-fold excess of NC(11-55) led to melting of the bottom of the cTAR stem, partially restoring the Rh6G fluorescence. (23) Therefore, NCIs designed to interfere with NC/nucleic acid interactions are expected to reverse the NC-promoted increase in Rh6G fluorescence. Addition of 100 μM concentration of AN1, AN2, and AN3 to the NC(11-55)/cTAR complex was found to induce 7%, 10%, and 28% decrease of the Rh6G emission, respectively (Figure 1B). A control experiment performed in the absence of NC excluded any direct effect of the compounds on Rh6G emission (data not shown). Thus, all three compounds are able to partially inhibit the NC(11-55)-induced destabilization of cTAR, AN3 being the most efficient NCI with an IC₅₀ of 140(±90) μM (Figure 1B, insert). The inhibition constant K_i was then calculated according to the Cheng and Prussof equation:(1)with [cTAR] = 100 nM and the dissociation constant (K_d(NC-cTAR) = 5.9 × 10^–8 M) of NC(11-55) for cTAR. (19) A K_i value of 50(±30) μM was obtained for AN3.

Figure 1. AN compounds and their effects on the NC(11-55)-induced cTAR destabilization. (A) Chemical structure of A10 and the three compounds selected by virtual screening. (B) Emission spectra of cTAR (0.1 μM) were recorded in the absence (black) and in the presence of 1 μM NC(11-55) before (red) and after addition of 100 μM **AN1** (blue), **AN2** (magenta), or **AN3** (green). Excitation wavelength was at 520 nm. The concentration of DMSO was less than 2% by volume. Insert: Inhibition of NC(11-55)-induced cTAR melting by **AN3**. The percentage of NC(11-55) activity is plotted as a function of the **AN3** concentration. An IC₅₀ value of 140 ± 90 μM was calculated for **AN3** from the fit of the experimental data (squares) to the following equation: % NC-induced cTAR destabilization = 1/(1 + (IC₅₀/[**AN3**])^p), where p = 1.8(±1) is the Hill coefficient.

Since AN3 showed the most efficient inhibition of NC chaperone activity, it was selected for further characterization and synthesized in larger quantities as outlined in Scheme 1. Compound 3 was obtained by nucleophilic substitution of commercially available methyl 4-(bromomethyl)benzoate 1 with 2′-hydroxyacetophenone 2 in a very good yield. Bromination at the α position of the ketone with copper(II) bromide afforded the monohalogenated compound 4 in moderate yield (60%); this latter was cyclized with thiourea in N,N-dimethylformamide to afford the 2-aminothiazole 5 quantitatively. (24) Finally the methyl ester was hydrolyzed with lithium hydroxide to afford AN3.

To confirm the direct binding of AN3 to NC as well as to rule out possible zinc ejection, we analyzed by mass spectrometry the changes in the mass of NC(11-55) after addition of AN3 to NC(11-55) at 1:1, 2:1, 5:1, and 10:1 molar ratio. While no peak indicates that AN3 could eject zinc, a peak corresponding to the sum of the masses of NC(11-55) and AN3 indicates the formation of a 1:1 complex with NC(11-55) (Supporting Information, Figure S2). Notably, the adduct between the reference fragment A10 and NC(11-55) was not detected by MS analysis. (14)

In a next step, the binding constant of AN3 to NC(11-55) was measured by monitoring the changes in the fluorescence anisotropy of AN3, which is intrinsically fluorescent, during its titration by NC(11-55) (Supporting Information, Figure S3). Assuming a 1:1 stoichiometry, we found an association constant K_NC-AN3 of (1.0 ± 0.2) × 10⁴ M^–1, which is well consistent with the K_i value determined in Figure 1B. We further characterized the binding properties of AN3 to NC(11-55) by isothermal titration calorimetry (ITC). Since marginal heat exchange accompanied the binding of AN3 to NC(11-55) even at high concentrations of both partners, indicating a mainly entropy-driven interaction, we performed a competition titration in which AN3 competed with cTAR for binding to NC(11-55). By monitoring the heat of reaction associated with the binding of NC to cTAR (Supporting Information, Figure S4), we obtained an association constant K_NC-AN3 = (1.4 ± 0.3) × 10⁴ M^–1, in good agreement with the above value.

Moreover, the ITC experiment confirmed that AN3 is a competitive inhibitor of NC/nucleic acids interaction. Taken together, our experiments consistently indicated that AN3 binds to NC(11-55) with a K_d value of ∼100 μM.

To check whether AN3 may directly interact with the Trp37 residue, which has been shown to be crucial for NC activity, (4, 19, 21, 25) we monitored the intrinsic fluorescence emission of the Trp37 residue in NC(11-55) upon the addition of 100 μM AN3, a concentration close to its K_d value. (26, 27)AN3 binding was found to decrease by 22% the fluorescence emission of Trp37, in line with a direct binding of AN3 to the Trp37 residue (Supporting Information, Figure S5). This direct interaction was also observed by molecular modeling, as the phenyl-aminothiazole moiety was found to be persistently over the Trp37 side chain along MD trajectories (Figure 2A). This nearly stacked conformation may well explain the decrease of Trp37 fluorescence emission, since Trp37 fluorescence was shown to be highly sensitive to stacking with oligonucleotide bases. (26, 28)

Figure 2. Binding of **AN3** to NC. (A) Representative MD structure: **AN3** is shown as cyan sticks, NC as cartoon and transparent surface. Residues highlighted by NMR and biophysical studies are shown as sticks and colored orange. (B) **AN3** protons involved in the interaction with NC by STD NMR experiment are shown on the STD spectrum. **AN3** concentration was 1 mM, whereas NC concentration was 30 μM. The STD experiment was recorded with selective saturation of the methyl group resonances of NC at 1 ppm, and with selective saturation in an empty spectral region (off-resonance). The subtraction of the spectra was performed internally via phase cycling after every scan to minimize the subtraction artifacts. The resulting subtracted spectrum contains only the signals of **AN3** interacting with NC. The signals of NC were suppressed with the use of a transverse relaxation filter applied before the acquisition.

NMR saturation-transfer difference (STD) (29) and chemical shift mapping experiments further confirmed the interaction of AN3 with NC. STD experiment (Figure 2B) clearly indicates that H5, H7, and the equivalent H16 and H18 protons of AN3 are involved in the interaction with NC. Moreover, NMR chemical shift mapping performed on N¹⁵-labeled NC suggests that residues Ala25, Asn17, and Met46 (as well as Gly4, Gln45, Cys18, Lys38, albeit to a lesser extent) of NC are involved in the binding with AN3 (Supporting Information, Figure S6). These findings nicely match with the binding mode of AN3 addressed by MD simulations (Figure 2A), thus facilitating further structure-based computer-aided ligand optimization studies. In particular, chemical optimization of AN3 should concentrate on improving intermolecular interactions with the NC to enhance its inhibitory potency, as well as optimizing the druggability of the small molecule. Concentrations of DMSO (50%) necessary for solubilizing AN3 during the NMR chemical shift mapping experiment prevented us from observing the interaction of AN3 with Trp37. Most probably, DMSO competed with AN3 for hydrophobic interactions with NC.

Antiretroviral activity against HIV-1 and cytotoxicity of AN3 were preliminarily evaluated as previously described. (17, 30) Briefly, HeLa P4.R5MAGI cells (expressing CD4, CCR5, and β-gal under the control of HIV-1 LTR) were infected by HIV-1Lai (MOI = 0.3) in the presence of serial dilutions of AN3 in medium for 24 h. After extensive washing, fresh medium with the respective AN3 concentrations was added. Viral supernatants were collected 24 h later, and viral titers were quantified on TZM-bl cells expressing firefly luciferase from an HIV-1 LTR promoter. Luciferase activity in cell lysates was measured by luminometry 44 h after infection. Compared to the DMSO control, which was set to 100% infection, AN3 clearly showed concentration-dependent antiviral activity with EC₅₀ = 95 μM (Figure 3). This value is highly comparable to the K_d value calculated by ITC or fluorescence anisotropy, as well as to the IC₅₀ value determined for the inhibition of NC(11-55)-induced cTAR destabilization, strongly suggesting that NC may be the primary target of the antiviral activity of AN3. Notably, complete inhibition was always achieved at 250 μM, a concentration where toxic effects were still low (25%) (Supporting Information, Figure S7).

Figure 3. **AN3** showed a concentration-dependent antiviral activity *in vitro*. Infectious viruses produced by P4.R5MAGI cells in the presence of **AN3** were quantified by luminometry on Tzm-bl cells. The mean of triplicates is shown with standard deviations. Data were normalized with respect to DMSO (100%). One of two representative experiments is shown.

In conclusion, starting from the previously identified NCI fragment A10, here we designed AN3 as an efficient inhibitor of the HIV-1 NC chaperone functions. Although the NC inhibitory activities of A10 and AN3 are comparable, AN3 showed antiviral activity in infected cells with a mechanism of action clearly indicating NC as the primary target. Structural requirements for AN3 binding to NC were elucidated by NMR, biophysical studies, and MD simulations, showing that AN3 interacts within the hydrophobic pocket of NC and competes with nucleic acids for this binding site. In agreement with MD simulations, a direct interaction of AN3 to the key residue Trp37 was observed, whereas NMR chemical shift mapping experiments further highlighted the involvement of Met46, Ala25, and Asn17, thus clearly identifying the hydrophobic pocket of NC as the binding site of AN3. Our multidisciplinary strategy demonstrates that the challenging goal of targeting the NC with specific small molecules is truly feasible, thus paving the way for the structure-based design of effective NCI. Overall, AN3 fulfills all expectations for an effective NCI, thus becoming a valuable starting point for further medicinal chemistry-oriented optimization.

Supporting Information

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Molecular dynamics, materials, mass spectrometry, general biophysics directions, isothermal titration calorimetry, general NMR directions, chemistry, and details of the antiretroviral and cytotoxicity assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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Corresponding Authors
- Yves Mely - Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, 74 route du Rhin, F-67401 Illkirch, France; Email: [email protected]
- Maurizio Botta - Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, via Aldo Moro 2, I-53019 Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, BioLife Science Bldg., Suite 333, 1900 N. 12th Street, Philadelphia, Pennsylvania 19122, United States; Email: [email protected]
Authors
- Mattia Mori - Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, via Aldo Moro 2, I-53019 Siena, Italy
- Alessandro Nucci - Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, via Aldo Moro 2, I-53019 Siena, Italy
- Maria Chiara Dasso Lang - Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, via Aldo Moro 2, I-53019 Siena, Italy
- Nicolas Humbert - Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, 74 route du Rhin, F-67401 Illkirch, France
- Christian Boudier - Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, 74 route du Rhin, F-67401 Illkirch, France
- Francois Debaene - Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, 74 route du Rhin, F-67401 Illkirch, France
- Sarah Sanglier-Cianferani - Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, 74 route du Rhin, F-67401 Illkirch, France
- Marjorie Catala - Laboratoire de Cristallographie et RMN Biologiques, CNRS, Paris Sorbonne Cité, 4 avenue de l’Observatoire, F-75006 Paris, France
- Patricia Schult-Dietrich - Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt, Germany
- Ursula Dietrich - Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt, Germany
- Carine Tisné - Laboratoire de Cristallographie et RMN Biologiques, CNRS, Paris Sorbonne Cité, 4 avenue de l’Observatoire, F-75006 Paris, France
Notes
The authors declare no competing financial interest.

Acknowledgment

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This work was supported by the European Project THINPAD “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance” (FP7–Grant Agreement 601969).

References

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Gorelick, R. J., Gagliardi, T. D., Bosche, W. J., Wiltrout, T. A., Coren, L. V., Chabot, D. J., Lifson, J. D., Henderson, L. E., and Arthur, L. O. (1999) Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences Virology 256, 92– 104
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Mori, M., Manetti, F., and Botta, M. (2011) Targeting protein-protein and protein-nucleic acid interactions for anti-HIV therapy Curr. Pharm. Des. 17, 3713– 3728
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Targeting protein-protein and protein-nucleic acid interactions for anti-HIV therapy
Mori, Mattia; Manetti, Fabrizio; Botta, Maurizio
Current Pharmaceutical Design (2011), 17 (33), 3713-3728CODEN: CPDEFP; ISSN:1381-6128. (Bentham Science Publishers Ltd.)
Protein-protein and protein-nucleic acid interactions are involved in many regulatory cellular pathways, playing a key role in cell growth and proliferation, as well as in the progression and development of various diseases such as infectious diseases. Esp. in the anti-AIDS research, protein-protein and protein-nucleic acid complexes are being considered as promising targets for pharmaceutical interventions aimed at overcoming the drug resistance obsd. for most of the classic enzyme inhibitors. Consequently, more and more protein-protein and protein-nucleic acid interaction inhibitors have being identified and developed as candidate agents for antiretroviral therapy. Here, we review the state of the art in the discovery and development of protein-protein and protein-nucleic acid interaction inhibitors that block the main steps of the HIV-1 replication cycle, giving a medicinal chem.-oriented view of strategies for inhibiting these regulatory interactions that are involved in the entry process, in the dimerization of reverse transcriptase and protease enzymes, and in the activity of the nucleocapsid protein by means of small mol. potential therapeutic agents.
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Shvadchak, V., Sanglier, S., Rocle, S., Villa, P., Haiech, J., Hibert, M., Van Dorsselaer, A., Mely, Y., and de Rocquigny, H. (2009) Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein Biochimie 91, 916– 923
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Goudreau, N., Hucke, O., Faucher, A. M., Grand-Maitre, C., Lepage, O., Bonneau, P. R., Mason, S. W., and Titolo, S. (2013) Discovery and structural characterization of a new inhibitor series of HIV-1 nucleocapsid function: NMR solution structure determination of a ternary complex involving a 2:1 inhibitor/NC stoichiometry J. Mol. Biol. 425, 1982– 1998
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Mori, M., Manetti, F., and Botta, M. (2011) Predicting the binding mode of known NCp7 inhibitors to facilitate the design of novel modulators J. Chem. Inf. Model. 51, 446– 454
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Predicting the Binding Mode of Known NCp7 Inhibitors To Facilitate the Design of Novel Modulators
Mori, Mattia; Manetti, Fabrizio; Botta, Maurizio
Journal of Chemical Information and Modeling (2011), 51 (2), 446-454CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
The HIV-1 nucleocapsid protein (NCp7) is an emerging target for antiretroviral therapy. Five hits have been reported to inhibit the NCp7-viral nucleic acids interaction at micromolar concns. We used two computationally refined structures of NCp7 as receptors to propose a reliable binding pose for these compds., by means of computational methods. Theor. binding modes are in agreement with available exptl. data. Results lay the foundations for a rationale development of more effective NCp7 inhibitors.
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Mori, M., Schult-Dietrich, P., Szafarowicz, B., Humbert, N., Debaene, F., Sanglier-Cianferani, S., Dietrich, U., Mely, Y., and Botta, M. (2012) Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein Virus Res. 169, 377– 387
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Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein
Mori, Mattia; Schult-Dietrich, Patrizia; Szafarowicz, Beata; Humbert, Nicolas; Debaene, Francois; Sanglier-Cianferani, Sarah; Dietrich, Ursula; Mely, Yves; Botta, Maurizio
Virus Research (2012), 169 (2), 377-387CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
The HIV-1 nucleocapsid protein (NC) is considered as an emerging drug target for the therapy of AIDS. Several studies have highlighted the crucial role of NC within the viral replication cycle. However, although NC inhibition has provided in vitro and in vivo antiretroviral activity, drug-candidates which interfere with NC functions are still missing in the therapeutic arsenal against HIV. Based on previous studies, where the dynamic behavior of NC and its ligand binding properties were investigated by computational methods, here the authors used a virtual screening protocol for discovering novel antiretroviral compds. which interact with NC. The antiretroviral activity of virtual hits was tested in vitro, whereas biophys. studies elucidated the direct interaction of most active compds. with NC(11-55), a peptide corresponding to the zinc finger domain of NC. Two novel antiretroviral small mols. capable of interacting with NC are presented here.
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Avilov, S. V., Piemont, E., Shvadchak, V., de Rocquigny, H., and Mely, Y. (2008) Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine Nucleic Acids Res. 36, 885– 896
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Beltz, H., Clauss, C., Piemont, E., Ficheux, D., Gorelick, R. J., Roques, B., Gabus, C., Darlix, J. L., de Rocquigny, H., and Mely, Y. (2005) 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. 348, 1113– 1126
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Godet, J., Ramalanjaona, N., Sharma, K. K., Richert, L., de Rocquigny, H., Darlix, J. L., Duportail, G., and Mely, Y. (2011) 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. 39, 6633– 6645
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Mori, M., Dietrich, U., Manetti, F., and Botta, M. (2010) Molecular dynamics and DFT study on HIV-1 nucleocapsid protein-7 in complex with viral genome J. Chem. Inf. Model. 50, 638– 650
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Molecular Dynamics and DFT Study on HIV-1 Nucleocapsid Protein-7 in Complex with Viral Genome
Mori, Mattia; Dietrich, Ursula; Manetti, Fabrizio; Botta, Maurizio
Journal of Chemical Information and Modeling (2010), 50 (4), 638-650CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
The HIV-1 nucleocapsid protein-7 (NCp7) is a highly basic, small zinc-binding protein involved in both DNA and RNA annealing and in viral particle maturation, including genome encapsidation; in addn., NCp7 exhibits a chaperone activity toward reverse transcriptase that promotes the two obligatory strand transfers during reverse transcription. Because of its interaction with highly conserved sequences of the HIV-1 genome, NCp7 is considered a new potential drug target, resistant to mutation, for antiviral activity. However, the high flexibility of this protein has limited the identification of structural determinants involved in the interaction with sequences of DNA and RNA. Here, we provide a quantum mechanics (d. functional theory, DFT) study of the zinc-binding motifs and a mol. dynamics (MD) simulation of the protein in complex with RNA and DNA, starting from available NMR structures. Results show that the interaction between the NCp7 and the viral genome is probably based on electrostatic interactions due to a cluster of basic residues, which is reinforced by the exploitation of nonelectrostatic contacts that further stabilize the complexes. Moreover, a possible mechanism for DNA destabilization that involves amino acids T24 and R26 is also hypothesized. Finally, a network of hydrophobic and hydrogen bond interactions for the stabilization of complexes with DNA and esp. with RNA is described here for the first time. The complexes between NCp7 and both DNA and RNA, resulting from computer simulations, showed structural properties that are in agreement with most of the currently available mol. biol. evidence and could be considered as reliable models (better than NMR structures currently available) for subsequent structure-based ligand design approaches.
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Cruceanu, M., Stephen, A. G., Beuning, P. J., Gorelick, R. J., Fisher, R. J., and Williams, M. C. (2006) Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein Anal. Biochem. 358, 159– 170
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Bernacchi, S. and Mely, Y. (2001) Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure Nucleic Acids Res. 29, E62– 62
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Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure
Bernacchi, Serena; Mely, Yves
Nucleic Acids Research (2001), 29 (13), e62/1-e62/8CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
Mol. beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramol. heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the resp. positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Forster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its assocd. absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of mol. beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.
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Zav’yalov, S. I., Kravchenko, N. E., Ezhova, G. I., Kulikova, L. B., Zavozin, A. G., and Dorofeeva, O. V. (2007) Synthesis of 2-aminothiazole derivatives Pharm. Chem. J. 41, 105– 108
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Synthesis of 2-aminothiazole derivatives
Zav'yalov, S. I.; Kravchenko, N. E.; Ezhova, G. I.; Kulikova, L. B.; Zavozin, A. G.; Dorofeeva, O. V.
Pharmaceutical Chemistry Journal (2007), 41 (2), 105-108CODEN: PCJOAU; ISSN:0091-150X. (Springer)
Ketones R1COCH2R2 (R1 = 4-MeC6H4, 4-ClC6H4, 4-MeOC6H4, R2 = H; R1 = Me, R2 = Ph; R1 = Me, Ph, R2 = EtO2C) or 5,5-dimethyl-1,3-cyclohexanedione were selectively brominated at the α-position with bromine in DMF in the presence of urea. The yield and purity of the product depends on the ketone/urea ratio with the optimal ratio being 1:(3-3.5). The obtained α-bromoketones were not isolated but converted into the corresponding 2-aminothiazoles on treatment with thiourea in DMF. The resulting 2-aminothiazoles were further modified by N-chloroacetylation followed by reaction with various heterocyclic thiols to provide several compds. with potential biol. activity.
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De Guzman, R. N., Wu, Z. R., Stalling, C. C., Pappalardo, L., Borer, P. N., and Summers, M. F. (1998) Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element Science 279, 384– 388
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Mely, Y., Piemont, E., Sorinas-Jimeno, M., de Rocquigny, H., Jullian, N., Morellet, N., Roques, B. P., and Gerard, D. (1993) Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study Biophys. J. 65, 1513– 1522
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Mely, Y., De Rocquigny, H., Morellet, N., Roques, B. P., and Gerad, D. (1996) Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy Biochemistry 35, 5175– 5182
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Zinc Binding to the HIV-1 Nucleocapsid Protein: A Thermodynamic Investigation by Fluorescence Spectroscopy
Mely, Y.; De Rocquigny, H.; Morellet, N.; Roques, B. P.; Gerard, D.
Biochemistry (1996), 35 (16), 5175-82CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be crit. in various NCp7 functions, esp. in the encapsidation process. To further understand the central role of zinc binding to NCp7, the authors closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivs. To this end, the fluorescence of either the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, resp. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with a 2.8×1014 M-1 binding const. about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this neg. cooperativity was shown to be related to the spatial proximity of the zinc-satd. finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were crit. and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding consts. to both sites. Taken together, the data provide further evidence for the nonequivalence of the two NCp7 finger motifs.
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Vuilleumier, C., Bombarda, E., Morellet, N., Gerard, D., Roques, B. P., and Mely, Y. (1999) Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study Biochemistry 38, 16816– 16825
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Mayer, M. and Meyer, B. (2001) Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor J. Am. Chem. Soc. 123, 6108– 6117
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Group Epitope Mapping by Saturation Transfer Difference NMR To Identify Segments of a Ligand in Direct Contact with a Protein Receptor
Mayer, Moriz; Meyer, Bernd
Journal of the American Chemical Society (2001), 123 (25), 6108-6117CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
A protocol based on satn. transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA120). As ligands we used Me β-D-galactoside and a biantennary decasaccharide. Anal. of the satn. transfer effects of Me β-D-galactoside showed that the H2, H3, and H4 protons are satd. to the highest degree, giving evidence of their close proximity to protons of the RCA120 lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the anal. of the binding specificities of many chem. modified galactose derivs. (Bhattacharyya, L.; Brewer, C. F., 1988). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over time-consuming chem. modifications. Efficient GEM benefits from a relatively high off rate of the ligand and a large excess of the ligand over the receptor. Even for a ligand like the biantennary decasaccharide with micromolar binding affinity, the binding epitopes could easily be mapped to the terminal β-D-Gal-(1-4)-β-D-GlcNAc (β-D-GlcNAc = N-acetyl-D-glucosamine) residues located at the nonreducing end of the two carbohydrate chains. The binding contribution of the terminal galactose residue is stronger than those of the penultimate GlcNAc residues. We could show that the GlcNAc residues bind "edge-on" with the region from H2 to H4, making contact with the protein. Anal. of STD NMR expts. performed under competitive conditions proved that the two saccharides studied bind at the same receptor site, thereby ruling out unspecific binding.
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Dietz, J., Koch, J., Kaur, A., Raja, C., Stein, S., Grez, M., Pustowka, A., Mensch, S., Ferner, J., Möller, L., Bannert, N., Tampe, R., Divita, G., Mely, Y., Schwalbe, H., and Dietrich, U. (2008) Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi ChemMedChem. 3, 749– 755
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Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal ψ
Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Moeller, Lars; Bannert, Norbert; Tampe, Robert; Divita, Gilles; Mely, Yves; Schwalbe, Harald; Dietrich, Ursula
ChemMedChem (2008), 3 (5), 749-755CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal ψ ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. The authors previously selected peptides binding to HIV-1 ψ-RNA or stem loops (SL) thereof by phage display. Herein, the authors describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize ψ-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the ψ packaging signal by NMR and tryptophan fluorescence quenching. Functional anal. revealed that psi-pepB caused a strong redn. of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compd. to develop new drugs targeting HIV-1.
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Savina Malancona, Mattia Mori, Paola Fezzardi, Marisabella Santoriello, Andreina Basta, Martina Nibbio, Lesia Kovalenko, Roberto Speziale, Maria Rosaria Battista, Antonella Cellucci, Nadia Gennari, Edith Monteagudo, Annalise Di Marco, Alessia Giannini, Rajhans Sharma, Manuel Pires, Eleonore Real, Maurizio Zazzi, Maria Chiara Dasso Lang, Davide De Forni, Francesco Saladini, Yves Mely, Vincenzo Summa, Steven Harper, Maurizio Botta. 5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein. ACS Medicinal Chemistry Letters 2020, 11 (5) , 766-772. https://doi.org/10.1021/acsmedchemlett.9b00608
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Virginia Pontecorvi, Mattia Mori, Francesca Picarazzi, Susi Zara, Simone Carradori, Amelia Cataldi, Andrea Angeli, Emanuela Berrino, Paola Chimenti, Alessia Ciogli, Daniela Secci, Paolo Guglielmi, Claudiu T. Supuran. Novel Insights on Human Carbonic Anhydrase Inhibitors Based on Coumalic Acid: Design, Synthesis, Molecular Modeling Investigation, and Biological Studies. International Journal of Molecular Sciences 2022, 23 (14) , 7950. https://doi.org/10.3390/ijms23147950
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J A Morales-Morales, A F Villamarin, E Florez-López, J J Rios-Acevedo. Study of the electrochemical oxidation of 2-Thiazolamine and 2-Oxazolamine on a platinum wire as working electrode. Journal of Physics: Conference Series 2018, 1119 , 012006. https://doi.org/10.1088/1742-6596/1119/1/012006
J.A Morales-Morales, J. J. Rios-Acevedo, E. Florez-Lopez. A preliminary study of cyclic voltammetry in glassy carbon electrode. Redox Behaviour of 2-Amino-4-phenylthiazoles. Journal of Physics: Conference Series 2018, 1119 , 012016. https://doi.org/10.1088/1742-6596/1119/1/012016
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Cite this: ACS Chem. Biol. 2014, 9, 9, 1950–1955

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Abstract
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Figure 1
Figure 1. AN compounds and their effects on the NC(11-55)-induced cTAR destabilization. (A) Chemical structure of A10 and the three compounds selected by virtual screening. (B) Emission spectra of cTAR (0.1 μM) were recorded in the absence (black) and in the presence of 1 μM NC(11-55) before (red) and after addition of 100 μM AN1 (blue), AN2 (magenta), or AN3 (green). Excitation wavelength was at 520 nm. The concentration of DMSO was less than 2% by volume. Insert: Inhibition of NC(11-55)-induced cTAR melting by AN3. The percentage of NC(11-55) activity is plotted as a function of the AN3 concentration. An IC₅₀ value of 140 ± 90 μM was calculated for AN3 from the fit of the experimental data (squares) to the following equation: % NC-induced cTAR destabilization = 1/(1 + (IC₅₀/[AN3])^p), where p = 1.8(±1) is the Hill coefficient.
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Scheme 1
Scheme 1. Synthesis of AN3^a
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Scheme aReaction and conditions: (i) K₂CO₃, DMF, rt, overnight; (ii) CuBr₂, CHCl₃/AcOEt (1:1), reflux, overnight; (iii) thiourea, DMF, rt, 1 h; (iv) LiOH·H₂O, THF/MeOH/H₂O (3:1:1), rt 5 h.
Figure 2
Figure 2. Binding of AN3 to NC. (A) Representative MD structure: AN3 is shown as cyan sticks, NC as cartoon and transparent surface. Residues highlighted by NMR and biophysical studies are shown as sticks and colored orange. (B) AN3 protons involved in the interaction with NC by STD NMR experiment are shown on the STD spectrum. AN3 concentration was 1 mM, whereas NC concentration was 30 μM. The STD experiment was recorded with selective saturation of the methyl group resonances of NC at 1 ppm, and with selective saturation in an empty spectral region (off-resonance). The subtraction of the spectra was performed internally via phase cycling after every scan to minimize the subtraction artifacts. The resulting subtracted spectrum contains only the signals of AN3 interacting with NC. The signals of NC were suppressed with the use of a transverse relaxation filter applied before the acquisition.
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Figure 3
Figure 3. AN3 showed a concentration-dependent antiviral activity in vitro. Infectious viruses produced by P4.R5MAGI cells in the presence of AN3 were quantified by luminometry on Tzm-bl cells. The mean of triplicates is shown with standard deviations. Data were normalized with respect to DMSO (100%). One of two representative experiments is shown.
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References
This article references 30 other publications.
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  A review. The role of nucleocapsid protein (NC) in the early steps of retroviral replication appears largely that of a facilitator for reverse transcription and integration. Using a wide variety of cell-free assay systems, the properties of mature NC proteins (e.g.HIV-1 p7NC or MLV p10NC) as nucleic acid chaperones HIV-1 p7NC or MLV p10NC have been extensively investigated. The effect of NC on tRNA annealing, reverse transcription initiation, minus-strand-transfer, processivity of reverse transcription, plus-strand-transfer, strand-displacement synthesis, 3' processing of viral DNA by integrase, and integrase-mediated strand-transfer has been detd. by a large no. of labs. Interestingly, these reactions can all be accomplished to varying degrees in the absence of NC; some are facilitated by both viral and non-viral proteins and peptides that may or may not be involved in vivo. What is one to conclude from the observation that NC is not strictly required for these necessary reactions to occur NC likely enhances the efficiency of each of these steps, thereby vastly improving the productivity of infection. In other words, one of the major roles of NC is to enhance the effectiveness of early infection, thereby increasing the probability of productive replication and ultimately of retrovirus survival.
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  Analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain
  Zhang, Yaqiang; Qian, Haoyu; Love, Zachary; Barklis, Eric
  Journal of Virology (1998), 72 (3), 1782-1789CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
  Previous studies have shown that in addn. to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.
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  Muriaux, Delphine; Mirro, Jane; Harvin, Demetria; Rein, Alan
  Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (9), 5246-5251CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as scaffolding in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjt1Onsbo%253D&md5=2fad56aa5e97a551725f135706cfcf28
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  Clever, Jared; Sassetti, Christopher; Parslow, Tristram G.
  Journal of Virology (1995), 69 (4), 2101-9CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
  The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (ψ site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 ψ element in vitro. This report aims at mapping features within the genetically defined ψ locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid deriv. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay contg. excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide ψ RNA element spanning the major splice donor and gag start codons but did not bind to antisense ψ transcripts. Quant. filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd ≃ 5 × 10-8 M), indicating that all major determinants of ψ binding affinity reside within the nucleocapsid portion of Gag. Chem. and RNase accessibility mapping, coupled with computerized sequence anal., suggested a model for ψ RNA structure comprising 4 independent stem-loops. Filter-binding studies revealed that RNAs corresponding to 3 of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with ∼4-fold-lower apparent affinity than the full-length ψ locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.
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  Gorelick, R. J., Gagliardi, T. D., Bosche, W. J., Wiltrout, T. A., Coren, L. V., Chabot, D. J., Lifson, J. D., Henderson, L. E., and Arthur, L. O. (1999) Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences Virology 256, 92– 104
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  Targeting protein-protein and protein-nucleic acid interactions for anti-HIV therapy
  Mori, Mattia; Manetti, Fabrizio; Botta, Maurizio
  Current Pharmaceutical Design (2011), 17 (33), 3713-3728CODEN: CPDEFP; ISSN:1381-6128. (Bentham Science Publishers Ltd.)
  Protein-protein and protein-nucleic acid interactions are involved in many regulatory cellular pathways, playing a key role in cell growth and proliferation, as well as in the progression and development of various diseases such as infectious diseases. Esp. in the anti-AIDS research, protein-protein and protein-nucleic acid complexes are being considered as promising targets for pharmaceutical interventions aimed at overcoming the drug resistance obsd. for most of the classic enzyme inhibitors. Consequently, more and more protein-protein and protein-nucleic acid interaction inhibitors have being identified and developed as candidate agents for antiretroviral therapy. Here, we review the state of the art in the discovery and development of protein-protein and protein-nucleic acid interaction inhibitors that block the main steps of the HIV-1 replication cycle, giving a medicinal chem.-oriented view of strategies for inhibiting these regulatory interactions that are involved in the entry process, in the dimerization of reverse transcriptase and protease enzymes, and in the activity of the nucleocapsid protein by means of small mol. potential therapeutic agents.
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  Mori, M., Manetti, F., and Botta, M. (2011) Predicting the binding mode of known NCp7 inhibitors to facilitate the design of novel modulators J. Chem. Inf. Model. 51, 446– 454
  16
  Predicting the Binding Mode of Known NCp7 Inhibitors To Facilitate the Design of Novel Modulators
  Mori, Mattia; Manetti, Fabrizio; Botta, Maurizio
  Journal of Chemical Information and Modeling (2011), 51 (2), 446-454CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
  The HIV-1 nucleocapsid protein (NCp7) is an emerging target for antiretroviral therapy. Five hits have been reported to inhibit the NCp7-viral nucleic acids interaction at micromolar concns. We used two computationally refined structures of NCp7 as receptors to propose a reliable binding pose for these compds., by means of computational methods. Theor. binding modes are in agreement with available exptl. data. Results lay the foundations for a rationale development of more effective NCp7 inhibitors.
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17. 17
  Mori, M., Schult-Dietrich, P., Szafarowicz, B., Humbert, N., Debaene, F., Sanglier-Cianferani, S., Dietrich, U., Mely, Y., and Botta, M. (2012) Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein Virus Res. 169, 377– 387
  17
  Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein
  Mori, Mattia; Schult-Dietrich, Patrizia; Szafarowicz, Beata; Humbert, Nicolas; Debaene, Francois; Sanglier-Cianferani, Sarah; Dietrich, Ursula; Mely, Yves; Botta, Maurizio
  Virus Research (2012), 169 (2), 377-387CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
  The HIV-1 nucleocapsid protein (NC) is considered as an emerging drug target for the therapy of AIDS. Several studies have highlighted the crucial role of NC within the viral replication cycle. However, although NC inhibition has provided in vitro and in vivo antiretroviral activity, drug-candidates which interfere with NC functions are still missing in the therapeutic arsenal against HIV. Based on previous studies, where the dynamic behavior of NC and its ligand binding properties were investigated by computational methods, here the authors used a virtual screening protocol for discovering novel antiretroviral compds. which interact with NC. The antiretroviral activity of virtual hits was tested in vitro, whereas biophys. studies elucidated the direct interaction of most active compds. with NC(11-55), a peptide corresponding to the zinc finger domain of NC. Two novel antiretroviral small mols. capable of interacting with NC are presented here.
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18. 18
  Avilov, S. V., Piemont, E., Shvadchak, V., de Rocquigny, H., and Mely, Y. (2008) Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine Nucleic Acids Res. 36, 885– 896
  There is no corresponding record for this reference.
19. 19
  Beltz, H., Clauss, C., Piemont, E., Ficheux, D., Gorelick, R. J., Roques, B., Gabus, C., Darlix, J. L., de Rocquigny, H., and Mely, Y. (2005) 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. 348, 1113– 1126
  There is no corresponding record for this reference.
20. 20
  Godet, J., Ramalanjaona, N., Sharma, K. K., Richert, L., de Rocquigny, H., Darlix, J. L., Duportail, G., and Mely, Y. (2011) 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. 39, 6633– 6645
  There is no corresponding record for this reference.
21. 21
  Mori, M., Dietrich, U., Manetti, F., and Botta, M. (2010) Molecular dynamics and DFT study on HIV-1 nucleocapsid protein-7 in complex with viral genome J. Chem. Inf. Model. 50, 638– 650
  21
  Molecular Dynamics and DFT Study on HIV-1 Nucleocapsid Protein-7 in Complex with Viral Genome
  Mori, Mattia; Dietrich, Ursula; Manetti, Fabrizio; Botta, Maurizio
  Journal of Chemical Information and Modeling (2010), 50 (4), 638-650CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
  The HIV-1 nucleocapsid protein-7 (NCp7) is a highly basic, small zinc-binding protein involved in both DNA and RNA annealing and in viral particle maturation, including genome encapsidation; in addn., NCp7 exhibits a chaperone activity toward reverse transcriptase that promotes the two obligatory strand transfers during reverse transcription. Because of its interaction with highly conserved sequences of the HIV-1 genome, NCp7 is considered a new potential drug target, resistant to mutation, for antiviral activity. However, the high flexibility of this protein has limited the identification of structural determinants involved in the interaction with sequences of DNA and RNA. Here, we provide a quantum mechanics (d. functional theory, DFT) study of the zinc-binding motifs and a mol. dynamics (MD) simulation of the protein in complex with RNA and DNA, starting from available NMR structures. Results show that the interaction between the NCp7 and the viral genome is probably based on electrostatic interactions due to a cluster of basic residues, which is reinforced by the exploitation of nonelectrostatic contacts that further stabilize the complexes. Moreover, a possible mechanism for DNA destabilization that involves amino acids T24 and R26 is also hypothesized. Finally, a network of hydrophobic and hydrogen bond interactions for the stabilization of complexes with DNA and esp. with RNA is described here for the first time. The complexes between NCp7 and both DNA and RNA, resulting from computer simulations, showed structural properties that are in agreement with most of the currently available mol. biol. evidence and could be considered as reliable models (better than NMR structures currently available) for subsequent structure-based ligand design approaches.
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22. 22
  Cruceanu, M., Stephen, A. G., Beuning, P. J., Gorelick, R. J., Fisher, R. J., and Williams, M. C. (2006) Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein Anal. Biochem. 358, 159– 170
  There is no corresponding record for this reference.
23. 23
  Bernacchi, S. and Mely, Y. (2001) Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure Nucleic Acids Res. 29, E62– 62
  23
  Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure
  Bernacchi, Serena; Mely, Yves
  Nucleic Acids Research (2001), 29 (13), e62/1-e62/8CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
  Mol. beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramol. heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the resp. positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Forster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its assocd. absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of mol. beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.
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24. 24
  Zav’yalov, S. I., Kravchenko, N. E., Ezhova, G. I., Kulikova, L. B., Zavozin, A. G., and Dorofeeva, O. V. (2007) Synthesis of 2-aminothiazole derivatives Pharm. Chem. J. 41, 105– 108
  24
  Synthesis of 2-aminothiazole derivatives
  Zav'yalov, S. I.; Kravchenko, N. E.; Ezhova, G. I.; Kulikova, L. B.; Zavozin, A. G.; Dorofeeva, O. V.
  Pharmaceutical Chemistry Journal (2007), 41 (2), 105-108CODEN: PCJOAU; ISSN:0091-150X. (Springer)
  Ketones R1COCH2R2 (R1 = 4-MeC6H4, 4-ClC6H4, 4-MeOC6H4, R2 = H; R1 = Me, R2 = Ph; R1 = Me, Ph, R2 = EtO2C) or 5,5-dimethyl-1,3-cyclohexanedione were selectively brominated at the α-position with bromine in DMF in the presence of urea. The yield and purity of the product depends on the ketone/urea ratio with the optimal ratio being 1:(3-3.5). The obtained α-bromoketones were not isolated but converted into the corresponding 2-aminothiazoles on treatment with thiourea in DMF. The resulting 2-aminothiazoles were further modified by N-chloroacetylation followed by reaction with various heterocyclic thiols to provide several compds. with potential biol. activity.
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25. 25
  De Guzman, R. N., Wu, Z. R., Stalling, C. C., Pappalardo, L., Borer, P. N., and Summers, M. F. (1998) Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element Science 279, 384– 388
  There is no corresponding record for this reference.
26. 26
  Mely, Y., Piemont, E., Sorinas-Jimeno, M., de Rocquigny, H., Jullian, N., Morellet, N., Roques, B. P., and Gerard, D. (1993) Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study Biophys. J. 65, 1513– 1522
  There is no corresponding record for this reference.
27. 27
  Mely, Y., De Rocquigny, H., Morellet, N., Roques, B. P., and Gerad, D. (1996) Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy Biochemistry 35, 5175– 5182
  27
  Zinc Binding to the HIV-1 Nucleocapsid Protein: A Thermodynamic Investigation by Fluorescence Spectroscopy
  Mely, Y.; De Rocquigny, H.; Morellet, N.; Roques, B. P.; Gerard, D.
  Biochemistry (1996), 35 (16), 5175-82CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be crit. in various NCp7 functions, esp. in the encapsidation process. To further understand the central role of zinc binding to NCp7, the authors closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivs. To this end, the fluorescence of either the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, resp. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with a 2.8×1014 M-1 binding const. about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this neg. cooperativity was shown to be related to the spatial proximity of the zinc-satd. finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were crit. and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding consts. to both sites. Taken together, the data provide further evidence for the nonequivalence of the two NCp7 finger motifs.
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28. 28
  Vuilleumier, C., Bombarda, E., Morellet, N., Gerard, D., Roques, B. P., and Mely, Y. (1999) Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study Biochemistry 38, 16816– 16825
  There is no corresponding record for this reference.
29. 29
  Mayer, M. and Meyer, B. (2001) Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor J. Am. Chem. Soc. 123, 6108– 6117
  29
  Group Epitope Mapping by Saturation Transfer Difference NMR To Identify Segments of a Ligand in Direct Contact with a Protein Receptor
  Mayer, Moriz; Meyer, Bernd
  Journal of the American Chemical Society (2001), 123 (25), 6108-6117CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  A protocol based on satn. transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA120). As ligands we used Me β-D-galactoside and a biantennary decasaccharide. Anal. of the satn. transfer effects of Me β-D-galactoside showed that the H2, H3, and H4 protons are satd. to the highest degree, giving evidence of their close proximity to protons of the RCA120 lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the anal. of the binding specificities of many chem. modified galactose derivs. (Bhattacharyya, L.; Brewer, C. F., 1988). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over time-consuming chem. modifications. Efficient GEM benefits from a relatively high off rate of the ligand and a large excess of the ligand over the receptor. Even for a ligand like the biantennary decasaccharide with micromolar binding affinity, the binding epitopes could easily be mapped to the terminal β-D-Gal-(1-4)-β-D-GlcNAc (β-D-GlcNAc = N-acetyl-D-glucosamine) residues located at the nonreducing end of the two carbohydrate chains. The binding contribution of the terminal galactose residue is stronger than those of the penultimate GlcNAc residues. We could show that the GlcNAc residues bind "edge-on" with the region from H2 to H4, making contact with the protein. Anal. of STD NMR expts. performed under competitive conditions proved that the two saccharides studied bind at the same receptor site, thereby ruling out unspecific binding.
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30. 30
  Dietz, J., Koch, J., Kaur, A., Raja, C., Stein, S., Grez, M., Pustowka, A., Mensch, S., Ferner, J., Möller, L., Bannert, N., Tampe, R., Divita, G., Mely, Y., Schwalbe, H., and Dietrich, U. (2008) Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi ChemMedChem. 3, 749– 755
  30
  Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal ψ
  Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Moeller, Lars; Bannert, Norbert; Tampe, Robert; Divita, Gilles; Mely, Yves; Schwalbe, Harald; Dietrich, Ursula
  ChemMedChem (2008), 3 (5), 749-755CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
  The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal ψ ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. The authors previously selected peptides binding to HIV-1 ψ-RNA or stem loops (SL) thereof by phage display. Herein, the authors describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize ψ-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the ψ packaging signal by NMR and tryptophan fluorescence quenching. Functional anal. revealed that psi-pepB caused a strong redn. of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compd. to develop new drugs targeting HIV-1.
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Supporting Information
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Molecular dynamics, materials, mass spectrometry, general biophysics directions, isothermal titration calorimetry, general NMR directions, chemistry, and details of the antiretroviral and cytotoxicity assays. This material is available free of charge via the Internet at http://pubs.acs.org.
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Functional and Structural Characterization of 2-Amino-4-phenylthiazole Inhibitors of the HIV-1 Nucleocapsid Protein with Antiviral Activity
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Functional and Structural Characterization of 2-Amino-4-phenylthiazole Inhibitors of the HIV-1 Nucleocapsid Protein with Antiviral ActivityClick to copy article linkArticle link copied!

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Functional and Structural Characterization of 2-Amino-4-phenylthiazole Inhibitors of the HIV-1 Nucleocapsid Protein with Antiviral Activity
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