Nucleocapsid Protein: A Desirable Target for Future Therapies Against HIV-1
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The currently available anti-HIV-1 therapeutics is highly beneficial to infected patients. However, clinical failures occur as a result of the ability of HIV-1 to rapidly mutate. One approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. In this respect, the nucleocapsid (NC) protein, a zinc finger protein, is particularly attractive, as it is highly conserved and plays a central role in virus replication, mainly by interacting with nucleic acids. The compelling rationale for considering NC as a viable drug target is illustrated by the fact that point mutants of this protein lead to noninfectious viruses and by the inability to select viruses resistant to a first generation of anti-NC drugs. In our review, we discuss the most relevant properties and functions of NC, as well as recent developments of small molecules targeting NC. Zinc ejectors show strong antiviral activity, but are endowed with a low therapeutic index due to their lack of specificity, which has resulted in toxicity. Currently, they are mainly being investigated for use as topical microbicides. Greater specificity may be achieved by using non-covalent NC inhibitors (NCIs) targeting the hydrophobic platform at the top of the zinc fingers or key nucleic acid partners of NC. Within the last few years, innovative methodologies have been developed to identify NCIs. Though the antiviral activity of the identified NCIs needs still to be improved, these compounds strongly support the druggability of NC and pave the way for future structure-based design and optimization of efficient NCIs.
KeywordsLong Terminal Repeat Virtual Screening Nuclear Magnetic Resonance Spectroscopy Isothermal Titration Calorimetry Nucleocapsid Protein
Human immunodeficiency virus
Long terminal repeat
Reverse transcriptase complex
Spacer peptide 1
Spacer peptide 2
Trans-activation response element
Trans-activator of transcription
Unique 5′ sequence
The clinical implementation of antiretroviral drugs (ARDs) for HIV-infected individuals resulted in shifting an acute and lethal disease, AIDS, to a clinically manageable condition. This combined use of ARDs as a therapy is based on small molecular weight inhibitors targeting either (i) the key viral enzymes required for HIV-1 replication, namely reverse transcriptase (RT), integrase (IN), and protease (PR) or, less commonly, (ii) the key proteins that control viral entry into the host cell. These therapies efficiently lower the systemic viral burden below the detection limit in the blood and strongly favor long-term survival of HIV-infected patients despite the persistence of latently infected cellular reservoirs. Nevertheless, emergence and circulation of multidrug-resistant HIV-1 strains are fueled by the high rates of HIV-1 mutation and recombination, thus emphasizing the continuous need for novel therapies and innovative strategies to overcome drug resistance (DR) (Richman 2014).
Accordingly, a highly selective inhibition of the interaction of NCp7 and Gag-NC with their nucleic acid (NA) partners should lead to a potent antiretroviral activity, in synergy with common ARDs, and greatly enhance the genetic barrier for resistance. In this context, through the pleiotropic functions of NCp7 in the whole viral life cycle, these NC inhibitors will offer the new possibility to affect the assembly, and budding steps, that have not been targeted so far, in addition to the viral steps already targeted by other ARDs.
2 Structure and Zinc-binding Properties of the Nucleocapsid Protein
In line with the importance of NC for viral function, the requirement to maintain a ZF-binding structure, and ability to interact with NAs, the amino acid sequence is highly conserved across B and non-B subtypes and in viral isolates from treated patients [Fig. 2 and (Darlix et al. 2011; Godet et al. 2012)]. When the variation indexes of NCp7 sequences are scrutinized (Fig. 2), it appears that the key residues Val13, Phe16, Thr24, Ala25, Trp37, Gln45, and Met46 of the hydrophobic plateau are invariant (Amarasinghe et al. 2000; Bazzi et al. 2011, 2012; Bourbigot et al. 2008; De Guzman et al. 1998; Morellet et al. 1998; Spriggs et al. 2008). Moreover, in most mutated sequences, the NCp7 consensus amino acids exchange with an amino acid of a similar profile. This strong requirement for amino acid conservation to maintain the structural integrity for function appears to provide few mutational options to escape inhibitors targeted against NC.
3 The Nucleocapsid Protein is Necessary for a Large Spectrum of Viral Activities
GagNC–actin interactions in relation to actin dynamics likely modify the local curvature of the membrane (Kerviel et al. 2013; Schiralli Lester et al. 2013; Wilk et al. 1999), in order to allow the formation of the budding particle. The cellular ESCRT machinery is recruited to allow the release of the budding particle (Van Engelenburg et al. 2014). GagNC is engaged in this recruitment by interacting with Alix-containing Bro1 domain in cooperation with the neighboring Gagp6 domain that binds to the Alix-V domain (Dussupt et al. 2009; Popov et al. 2008). Moreover, it has been proposed that NC–Bro1 interactions depend on RNA in the cell (Sette et al. 2012). Similar to GagNC-Bro1 interaction, GagNC interacts with Tsg101 in the ESCRT I complex to support budding, which in turn, maintains gRNA integrity for packaging by preventing premature reverse transcription assembly due to budding defects (Chamontin et al. 2015).
4 The Nucleocapsid Protein Interacts with Self and Host-cell Proteins
The viral proteins RT (Druillennec et al. 1999; Lener et al. 1998), Vif (Bouyac et al. 1997), Vpr (de Rocquigny et al. 1997; Li et al. 1996), and Tat (Boudier et al. 2010) have been proposed to interact with NC. In the case of Vif, GagNC is likely the main target, while the main partner of RT is NCp7. Within Gag, the NC domain is also suspected to interact with its neighboring domain, p6. GagNC has also been shown to interact with cellular factors such as the actin cytoskeleton (Liu et al. 1999), the dsRNA-binding protein Staufen (Chatel-Chaix et al. 2007, 2008), the IGF-II mRNA-binding protein 1 (Zhou et al. 2008), the cellular ATP-binding protein ABCE1 (also termed HP68) (Lingappa et al. 2006), and Alix (Popov et al. 2008). These protein–protein interactions, notably with Alix, are thought to participate in HIV-1 assembly and budding. Moreover, most of these cellular proteins are packaged into viral particles (Alce and Popik 2004; Mouland et al. 2000; Ott et al. 1996; Zhou et al. 2008). In the case of Alix, a ternary complex has been recently proposed to form between GagNC, RNA, and the Bro domain of Alix, suggesting that GagNC–RNA interactions could be useful to recruit cellular proteins (Sette et al. 2012).
5 The Nucleocapsid Protein is Key for HIV-1 Nucleic Acids Regulation
NCp7 binds both specifically and nonspecifically to a large panel of NA sequences of sufficient length (5–8 nt.), with a reverse binding polarity between RNA and ssDNA [for a review, see (Darlix et al. 2011)]. The binding constants can vary by several orders of magnitude depending on the nature, the sequence, and the folding of the interacting sequences (Fisher et al. 1998; Vuilleumier et al. 1999), so that NCp7 can exert different functions, depending on the respective concentrations of the protein and the NA sequences. As a consequence of its basic character and its millimolar range concentration in the virus, NCp7 molecules can likely coat the complete gRNA (Chen et al. 2009a, b; Chertova et al. 2006), ensuring its protection against cellular nucleases (Krishnamoorthy et al. 2003). NCp7 also exhibits sequence-specific binding properties to defined single-stranded sequences. These specific and strong binding properties notably play a critical role in the recognition by the NC domain of Gag of the Ψ-encapsidation signal of the gRNA, enabling its specific recognition and selection among a large excess of cellular RNAs during virus assembly (Aldovini and Young 1990; Cimarelli and Darlix 2002; Lever et al. 1989; Muriaux and Darlix 2010; Muriaux et al. 2004).
Through its binding to NA, NCp7 exerts a role as a NA chaperone, which allows the protein to direct the rearrangement of NAs into their most stable conformation, and to promote the annealing of complementary sequences (Godet and Mely 2010; Levin et al. 2005; Rein et al. 1998). These NA chaperone properties rely on the ability of NCp7 to transiently destabilize the NA secondary structure (Azoulay et al. 2003; Beltz et al. 2003, 2004; Bernacchi et al. 2002; Cosa et al. 2006; Egele et al. 2004; Godet et al. 2011, 2013; Liu et al. 2005; Williams et al. 2001). This destabilization is mainly mediated by the hydrophobic region located on the top of the folded ZFs and strongly depends on the NA stability and structure, suggesting a co-evolutionary relationship between NCp7 and its NA targets (Beltz et al. 2003, 2005; Godet et al. 2011, 2013; Hergott et al. 2013). Guanosine is the pivotal nucleoside to be trapped (Grohman et al. 2013). This destabilization is further accompanied by the exposure and freezing of the local mobility of the bases where NCp7 is bound (Avilov et al. 2008; Bourbigot et al. 2008; Godet et al. 2011, 2013), a feature which is thought to be critical for the recognition of the complementary oligonucleotide sequence in the annealing reaction. A second major component of the NCp7 chaperone properties relies on its ability to promote the rapid annealing of complementary NA sequences (Darlix et al. 1993; Godet et al. 2006; Hargittai et al. 2004; Liu et al. 2007; Ramalanjaona et al. 2007; Vo et al. 2006, 2009; You and McHenry 1994). This component mainly depends on the N-terminal basic domain and its NA aggregation properties, which provide the highly dynamic macromolecular context to favor efficient strand exchange (Mirambeau et al. 2006; Stoylov et al. 1997). The ZFs and the hydrophobic plateau are also instrumental in the annealing reaction, by promoting specific pathways which are notably required to faithfully and specifically chaperone the two obligatory strand transfers, during reverse transcription (Godet et al. 2011, 2013). Effective strand annealing activity is further correlated with NCp7’s ability to rapidly bind and dissociate from NAs. Indeed, NC variants with slow on/off rates are poorly efficient in rearranging NAs, even though they are still capable of promoting aggregation of NAs (Cruceanu et al. 2006a, b; Stewart-Maynard et al. 2008). Comparison of the various forms of NC further revealed that Gag is a less efficient NA chaperone than NCp7 (Cruceanu et al. 2006a, b) and that NCp15 appears much weaker for NA aggregation compared to NCp9 and NCp7 (Mirambeau et al. 2006, 2007; Wang et al. 2014).
6 Zinc Ejectors as Nucleocapsid Protein Inhibitors
Due to their key involvement at many critical points in the HIV-1 replication cycle and their strong conservation among HIV-1 strains, the ZFs of NCp7 were naturally selected as the primary target for the development of inhibitors. To properly exert their functions, the ZFs of NC crucially rely on the binding of zinc atoms that are required to fold them into their highly constrained structures. As a consequence, molecules able to eject the zinc atoms from the fingers were naturally developed as the first NC inhibitors. As anticipated, these molecules were found to induce NC unfolding as well as a full loss of HIV-1 infectivity.
Since the development of the first zinc ejectors in 1993 (Rice et al. 1993), a number of different classes of compounds were designed [for a review, see (de Rocquigny et al. 2008; Goldschmidt et al. 2010; Musah 2004; Turpin et al. 2008)]. Most of these compounds exhibited strong antiviral activity and elicited little viral resistance, clearly underlining the relevance of NC as an appropriate target for an antiviral therapy. Unfortunately, these compounds appeared also quite toxic, so that their use for systemic administration was prevented. Currently, efforts are underway to use them as topical microbicides, in order to prevent HIV-1 transmission.
6.1 Zinc Ejectors: Structure and Mechanism of Action
The mechanism of action of several of these compounds was carefully investigated to identify the NC chemical groups targeted by these compounds and the sequence of chemical reactions that results in zinc ejection. The mechanisms of inactivation of NC ZFs by these compounds can be classified into three main groups: (i) electrophilic attack of the zinc fingers, (ii) zinc ejection through chelation, and (iii) covalent binding of the Cys residues by Pt.
In both ZFs, the nucleophilic cysteine thiolates appear as the primary targets for electrophilic attack. Though both fingers contain the same CysX2CysX4HisX4Cys motif, zinc ejectors were found to preferentially react with the distal finger motif. Computational studies (Loo et al. 1996; Maynard and Covell 2001) indicated that this increased reactivity was at least partly related to the better accessibility of the Cys residues in this finger. Electrophilic attack may be accompanied by either formation of intra- or inter-molecular disulfide bonds or acylation of cysteine and then lysine residues. The oxidative mechanism leading to disulfide bonds was observed for compounds of the NOBA and DIBA families (Loo et al. 1996; Yu et al. 1995). For instance, when NCp7 was incubated with NOBA, three intermolecular disulfide bonds, Cys15-Cys18, Cys28-Cys36, and Cys39-Cys49, formed (Yu et al. 1995). Similarly, DIBA was found to initiate the formation of intra- and inter-molecular disulfide bonds by preferentially attacking Cys36 and Cys49 residues (Loo et al. 1996). Formation of three disulfide bridges was also observed with the recently discovered WDO-217 compound, though in this case, the preferential sites of attack were not identified (Vercruysse et al. 2012). An acylation mechanism is observed with PATEs and SAMTs. It involves the nucleophilic attack by a zinc-coordinated cysteine of the carbonyl carbon of the inhibitor. This results in the covalent modification of the cysteine sulfur via an acyl transfer mechanism. Subsequently, additional acyl transfer reactions occur with other cysteine and lysine residues of NCp7 that will further decrease the affinity for zinc and finally lead to zinc ejection. Cys36 and Cys49 are the primary targets of PATEs, while Cys36 is the primary target of SAMT analogs (Basrur et al. 2000; Miller Jenkins et al. 2007). The preferential susceptibility of the Cys49 residue to electrophilic attack is likely related to its rather high pKa value in the zinc-bound protein, which confers it a role of a switch in the dissociation of zinc (Bombarda et al. 2002).
A different mechanism was inferred for NV038. Indeed, based on its structure, this compound is likely unable to allow acyl transfer or thiol-disulfide interchange. In fact, molecular modeling suggests that NV038 may act as a zinc chelator that binds one zinc ion through the two carbonyl oxygens of its ester groups (Pannecouque et al. 2010).
The third mode of action is represented by platinum nucleobase compounds that act through a two-step mechanism (Anzellotti et al. 2006; Quintal et al. 2011). They first recognize the Trp37 residue of NCp7 through π–π stacking and then form a Zn-S–Pt covalent bond, which results in zinc ejection. As for electrophilic zinc ejectors, the primary target of platinum nucleobase compounds is Cys49 in the C-terminal zinc finger.
6.2 Antiviral Activity In Vitro
Antiviral activity and cytotoxicity of zinc ejectors
Huang et al. (1998)
Huang et al. (1998)
Turpin et al. (1999)
Srivastava et al. (2004)
Pannecouque et al. (2010)
Vercruysse et al. (2012)
Sartori et al. (2000)
The activity of zinc ejectors is related to their ability to decrease the affinity of NCp7 for its target nucleic acids, as for example the ψ RNA sequence (Huang et al. 1998; Jenkins et al. 2005; Tummino et al. 1996). This effect depends on the concentration of the zinc ejector and on the order of addition of the partners. While SAMTs and PATEs were able to strongly inhibit RNA binding when preincubated with NCp7, they exhibit nearly no effect on metal coordination and RNA binding when they were added to preformed NCp7-RNA complexes. Likely, RNA protects the zinc-coordinating residues of NCp7 from the inhibitors (Chertova et al. 1998; de Rocquigny et al. 2008; Jenkins et al. 2005). Noticeably, WDO-217 appears quite unique in this respect, as it was found to efficiently eject zinc ions from NCp7, even in complexes with nucleic acids (Vercruysse et al. 2012). In addition, WDO-217 was observed to change the binding mode of NCp7 to oligonucleotides, but with no dramatic change in the binding constant. As the result of their reaction with NCp7, zinc ejectors were found to affect reverse transcription (Morcock et al. 2005; Pannecouque et al. 2010; Rice et al. 1995; Rice and Turpin 1996; Sharmeen et al. 2001), likely by altering the nucleic acid chaperone properties of NCp7 (Pannecouque et al. 2010; Vercruysse et al. 2012) that critically depend on the binding of zinc (Avilov et al. 2008; Beltz et al. 2005; Bernacchi et al. 2002; Godet et al. 2011). In addition, zinc ejectors affect also the late steps of the viral life cycle, since DIBAs and PATEs (Turpin et al. 1996, 1999), SRR-SB3 (Mahmood et al. 1998) and SAMTs (Miller Jenkins et al. 2010), but not WDO-217 (Vercruysse et al. 2012), were found to induce accumulation of aggregated and unprocessed Gag polyproteins (Turpin et al. 1996, 1999) that lead to the release of noninfectious virus particles. This aggregation is likely due to intermolecular bridging of the NC domains of neighbor Gag polyproteins. Zinc ejectors also fully inactivate cell-free HIV-1 virions, by promoting NCp7 oligomerization (Rice et al. 1995) or acylation (Basrur et al. 2000; Jenkins et al. 2005). Furthermore, WDO-217 was found to relieve the protection of the viral RNA from the NCp7 proteins in cell-free virions, through a still unknown mechanism (Vercruysse et al. 2012). Finally, zinc ejectors were also shown to inhibit HIV-1 transmission from infected cells to uninfected ones (Srivastava et al. 2004; Vercruysse et al. 2012).
Cytotoxicity of zinc ejectors is likely related to their limited selectivity for NCp7 over zinc finger-containing host proteins, such as poly(ADP-ribose) polymerase (PARP) (with two CCHC zinc fingers), SP1 (with three CCHH-type Zn fingers), and GATA-1 (with two CCCC-type Zn fingers). For instance, NOBA shows only poor selectivity for NCp7, as it inhibits the enzymatic activity of PARP and blocks GATA-1 binding to their target DNA sequences (Huang et al. 1998). On the contrary, DIBA, ADA, and dithiane did not show any significant reactivity on either PARP or SP1 and GATA-1, which may likely explain their lower cytotoxicity (Huang et al. 1998). Likewise, the poorly cytotoxic PATE compounds did not show any reactivity on SP1 (Turpin et al. 1999). Finally, SAMTs did not react with CCHH zinc finger proteins and RING-like zinc-binding domains, but showed some reactivity toward Friend of GATA-1 (FOG-1) and GATA-1 (Jenkins et al. 2006).
6.3 Evaluation of Zinc Ejectors for Therapeutic Applications
Due to their potent antiviral activity in vitro, several attempts were made to evaluate the potential therapeutic use of zinc ejectors in vivo. To our knowledge, only two zinc ejectors, namely ADA and benzisothiazolone, were tested in clinical studies. Due to its toxicity, assays with the second compound were rapidly stopped (Turpin 2003). Preclinical tolerance assays showed that oral doses of 1.5 g ADA daily for 1 month were well tolerated, with no evidence of adverse effects (Vandevelde et al. 1996). Then, ADA was administrated three times daily during 3 months in addition to other antiviral therapy to fifteen individuals with advanced AIDS within a Phase I/II clinical trial. Unfortunately, serious nephrotoxicity as well as glucose intolerance appeared during the treatment, a serious enough event so that several patients dropped out of the clinical trial (Goebel et al. 2001). Moreover, ADA showed only a modest efficacy, as evidenced by an increase in T cell CD4+ counts and a reduction in the viral load in less than half of the treated patients (Goebel et al. 2001). On a more positive note, no ADA resistant virus could be isolated from ADA-treated patients. Unfortunately, the clinical trial was not conclusive, most likely since ADA is clearly not the most efficient antiviral compound in vitro (Table 1) and shows a number of off-target effects, such as inhibition of lymphocyte cytokine production (Rice et al. 1997a, b; Tassignon et al. 1999) and ribonucleotide reductase activity (Fagny et al. 2002). The systemic activity of zinc ejectors was also tested with SAMT compounds on an HIV-1 transgenic mouse model (Schito et al. 2003). These compounds reduced by 2–3 logs the infectivity of viruses expressed from the spleen cells of the transgenic mice and had no effect on immune cell cytokine production. Furthermore, sub-dermal delivery of a SAMT lead compound in cynomolgus macaques infected with SIV/DeltaB670 virus lowered the levels of infectious virus in peripheral blood mononuclear cells, but did not affect the virus load (Schito et al. 2006). Importantly, the SAMT lead compound was well tolerated and did not alter liver, kidney, or immunologic function of the treated monkeys. Though these data suggest that SAMT compounds may be safe in a primate model, it still remains to be demonstrated whether, due to their limited selectivity, zinc ejectors could be reasonably used as a long-term systemic therapeutics in patients.
Due to their potent activity and potential safety concerns, the application of zinc ejectors as topical microbicides appears more promising. The proof of concept for this application was demonstrated with SAMTs, which were shown to prevent HIV transmission from infected cells to uninfected cells, with EC50 values below 0.1 µM (Srivastava et al. 2004). Later, SAMTs were shown in the cervical explant model to inhibit the infection of target cells in the explant tissue and the dissemination of the infection by immune cells migrating out of the explant (Wallace et al. 2009). Interestingly, no virus infectivity was observed up to one week after SAMTs removal. Moreover, SAMTs antiviral activity was retained in both synthetic cervical mucous and human seminal plasma. Finally, the SAMT compounds were shown to induce no significant histology changes and irritation in the rabbit vaginal irritation model (Tien et al. 2005; Wallace et al. 2009). The SAMTs were further evaluated in rhesus macaques to determine their ability to prevent vaginal transmission of the simian-human immunodeficiency virus (SHIV) (Wallace et al. 2009). The monkeys were treated vaginally with 1 % SAMT in hydroxyethylcellulose universal placebo gel 20 min prior to challenge with a mixed CXCR4-tropic and CCR5-tropic SHIV virus inoculum (Wallace et al. 2009). Five out of six macaques were protected from infection, while only one infected animal expressed the CCR5-tropic SHIV. These findings strongly support the use of SAMTs as potential topical microbicides to prevent HIV transmission. Since WDO-217 at low micromolar concentrations was recently shown to inactivate HIV-1 captured by DC-SIGN-expressing cells and prevent their transmission to CD4+ T lymphocytes (Vercruysse et al. 2012), it is anticipated that WDO-217 may also be a valuable candidate for the development of topical microbicide formulations.
In conclusion, zinc ejectors show potent antiviral activity against a large spectrum of HIV-1 strains, without eliciting resistance. However, their limited selectivity raises toxicity concerns, limiting this class of NC inhibitors to microbicide formulations. Alternatively, due to their ability to inactivate HIV-1 efficiently without compromising viral surface antigens, they may have promise for use in vaccine strategies (Arthur et al. 1998; Chertova et al. 1998, 2006).
7 Inhibitors Targeting Nucleocapsid Protein Interaction with Nucleic Acids
In addition to zinc ejectors, a number of non-covalent NC inhibitors (NCIs) were identified during the past decade and used both as tools to increase our understanding of the biological and pathological functions of NC, as well as hit/lead candidates for the development of potential innovative antiretroviral therapeutics. However, the discovery of NCIs that demonstrate potent antiretroviral activity in vitro and in vivo still remains a considerable challenge. Indeed, only a few of the NCIs disclosed to date were found to inhibit HIV-1 replication in cell-based antiretroviral assays and none reached yet the preclinical phases of pharmaceutical evaluation. Since non-covalent NCIs are thought to show a greater specificity than zinc ejectors, and thus be presumably less toxic, these properties may well be superior for clinical translation, which makes this class of NCIs a desirable pharmaceutical goal. Since pioneering studies on the discovery and preliminary characterization of non-covalent NCIs have been reviewed recently (de Rocquigny et al. 2008; Goldschmidt et al. 2010; Mori et al. 2011a, b), we will mainly focus on novel strategies undertaken since 2009 that have identified small molecules endowed with two different mechanisms of action: (i) non-covalent NCIs binding to NC and (ii) non-covalent NCIs binding to nucleic acid partners of NC.
7.1 Non-covalent NCIs Binding to the Nucleocapsid Protein
In an attempt to provide structural hints on the binding of these fragments to NC, an in-depth molecular modeling study was performed by Mori and colleagues (Mori et al. 2011a, b). NCI fragments were docked toward two computationally refined structures of NCp7 (Mori et al. 2010), showing that these molecules may preferentially bind to the Trp37 residue on the ZF hydrophobic platform (Fig. 3). The good correlation between experimental and theoretical findings corroborated the reliability of the computational model, thus paving the way for possible structure-based drug design approaches.
The HTS assay methodology, discussed above, was also used to characterize a methylated oligoribonucleotide NCI (Avilov et al. 2012; Grigorov et al. 2011). Although modified oligoribonucleotides may be considered at the boundary between small molecules and biomolecules, the findings of this study have significantly contributed to the understanding of the molecular basis of NC inhibition and theoretical design of NCIs. Based on the evidence that NCp7 chaperones reverse transcription, methylated oligoribonucleotides (mODNs) mimicking the long terminal repeat end sequences of proviral DNA were synthesized and evaluated in vitro and ex vivo. Inhibition of the NCp7 chaperone activity was monitored through the fluorescence of the Rh6G-5′-cTAR-3′-Dabcyl DNA sequence (Shvadchak et al. 2009). Further tests revealed that mODN-11, having the sequence 2′-O-Me-(GGUUUUUGUGU-NH2), was the most potent oligoribonucleotide among the test set, inhibiting HIV-1 replication in MT4 cells at sub-nanomolar concentrations (IC50 = 0.3 nM) and also showing low cytotoxicity (CC50 = 7.7−13.4 µM). Time of addition experiments further revealed that mODN-11 inhibited HIV-1 replication with the same time frame as the reverse transcriptase (RT) inhibitor AZT, thus suggesting that the reverse transcription complex may be the target of the oligoribonucleotide. In fact, AZT and mODN-11 provided a synergistic inhibition of HIV-1 replication, further reinforcing the hypothesis, already verified in vitro, that mODN-11 targets NCp7 and that NCp7 is an indispensable partner of RT. The mechanism of action of mODNs was further investigated by isothermal titration calorimetry and fluorescence-based techniques and compared to unmodified oligoribonucleotides (Avilov et al. 2012). Interestingly, this study showed that mODNs bearing repeats of GU or GT pairs tightly bind to NCp7 through nonelectrostatic interactions and compete with NAs for the binding to the NCp7 hydrophobic pocket, suggesting that the mODNs may impair the RT-directed viral DNA synthesis by sequestering NCp7 molecules.
Based on these results, one may speculate that the methylation of the GU- or GT-rich oligoribonucleotides improves their lipophilicity and, therefore, their affinity for the small hydrophobic pocket of NCp7. Indeed, although NCp7 is a highly basic protein that interacts with NAs by means of electrostatic interactions, hydrophobicity appears as a key feature for potent and effective NCIs. In agreement with the several studies that emphasized the crucial role of NCp7 aromatic residues Trp37 and Phe16, the ideal NCI should be able to compete for the binding of NAs by interacting with the NCp7 hydrophobic platform. Consistent with this hypothesis, in recent medicinal chemistry-oriented studies, a number of NCIs endowed with hydrophobic/aromatic groups have been discovered by means of different techniques, including virtual screening and HTS. Moreover, the three-dimensional structure of NCp7 in complex with a NCI confirmed the key role of the aromatic residues in the interaction. Highlights of these studies are reported below.
Overall, the study provided a new HTS for identifying NCIs with a specific mechanism of action, which was exemplified by the identification of two low molecular weight NCIs with modest antiretroviral activity in ex vivo cell assays. These compounds provide a starting point from which to rationally optimize their NCI efficacy through directed medicinal chemistry effort. Notably, CMPD-8 shares a significant pharmacophoric similarity with EO3 and HO2 fragments previously identified (Shvadchak et al. 2009), thus suggesting that this molecular scaffold may be highly promising for the development of effective NCIs.
The optimization of the above-discussed NCIs for increased antiviral efficacy is hampered by the lack of structural details on their respective adducts with NCp7. This could be partially attributed to the high flexibility of NCp7, which makes it not suitable for high-throughput techniques such as X-ray crystallography. To this point, all published structures of NCp7 to date have been solved by NMR spectroscopy. Although the conformation of NCp7 in complex with a small hydrophobic NCI was unknown at the time of these works, molecular modeling studies have generally assumed that NCp7 in complex with a small hydrophobic NCI may be similar to the conformation adopted in binding to NAs.
In summary, these reports provide both an important step forward bettering the understanding of the molecular basis for NC inhibition by small molecules as well as strongly supporting the druggability of NCp7. Moreover, the high-resolution details of NCp7 in complex with a guanosine mimicking NCI may be used for future structure-based design and optimization of more efficient and drug-like NCIs.
7.2 Non-covalent NCIs Binding to Nucleic Acid Partners of the Nucleocapsid Protein
In the attempt to identify non-covalent NCIs, another strategy is to design small molecules that bind to the NA partners of NC, in order to prevent the interaction between NC and NAs or to disrupt the already formed complexes. As a proof of concept, in 2009, Turner and colleagues used a series of non-covalent molecular probes to investigate the structural features involved in the NC-mediated dimerization of HIV-1 genomic RNA (Turner et al. 2009). To this end, the authors used general intercalators, minor groove binders, mixed-mode intercalator/groove binders, and multifunctional polycationic aminoglycosides that, notably, have shown to not bind NC. The polycationic aminoglycosides were found to prevent the NC chaperone activity by binding to specific sites of the RNA stem loop 1 (SL1) mostly by mimicking the RNA-binding properties of the NC through electrostatic interactions, whereas all other molecules reduced the efficiency of NC-mediated isomerization by stabilizing double-stranded RNA structures. Although these studies were performed with molecular probes that are rather far from being considered as candidate therapeutics, these findings point out that inhibition of NC chaperone activity in vitro could be accomplished using small molecules binding to nucleic acids partners of NC, an important precedent.
8 Concluding Remarks
We have provided evidence that novel screening methodologies and chemical libraries have resulted in the identification of novel compounds that show inhibitory activity against GagNC/NCp7 (NCIs). Protease inhibitors (PIs) are very effective and demonstrate highly cooperative dose-response curves, which can be explained by the capacity of these inhibitors to independently affect multiple discrete steps in the viral life cycle, such as entry, RT, and post-reverse transcription steps (Rabi et al. 2013). In a parallel capacity to PIs, and as we have discussed in our review, NCIs have the potential to affect multiple discrete viral pathways, similar to PIs. We propose that NCIs will have similar properties to PIs in regard to demonstrating highly cooperative dose-response curves. Most importantly, and in contrast to protease, NCIs should not tolerate mutational changes without considerable loss of function. Therefore, the apparent strong genetic barrier necessary for NCI resistance and the fact that NCIs inhibit a viral protein with multiple key functions throughout the HIV-1 life cycle strongly supports the continued research on identifying and optimizing NCIs as well as investigations into their antiviral mechanisms.
This work was supported by the European Project THINPAD “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance” (FP7—Grant Agreement 601969), ANRS, NIGMS (P50GM103368), and the HIVE Center. G.M. and S.L. are greatly indebted to Jose Maria Gatell for his kind hosting and support of the AIDS Research Group (IDIBAPS).
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