Several cellular components may serve as potential targets for antiviral chemotherapy [44]. The complex P-TEFb is one of them, but finding a highly selective and non-cytotoxic CDK9 inhibitor is a difficult task due to its role in cellular transcription [45,46]. Nevertheless, CDK9 has been the focus of several studies (Reviewed in [46,47,48]). Among the principal inhibitors known to block CDK9 kinase activity is flavopiridol [49,50] and derivatives [51], indirubin-3'-monoxime [52,53], a nucleotide analog DRB [54], and R-roscovitine (CYC202) [54]; however, this last one is also known to inhibit other CDKs, such as CDK2 [55]. Recently, iron chelators have also been shown to block HIV-1 transcription by targeting both CDK9 and CDK2 [56].

Cyclin T1 has also been the target of several inhibitors such as anti-human Cyclin T1 intrabodies targeting the Cyclin T1/Tat interaction [57], microRNAs targeting Cyclin T1 expression [58], dominant negative mutants of Cyclin T1 that act by either specifically degrading Tat [59] or forming kinase inactive complexes with Tat and CDK9 [60]. The growth factor granulin and some of its granulin cysteine-rich motif repeats were able to inhibit Tat transactivation by either binding Tat or the histidine rich domain of Cyclin T1 [61,62]. Potent inhibition of Tat transactivation is obtained by the overexpression of HEXIM1 (which sequesters P-TEFb in an inactive form) or its paralog HEXIM2 by binding to Cyclin T1 [63]. Furthermore, the human I-mfa domain-containing protein (HIC), as well as its I-mfa domain alone, can act as a dominant negative repressor [64].

CDK2 is another possible target to block HIV transcription. Indeed, Tat interacts with both the CTD of RNAPII and CDK2/Cyclin E, and hence helps CTD phosphorylation at Ser2 by CDK2. This kinase was suggested to be required for Tat-dependent transcription in vitro [65,66]. Among CDK2 inhibitors that blocked HIV-1 transcription were CYC202 ([55]; reviewed in [67]), Alsterpaullone [68], a CDK2 RNAi [69], and small peptide inhibitors [70,71]. CDK2/Cyclin E was also suspected to be implicated in the HIV transcription inhibition by an N-aminoimidazole derivative NR-818 [72].

Transcription factors such as Sp1 and NF-kB play a critical role in initiation of HIV transcription in newly infected cells when Tat has not yet been made [73]. They are potential targets to block HIV basal transcription [74]; however, off target effects might be observed due to the shared nature of these transcription factors with cellular genes. A plant lignan, isolated from Larrea tridentata [75] and derivatives [76,77], inhibited basal transcription by preventing interaction of Sp1 to its DNA binding site as well as Tat-dependent HIV replication in peripheral blood mononuclear cells (PBMCs). Some compounds have also been reported to interfere with both Sp1 and NF-kB [78,79,80]. Several studies have identified and characterized compounds targeting NF-kB-mediated activation of HIV-1 transcription, but none of them have reached the necessary selectivity (Reviewed in [74,81]). More recently, two inhibitors of tumor necrosis factor α (TNF-α) production, piperidylpyrimidine [82] and LMP-420 [83], have been shown to inhibit respectively HIV-1 LTR transactivation or/and HIV replication in PBMCs. Another known inhibitor of TNF-a production and HIV replication, pentoxifylline, initially found to inhibit HIV by blocking the binding of NF-kB to its motif [84,85], was shown to block HIV by inhibition of a cellular DNA damage sensing and repair protein, ATR [86,87]. Finally, noraristeromycin, a carbocyclic adenine nucleoside, inhibits IkBa phosphorylation and degradation, thus preventing phosphorylation of p65, one of the NF-kB subunits [88].

P300/CBP is also a potential target due to its role as co-activator of Tat transcription, as well as in transcription reactivation by acetylating Tat and by reversing effects of histone deacetylation [30,33]. A specific inhibitor of p300/CBP HAT activity, such as curcumin, blocked acetylation of Tat in vitro and the proliferation of the virus by reducing HIV transcription [89]. Another inhibitor, LTK-14, was shown to be more specific and less toxic than its parent garcinol by inhibiting only p300 and not PCAF, but did display some toxicity by altering global gene expression [90,91]. LTK-14 blocked histone acetylation in HIV infected cells and restricted HIV at micromolar concentrations. Another potential cellular target is PCAF, a Tat recruited HAT, which is involved in reactivation from latency. A polyclonal PCAF BRD antibody blocked Tat transactivation [92]. A few thousand compounds have been tested on an NMR-based chemical screen yielding one lead compound, and upon structure activity relationship (SAR), N1-aryl-propane-1,3-diamine was found to selectively bind to the BRD domain of PCAF [93]. Using a structure-based lead optimization of small molecule, Pan et al. found a compound able to disrupt Tat/PCAF interaction and Tat-dependent transactivation in the micromolar range [94].

Tat activates NF-kB via oxidative stress-induced cell signaling pathways like the PI3K/Akt signaling pathway [95]. Epigallocatechin-3-gallate by activating both AMPK and Nrf2 antioxidant signaling pathways, leads to the inhibition of the AKT signaling pathway preventing both NF-κB activation and Tat induced HIV-1 transactivation [96]. Another compound targeting the Akt pathway is the coumarin derivative BPRHIV001, displaying a therapeutic index (TI) (half-maximal cytotoxic concentration (CC₅₀)/half-maximal inhibitory concentration (IC₅₀)) over 1,000 in acutely infected PBMCs [97]. This compound decreases the phosphorylation of PDPK1, a known Akt activator, which in turn blocks Akt phosphorylation, leading to a destabilization of p300 protein, thus negatively influencing Tat-dependent transcription. Alternatively, 9-aminoacridine activates the Akt pathway and phosphorylation of p21WAF1, reactivating p53 and p21WAF1 pathways, leading to HIV replication inhibition. Indeed, p21WAF1 associate with P-TEFb, which leads to a decrease of CDK9 on the HIV LTR and inhibits Tat-dependent transcription [98].

Overexpression of NIPP1, the major nuclear inhibitor of the host Ser/Thr phosphatase-1 (PP1) [99], or stable expression of its central domain [100] increased CDK9 phosphorylation on Thr186 and the association of CDK9 with 7SK RNA, as well as decreased binding of PP1 to Tat. Compound 1H4, found by screening a library of 300,000 compounds, decreases transcription and HIV replication by its ability to bind a RVxF motif on PP1 and to disrupt PP1/Tat interaction at micromolar concentration, which are unfortunately close to being toxic [101]. Finally, resveratrol, a polyphenolic phytostilbene found in grapes and berries, was able to decrease Tat-induced HIV-1 LTR transactivation in a TAR-independent manner by increasing NAD⁺ and SIRT1 production [102,103,104]. Indeed, SIRT1, a class III HDAC, is known to recycle Tat into an inactive form by deacetylating Lys50 [105]. Tat is also known to inhibit SIRT1 activity, that in turn blocks the deacetylation of the NF-κB p65 subunit, resulting in transcription hyperactivity [105,106].

Oligonucleotide derivatives such as TAR decoys, aptamers, ribozymes and RNA interference (RNAi) have been the focus of numerous studies to inhibit Tat-dependent HIV transcription (reviewed in [107,108]). Recently, several clinical trials have been done targeting total or partial HIV transcription using TAR decoys, aptamers, ribozymes and/or small interference RNAs (siRNAs) [109]. The hammerhead ribozyme (OZ1) targeting the overlapping HIV vpr and Tat coding sequence has been delivered into primary cells using a gammaretroviral vector, an approach that is currently in phase II clinical trials ([110,111]; reviewed in [112,113]). RNAs have been designed to target conserved viral sequences in order to silence gene expression at the post-transcriptional level [114,115]. One of the major difficulties is delivery of the siRNAs into the appropriate target cells. To circumvent this issue, chimeric RNAs containing a CD4 aptamer and an siRNA have been designed allowing for the specific delivery into CD4 expressing cells, and via receptor competition, inhibit HIV entry, as well as gene expression. Others, such as anti-gp120 aptamer-siRNA chimeras, also display this inhibitory behavior (reviewed in [115]). An alternative delivery approach is to use RNAi-based gene therapy using lentiviral vectors to transduce RNA based inhibitors into non-dividing cells and thus, permanently deliver the transgene. To avoid the selection of resistant mutants, several RNA-based inhibitors can be used together. RNA-based hematopoietic cell gene therapies for treatment of HIV infection using ribozymes, aptamers and siRNA have been reviewed [116]. A lentiviral vector encoding a combination of a TAR-decoy (small nucleolar RNA that binds the Tat protein), a CCR5 targeting hammerhead ribozyme and an shRNA targeting Tat-Rev mRNAs suppressed HIV replication for over 42 days in primary CD34⁺ hematopoietic stem cells ([117]; reviewed in [114]). Preliminary proofs of safety using RNA-based hematopoietic cell gene therapy have been obtained; a study showed that transgenic T cells generated in SCID-hu-mouse have normal T-cell phenotypes [118]. This lentiviral vector has been used in phase I clinical trials, via ex vivo gene therapy, using hematopoietic stem cell engraftment in human patients [119]. Foamy virus vectors, another type of virus based vector, is also being explored. A foamy virus combinatorial anti-HIV vector that expresses three anti-HIV transgenes (HIV fusion inhibitor and two shRNAs against either Tat or Rev) was successfully transduced into CD34⁺ cells and engrafted into SCID mice [120]. Despite lingering challenges in delivery and off-target toxicity, RNA-based hematopoietic cell gene therapies are promising approaches to fight HIV.

Peptide-related inhibitors, such as peptoids [121] and Tat-mimicking peptides, have been developed to block Tat-dependent HIV transcription (reviewed in [74,107]). More recently, L50 a cyclic b-hairpin peptide was found to bind to TAR with nanomolar affinity and induce a conformational change in the bulge region that was incompatible with Tat binding [122,123,124]. However, L50 inhibited HIV replication in PBMCs in the high namolar range and affected reverse transcription and HIV transcription in the high micromolar range. So far, no peptide-based inhibitor has entered clinical trials. Interestingly, T-U2AF65, the fusion of a splicing factor with the Tat activation domain, showed a dominant negative effect on HIV transcription. This inhibitor binds to RNAPII CTD and to Cyclin T1, blocking the interaction of wild type Tat to the CTD in a TAR-independent fashion [125]. Expression of T-U2AF65 slowed down virus replication in a lymphocytary cell line, which eventually became chronic, but replicated poorly.

Besides its important role in viral transactivation, extracellular Tat is an important factor in viral pathogenesis. Strategies to develop a vaccine using Tat as an epitope have been reviewed [107,126,127]. Several clinical trials have been done to assess therapeutic and preventive Tat-based vaccinations [128,129,130,131,132]. TUTI-16, a synthetic anti-Tat epitope, successfully induced high titer antibodies against Tat variants from all clades and was tested on asymptomatic HIV infected subjects in phase I/IIa clinical trials [133,134]. TUTI-16 significantly reduced viral load at low vaccine doses, but not at higher doses due to activation of cytokines by the adjuvant components. In another phase I/IIa clinical trial, HIV-infected patients were vaccinated with mRNA electroporated dendritic cells expressing Tat, Rev and Nef. This vaccine was shown to be safe and feasible, but had no significant impact on clinical parameters [135]. The HIV-1 Oyi strain was identified in HIV infected patients in Africa who did not progress to AIDS and, though genetically similar to common HIV-1 strains, contained mutations in the Tat gene not found in other Tat variants. Using Tat Oyi, Mediouni et al. successfully developed a monoclonal antibody named 7G12 that has the capacity to recognize heterologous Tat variants by a common 3D epitope [136]. This antibody was able to block the uptake of extracellular Tat, resulting in the inhibition of all Tat biological activities. Despite several trials, vaccine based therapies have been met with limited success.

Natural aminoglycoside antibiotics such as neomycin have been shown to interact with TAR and to inhibit HIV, but non-specifically [151]. To increase affinity and selectivity to TAR, derivatives have been made by adding to the aminoglycoside backbones Arg or guanidino groups [184,195,196,197]. The best compound of the family is NeoR, an aminoglycoside derivative with a TI of 125 in PBMCs (Table 1) [197]. However, L-Arg-aminoglycoside conjugates were not completely selective in inhibiting HIV transcription, since they also blocked the virus entry into cells by interacting with the CXCR4 coreceptor [198,199]. Mechanisms of inhibition by amino-glycoside derivatives of virus entry or HIV transcription, as well as comparison to the compounds available, have been recently reviewed [194]. Moreover, aminoglycoside-Arg conjugates seemed to also be general inhibitors of eukaryote and prokaryote translation [200].

Temacrazine, a nanomolar active bis-triazoloacridone compound with low toxicity, was initially tested because of its anti-transcriptional activity in cancer and was found to inhibit HIV in acute, chronic and latent cells (Table 1) [201]. It is a selective inhibitor of transcription and blocked several strains of HIV and RT resistant mutants; however, it did not affect HIV-2 or SIV. Moreover, the mechanism is unknown, since it did not seem to affect Tat/TAR, Rev/RRE interactions and TNF-a pathway. It was suggested it affects a still unidentified highly specific viral target required for HIV-1 transcription. Besides this publication, no further work on HIV inhibition by temacrazine was ever reported, suggesting that this compound might present some undesirable properties to be pursued as an anti-HIV drug.

Several reports show that quinolone derivatives presented anti-HIV properties (reviewed in [202]). One of the early reports identified fluoroquinoline, K12, as a selective inhibitor of HIV transcription in acute, chronic and latent cells [203,204]. However, its mechanism of action was TAR independent and did not seem to be mediated by TNF-a, Tat or Sp1 [205]. WM5, a 6-aminoquinolone, was able to inhibit Tat-dependent LTR-driven transcription at the micromolar range, as well as specifically blocking Tat/TAR interaction by binding to TAR (Table 1) [187,206,207]. Using WM5, a 6-aminoquinolone, as a lead compound, several derivatives have been synthesized during several successive SAR (reviewed in [202]). The best quinolone based inhibitors of Tat-dependent transcription described so far are HM13N and NM13 (Table 1) [186,208]. HM13N, a drug-like compound, was obtained by replacing the quinolone ring with a naphthyridone core. This naphthyridone derivative was able to inhibit both HIV-1 and HIV-2 in MT-4 cells and displayed a reasonably good TI in chronically and latently infected cell lines, but was less potent in acutely infected Jurkat cells and PBMCs. The second compound, NM13, was selective only to HIV-1 with an IC₅₀ of 80 nM and a therapeutic index ³3,707 in MT-4 cells [208]. However, this compound presents a low solubility as compared to HM13N. It is interesting to note that two related 6-desfluoroquinolones, HM12 and HM13, were also able to inhibit in vivo TNF-a reactivation of latently infected OM-10.1 cells engrafted in hu-SCID mice [209,210]. However, to our knowledge, after more than 10 years of quinolone derivative optimizations, none of these compounds ever reached clinical trials, due in part to either solubility or cytoxicity issues.

Tat inhibitors were also screened from natural products of microbial fermentation extracts [165]. Durhamycin A, from the aureolic acid antibiotics family, is closely related structurally to the anti-tumor antibiotic UCH9. When tested on Jurkat cells infected with a defective HIV-1 virus needing co-transfection of Tat, the viral replication was inhibited in the low nanomolar range with a TI > 5,000 (Table 1). However, this molecule, with a MW over 2,000, is quite large, limiting its utility as an anti-HIV drug.

We have recently found that dCA, an analogue of a natural steroidal alkaloid from a marine sponge, inhibits Tat-mediated trans-activation of the integrated provirus by binding specifically to the TAR-binding domain of Tat (Figure 2A,B) (Table 1) [146]. dCA represses transcriptional activation when transfected Tat or purified recombinant Tat is added to cells stably expressing the 5' LTR promoter driving Luciferase expression. When transfected, Tat localizes in the nucleus and specifically accumulates in the nucleolus. dCA causes redistribution of Tat to the periphery of the nucleolus, forming a distinctive ring-like structure determined by confocal fluorescence microscopy of HeLa-CD4 cells expressing HIV-1 Tat-flag (Figure 2C). Without a TAR binding domain, the basic region-mutant Tat-1-BRM is excluded from the nucleolus, again confirming that dCA binds Tat via its TAR binding domain. By chromatin immunoprecipitation (ChIP), we demonstrate that dCA inhibits transcription initiation and elongation from the viral promoter in chronically infected cells [146].

Working at subnanomolar concentrations, dCA inhibits wild type HIV-1 and HIV-2 replication in acutely and chronically infected cells at an IC₅₀ as low as 0.7 pM. By comparison, selected compounds reported in Table 1 inhibit HIV only in the low-to-high nanomolar range (Table 1). Consistent with this mode of action, dCA treatment for 10 or 60 days of chronically infected cells reduces viral RNAs to undetectable levels with an IC₅₀ of »0.1 nM and IC₉₀ of <10 nM [146]. Moreover, dCA provides a significant additive effect with other ARVs when added to primary CD4⁺ T cells from HIV-viremic subjects (Figure 2D). Importantly, dCA abrogates spontaneous residual viral particle release from CD4⁺ T cells from virally suppressed subjects on highly active antiretroviral therapy (HAART) (Figure 2E). Finally, dCA discontinuation does not result in viral rebound [146]. Indeed, after terminating dCA treatment of HeLa-CD4 cells chronically infected with pNL43, no virus rebound was observed even 27 days post treatment arrest, contrary to what is observed with ARVs, suggesting that dCA promotes rapid and prolonged silencing of the promoter. Thus, dCA defines a unique class of anti-HIV drugs that may inhibit viral production from stable reservoirs and reduce residual viremia during HAART.