Protein Phosphatase-1 -targeted Small Molecules, Iron Chelators and Curcumin Analogs as HIV-1 Antivirals.

BACKGROUND: Despite efficient suppression of HIV-1 replication, current antiviral drugs are not able to eradicate HIV-1 infection. Permanent HIV-1 suppression or complete eradication requires novel biological approaches and therapeutic strategies. Our previous studies showed that HIV-1 transcription is regulated by host cell protein phosphatase-1. We also showed that HIV-1 transcription is sensitive to the reduction of intracellular iron that affects cell cycle-dependent kinase 2. We developed protein phosphatase 1-targeting small molecules that inhibited HIV-1 transcription. We also found an additional class of protein phosphatase-1-targeting molecules that activated HIV-1 transcription and reported HIV-1 inhibitory iron chelators and novel curcumin analogs that inhibit HIV-1. Here, we review HIV-1 transcription and replication with focus on its regulation by protein phosphatase 1 and cell cycle dependent kinase 2 and describe novel small molecules that can serve as future leads for anti-HIV drug development. RESULTS: Our review describes in a non-exhaustive manner studies in which HIV-1 transcription and replication are targeted with small molecules. Previously, published studies show that HIV-1 can be inhibited with protein phosphatase-1-targeting and iron chelating compounds and curcumin analogs. These results are significant in light of the current efforts to eradicate HIV-1 through permanent inhibition. Also, HIV-1 activating compounds can be useful for "kick and kill" therapy in which the virus is reactivated prior to its inhibition by the combination antiretroviral therapy. CONCLUSION: The studies described in our review point to protein phosphatase-1 as a new drug target, intracellular iron as subject for iron chelation and novel curcumin analogs that can be developed for novel HIV-1 transcription- targeting therapeutics.

Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1.

BACKGROUND: HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1. RESULTS: Here we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9's Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1. CONCLUSION: We identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs.

1E7-03, a low MW compound targeting host protein phosphatase-1, inhibits HIV-1 transcription.

BACKGROUND AND PURPOSE: HIV-1 transcription is activated by the Tat protein which recruits the cyclin-dependent kinase CDK9/cyclin T1 to TAR RNA. Tat binds to protein phosphatase-1 (PP1) through the Q(35) VCF(38) sequence and translocates PP1 to the nucleus. PP1 dephosphorylates CDK9 and activates HIV-1 transcription. We have synthesized a low MW compound 1H4, that targets PP1 and prevents HIV-1 Tat interaction with PP1 and inhibits HIV-1 gene transcription. Here, we report our further work with the 1H4-derived compounds and analysis of their mechanism of action. EXPERIMENTAL APPROACH: Using the 1H4-PP1 complex as a model, we iteratively designed and synthesized follow-up libraries that were analysed for the inhibition of HIV-1 transcription and toxicity. We also confirmed the mechanism of action of the PP1-targeting molecules by determining the affinity of binding of these molecules to PP1, by analysing their effects on PP1 activity, disruption of PP1 binding to Tat and shuttling of PP1 to the nucleus. KEY RESULTS: We identified a tetrahydroquinoline derivative, compound 7, which disrupted the interaction of Tat with PP1. We further optimized compound 7 and obtained compound 7c, renamed 1E7-03, which inhibited HIV-1 with low IC50 (fivefold lower than the previously reported compound, 1H4), showed no cytotoxicity and displayed a plasma half-life greater than 8 h in mice. 1E7-03 bound to PP1 in vitro and prevented shuttling of PP1 into the nucleus. CONCLUSIONS AND IMPLICATIONS: Our study shows that low MW compounds that functionally mimic the PP1-binding RVxF peptide can inhibit HIV-1 transcription by deregulating PP1.

Iron chelators of the di-2-pyridylketone thiosemicarbazone and 2-benzoylpyridine thiosemicarbazone series inhibit HIV-1 transcription: identification of novel cellular targets--iron, cyclin-dependent kinase (CDK) 2, and CDK9.

HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 transcription. Here we have analyzed a group of novel di-2-pyridylketone thiosemicarbazone- and 2-benzoylpyridine thiosemicarbazone-based iron chelators that exhibit marked anticancer activity in vitro and in vivo (Proc Natl Acad Sci USA 103:7670-7675, 2006; J Med Chem 50:3716-3729, 2007). Several of these iron chelators, in particular 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), inhibited HIV-1 transcription and replication at much lower concentrations than did 311 and ICL670. Neither Bp4aT nor Bp4eT were toxic after a 24-h incubation. However, longer incubations for 48 h or 72 h resulted in cytotoxicity. Analysis of the molecular mechanism of HIV-1 inhibition showed that the novel iron chelators inhibited basal HIV-1 transcription, but not the nuclear factor-κB-dependent transcription or transcription from an HIV-1 promoter with inactivated SP1 sites. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1, suggesting that inhibition of CDK9 may contribute to the inhibition of HIV-1 transcription. Our study suggests the potential usefulness of Bp4aT or Bp4eT in antiretroviral regimens, particularly where resistance to standard treatment occurs.

Regulation of HIV-1 transcription at 3% versus 21% oxygen concentration.

HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-kappaB, and others. The cells of most of the body's organs are exposed to approximately 3-6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O(2), and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O(2) compared to 21% O(2). HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O(2) compared to 21% O(2). This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O(2), the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-kappaB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O(2) and also expression of CDK9/cyclin T1-dependent IkappaB inhibitor alpha was repressed. Our results suggest that lower HIV-1 transcription at 3% O(2) compared to 21% O(2) may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O(2) and that additional host cell factors such as CDK2 and NF-kappaB might be major regulators of HIV-1 transcription at low O(2) concentrations.

Iron chelators ICL670 and 311 inhibit HIV-1 transcription.

HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.

Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.

BACKGROUND: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription. RESULTS: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease. CONCLUSION: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

Inhibition of PP2A by LIS1 increases HIV-1 gene expression.

BACKGROUND: Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR. RESULTS: Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity. CONCLUSION: Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A.

Nuclear targeting of protein phosphatase-1 by HIV-1 Tat protein.

Transcription of human immunodeficiency virus (HIV)-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val36 and Phe38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat.

RNA interference directed to CDK2 inhibits HIV-1 transcription.

We previously reported that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro. In the present study, CDK2-specific RNA interference in cultured HEK293T cells inhibited CDK2 expression and Tat-induced HIV-1 transcription from non-integrated HIV-1 promoter but not basal HIV-1 transcription or transcription from CMV or beta-actin promoters. Also, CDK2-specific RNA interference inhibited Tat-induced transcription from the integrated HIV-1 promoter in HeLa-CD4-LTR-beta-gal cells and potently blocked TNFalpha-induced HIV-1 viral replication in OM10.1 cells. CDK2-specific RNA interference did not have an effect on cell cycle progression, but it augmented TNFalpha-induced apoptosis of OM10.1 cells. Our results indicate that CDK2 participates in Tat-mediated HIV-1 transcription and may serve as a potential therapeutic target.

Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription.

BACKGROUND: HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo. RESULTS: In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9. CONCLUSION: Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription.

HIV-1 Tat interacts with LIS1 protein.

BACKGROUND: HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization. RESULTS: We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system. CONCLUSION: Our results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation.

Nuclear protein phosphatase-1 regulates HIV-1 transcription.

We recently reported that protein phosphatase 1 (PP1) dephosphorylates RNA polymerase II C-terminal repeats and regulates HIV-1 transcription in vitro. Here we provide evidence that PP1 is also required for Tat-induced HIV-1 transcription and for viral replication in cultured cells. Inhibition of PP1 by overexpression of nuclear inhibitor of PP1 (NIPP1) inhibited Tat-induced HIV-1 transcription in transient transfection assays. A mutant of NIPP1 that was defective in binding to PP1 did not have this effect. Also the co-expression of PP1 gamma reversed the inhibitory effect of NIPP1. Adeno-associated virus-mediated delivery of NIPP1 significantly reduced HIV-1 transcription induced by Tat-expressing adenovirus in CD4+ HeLa cells that contained an integrated HIV-1 promoter (HeLa MAGI cells). In addition, infection of HeLa MAGI cells with adeno-associated virus-NIPP1 prior to the infection with HIV-1 significantly reduced the level of HIV-1 replication. Our results indicate that PP1 might be a host cell factor that is required for HIV-1 viral transcription. Therefore, nuclear PP1 may represent a novel target for anti-HIV-1 therapeutics.

Protein phosphatase-1 dephosphorylates the C-terminal domain of RNA polymerase-II.

Transcription by RNA polymerase-II (RNAPII) is controlled by multisite phosphorylation of the heptapeptide repeats in the C-terminal domain (CTD) of the largest subunit. Phosphorylation of CTD is mediated by the cyclin-dependent protein kinases Cdk7 and Cdk9, whereas protein serine/threonine phosphatase FCP1 dephosphorylates CTD. We have recently reported that human immunodeficiency virus-1 (HIV-1) transcription is positively regulated by protein phosphatase-1 (PP1) and that PP1 dephosphorylates recombinant CTD. Here, we provide further evidence that PP1 can dephosphorylate RNAPII CTD. In vitro, PP1 dephosphorylated recombinant CTD as well as purified RNAPII CTD. HeLa nuclear extracts were found to contain a species of PP1 that dephosphorylates both serine 2 and serine 5 of the heptapeptide repeats. In nuclear extracts, PP1 and FCP1 contributed roughly equally to the dephosphorylation of serine 2. PP1 co-purified with RNAPII by gel filtration and associated with RNAPII on immunoaffinity columns prepared with anti-CTD antibodies. In cultured cells treated with CTD kinase inhibitors, the dephosphorylation of RNAPII on serine 2 was inhibited by 45% by preincubation with okadaic acid, which inhibits phosphatases of PPP family, including PP1 but not FCP1. Our data demonstrate that RNAPII CTD is dephosphorylated by PP1 in vitro and by PPP-type phosphatase, distinct from FCP1, in vivo.

HIV-1 Tat interaction with RNA polymerase II C-terminal domain (CTD) and a dynamic association with CDK2 induce CTD phosphorylation and transcription from HIV-1 promoter.

Human immunodeficiency virus, type 1 (HIV-1), Tat protein activates viral gene expression through promoting transcriptional elongation by RNA polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of RNAPII by activating cell cycle-dependent kinases (CDKs) associated with general transcription factors of the promoter complex, specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived kinase, which contained CDK2. Here, we provide further evidence that CDK2 is involved in Tat-mediated CTD phosphorylation and in HIV-1 transcription in vitro. Tat-mediated CTD phosphorylation by CDK2 required cysteine 22 in the activation domain of Tat and amino acids 42-72 of Tat. CDK2 phosphorylated Tat itself, apparently by forming dynamic contacts with amino acids 15-24 and 36-49 of Tat. Also, amino acids 24-36 and 45-72 of Tat interacted with CTD. CDK2 associated with RNAPII and was found in elongation complexes assembled on HIV-1 long-terminal repeat template. Recombinant CDK2/cyclin E stimulated Tat-dependent HIV-1 transcription in reconstituted transcription assay. Immunodepletion of CDK2/cyclin E in HeLa nuclear extract blocked Tat-dependent transcription. We suggest that CDK2 is part of a transcription complex that is required for Tat-dependent transcription and that interaction of Tat with CTD and a dynamic association of Tat with CDK2/cyclin E stimulated CTD phosphorylation by CDK2.