Design,synthesis, and biological evaluation of novel quinoline derivatives as HIV-1 Tat–TAR interaction inhibitors

Thirty-two quinoline derivatives were designed and synthesized as HIV-1 Tat–TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Nine of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells, indicating effective inhibitory activities of blocking the Tat–TAR interaction. Molecular modeling experiments indicated that these compounds may inhibit Tat–TAR interaction by binding to Tat protein instead of TAR RNA.     

Design and SAR of new substituted purines bearing aryl groups at N9 position as HIV-1 Tat-TAR interaction inhibitors

Twenty-four purine derivatives bearing aryl groups at N9 position were designed and synthesized as HIV-1 Tat-TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Ten of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells at a concentration of 30 microM, indicating effective inhibitory activities of blocking the Tat-TAR interaction. The aryl groups at N9 position affected the binding affinities between compounds and TAR RNA, showing some specificities of aryl groups to TAR RNA.     

Alpha,alpha-trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6'-dideoxy-alpha,alpha-trehalose was obtained from selective bromination of, alpha,alpha-trehalose at C-6,6', followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel alpha,alpha-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.     

Pharmacokinetic analysis of polyamide nucleic-acid-cell penetrating peptide conjugates targeted against HIV-1 transactivation response element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.     

抗HIV-1活性的大环多胺类化合物与RNA的识别作用及其对细胞凋亡的影响(英)

研究了具有抗HIV-1活性的大环多胺类化合物与RNA的识别作用，以及其对cos-7细胞凋亡的影响，以进一步探讨其抗HIV-1的作用机理.实验采用琼脂糖凝胶电泳方法, 观察化合物与RNA的识别作用；通过流式细胞计数法探讨其对cos-7细胞凋亡的影响；运用计算机分子模型, 从理论上Docking计算化合物与TAR RNA结合的可能性.结果表明, 大环多胺类化合物MP-1、MP-2和MP-3不仅具有断裂RNA的作用，并可抑制Tat-RNA的相互作用, 还可影响cos-7细胞亚二倍体的含量; 理论化学计算数据与实验结果基本一致.这一结果提示化合物的抗HIV-1活性可能通过作用于病毒基因组RNA而发挥作用，是多靶作用的结果.     

Lycopene induces apoptosis in immortalized fibroblasts exposed to tobacco smoke condensate through arresting cell cycle and down-regulating cyclin D1, pAKT and pBad

There is a lot of interest in the health benefits of dietary carotenoids and on the relationship of these compounds with smoke. In particular, it is unknown if the enhanced cancer risk observed in smokers following β-carotene supplementation can be also found using other carotenoids. Here, we studied the effects of the tomato carotenoid lycopene on molecular pathways involved in cell cycle progression, apoptosis and survival in immortalized RAT-1 fibroblasts exposed to cigarette smoke condensate (TAR). Lycopene (0.5–2.0 μM) inhibited cell growth in a dose-and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis in cells exposed to TAR. The arrest of cell cycle was independent of p53 and of 8-OH-dG DNA damage and related to a decreased expression of cyclin D1. Moreover, the carotenoid up-regulated apoptosis and down-regulated the phosphorylation of AKT and Bad in cells exposed to TAR. Such an effect was associated to an inhibition of TAR-induced expression of Cox-2 and hsp90, which is known to maintain AKT activity. This study suggests that lycopene, differently from β-carotene, can exert protective effects against cigarette smoke condensate.     

抗HIV—1活性的大环多胺类化合物与RNA的识别作用及其对细胞凋亡的影响

研究了具有抗HIV－1活性的大环多胺类化合物与RNA的识别作用,以及其对cos-7细胞凋亡的影响,以进一步探讨其抗HIV－1的作用机理。实验采用琼脂糖凝胶电泳方法。观察化合物与RNA的识别作用。通过流式细胞计数法探讨其对cos-7细胞凋亡的影响。运用计算机分子模型,从理论上Docking计算化合物与TAR RNA结合的可能性。结果表明,大环多胺类化合物MP－1、MP－2和MP－3不仅具有断裂RNA的作用,并可抑制Tat-RNA的相互作用,还可影响cos-7细胞亚二倍体的含量;理论化学计算数据与实验结果基本一致,这一结果提示合物的抗HIV－1活性可能通过作用于病毒基因组RNA而发挥作用,是多靶作用的结果。     

Polyamide amino acids trimers as TAR RNA ligands and anti-HIV agents

Based on a split-and-mix strategy, a library of trimeric Polyamide Amino Acids (PAA) incorporating four different amino acids (Lys, Ala, Arg, and Phe) has been prepared. Screening of the batches for HIV TAR RNA binding in a fluorescent assay allowed the identification of several components that interact with TAR RNA at a micromolar concentration, with a good TAR versus tRNA specificity. Some of these compounds compete efficiently with the association of TAR and Tat protein. In cell cultures, these compounds display a moderate antiviral activity, associated nevertheless with some toxicity. Overall, these results confirm that this new family can be a basis for the design of novel RNA targeting drugs.     

Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS.     

HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.     

Thrombocytopenia-absent radius (tar) syndrome: a case with agenesis of corpus callosum, hypoplasia of cerebellar vermis and horseshoe kidney

The thrombocytopenia-absent radius (TAR) syndrome (MIM 274000) is a congenital malformation syndrome characterised by bilateral absence of the radii with present thumbs, hypomegakaryocytic thrombocytopenia and a number of additional features including skeletal and cardiac anomalies. Mental retardation, reported in about 7% of patients, is usually secondary to intracranial hemorrhage. In 1994 there was a single report of a girl with TAR syndrome and hypoplasia of the cerebellar vermis and corpus callosum and in 2003 another case of TAR syndrome with cerebellar dysgenesis has been reported. In 2000 there was first report of horseshoe kidney in association with TAR syndrome followed by a clinical study of 34 cases with TAR syndrome in 2002 where horseshoe kidney was noted in two cases. Here we report of a girl with TAR syndrome, severe mental retardation, agenesis of corpus callosum, hypoplasia of cerebellar vermis and horseshoe kidney. There is no previous report of a child with TAR syndrome and all those associated anomalies in the same patient.     

TAR loop-dependent human immunodeficiency virus trans activation requires factors encoded on human chromosome 12.

The trans-activator response region (TAR) RNA in the human immunodeficiency virus type 1 (HIV-1) and HIV-2 long terminal repeat forms stem-loop secondary structures in which the loop sequence is essential for trans activation. We investigated how the HIV trans-activation mechanism encoded on human chromosome 12 relates to the TAR RNA loop-dependent pathway. DNA transfection experiments showed that trans activation in human-hamster hybrid cells with the single human chromosome 12 and human T-cell lines was highly dependent on the native sequences of the HIV-1 TAR loop and the HIV-2 5' TAR loop. In nonhuman cell lines or hybrid cells without chromosome 12 that supported trans activation, the cellular mechanism was independent of the HIV-1 TAR loop and the response to mutations in the HIV-2 TAR loops differed from that found in human T-cell lines and human-hamster hybrid cells with chromosome 12. Our results suggest that the human chromosome 12 mechanism interacts directly with the TAR RNA loop or indirectly by regulating TAR RNA-binding proteins.     

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.     

Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ～23.1 μM).     

Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.