Nanosome-Mediated Delivery Of Hdac Inhibitors and Oxygen Molecules for the Transcriptional Reactivation of Latent Hiv-Infected Cd4+ T Cells.

The treatment of human immunodeficiency virus (HIV) infection is notoriously difficult due to the ability of this virus to remain latent in the host's CD4+ T cells. Histone deacetylases (HDACs) interfere with DNA transcription in HIV-infected hosts, resulting in viral latency. Therefore, HDAC inhibitors can be used to activate viral transcription in latently infected cells, after which the virus can be eliminated through a shock-and-kill strategy. Here, a drug delivery system is developed to effectively deliver HDAC inhibitors to latent HIV-infected cells. Given that the efficacy of HDAC inhibitors is reduced under hypoxic conditions, oxygen-containing nanosomes are used as drug carriers. Oxygen-containing nanosomes can improve the efficiency of chemotherapy by delivering essential oxygen to cells. Additionally, their phospholipid bilayer structure makes them uniquely well-suited for drug delivery. In this study, a novel drug delivery system is developed by taking advantage of the oxygen carriers in these oxygen nanosomes, incorporating a multi-drug strategy consisting of HDAC inhibitors and PKA activators, and introducing CXCR4 binding peptides to specifically target CD4+ T cells. Oxygen nanosomes with enhanced targeting capability through the introduction of the CXCR4 binding peptide mitigate drug toxicity and slow down drug release. The observed changes in the expression of p24, a capsid protein of HIV, indirectly confirm that the proposed drug delivery system can effectively induce transcriptional reactivation of HIV in latent HIV-infected cells.

A Novel Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of Small-Molecule Inhibitors of HIV-1 Tat-Regulated Transcription.

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.

Identification of 3-Oxindole Derivatives as Small Molecule HIV-1 Inhibitors Targeting Tat-Mediated Viral Transcription.

The heterocyclic indole structure has been shown to be one of the most promising scaffolds, offering various medicinal advantages from its wide range of biological activity. Nonetheless, the significance of 3-oxindole has been less known. In this study, a series of novel 3-oxindole-2-carboxylates were synthesized and their antiviral activity against human immunodeficiency virus-1 (HIV-1) infection was evaluated. Among these, methyl (E)-2-(3-chloroallyl)-4,6-dimethyl-one (6f) exhibited the most potent inhibitory effect on HIV-1 infection, with a half-maximal inhibitory concentration (IC50) of 0.4578 µM but without severe cytotoxicity (selectivity index (SI) = 111.37). The inhibitory effect of these compounds on HIV-1 infection was concordant with their inhibitory effect on the viral replication cycle. Mode-of-action studies have shown that these prominent derivatives specifically inhibited the Tat-mediated viral transcription on the HIV-1 LTR promoter instead of reverse transcription or integration. Overall, our findings indicate that 3-oxindole derivatives could be useful as a potent scaffold for the development of a new class of anti-HIV-1 agents.

Identification of novel compounds against Tat-mediated human immunodeficiency virus-1 transcription by high-throughput functional screening assay.

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2'-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase. Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.

Selection of biomarkers for HIV-1 latency by integrated analysis.

A major obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1) is its ability to establish latent infection. To find novel biomarkers associated with the mechanism of HIV-1 latent infection, we identified 70 candidate genes in HIV-1 latently infected cells through the integrated analysis in a previous study. It is important to select more effective biomarkers among 70 candidates and to verify the possibility of selected biomarkers for HIV-1 latency. We identified the 24 and 25 genes from 70 candidate genes in significantly enriched categories selected by Database for Annotation, Visualization and Integrated Discovery (DAVID) software and Gene Set Enrichment Analysis (GSEA) software, respectively. Also, we investigated genes regulated in both HIV-1 latently infected cell lines and PBMCs from HIV-1 infected patients and found the genes with a common pattern of expression levels in both cell lines and PBMCs. Consequently, we identified nine genes, APBB2, GMPR, IGF2BP3, LRP1, MAD2L2, MX1, OXR1, PTK2B, and TNFSF13B, via integrated analysis. Especially, APBB2 and MAD2L2 were identified in both DAVID and GSEA software. Our findings suggest that nine genes were identified via integrated analysis as potential biomarkers and in particular, APBB2 and MAD2L2 may be considered as more significant biomarkers for HIV-1 latency.

Identification of novel genes associated with HIV-1 latency by analysis of histone modifications.

BACKGROUND: A reservoir of HIV-1 is a major obstacle in eliminating HIV-1 in patients because it can reactivate in stopping antiretroviral therapy (ART). Histone modifications, such as acetylation and methylation, play a critical role in the organization of chromatin domains and the up- or downregulation of gene expression. Although many studies have reported that an epigenetic mechanism is strongly involved in the maintenance of HIV-1 transcriptional latency, neither the epigenetic control of viral replication nor how HIV-1 latency is maintained is not fully understood. RESULTS: We re-analyzed a high throughput parallel DNA sequencing (ChIP-seq) data from previous work to investigate the effect of histone modifications, H3K4me3 and H3K9ac, on HIV-1 latency in terms of chromosome distribution. The outputs of ChIP-seq from uninfected CD4+ T cell lines and HIV-1 latently infected cells were aligned to hg18 using bowtie and then analyzed using various software packages. Certain chromosomes (16, 17, 19, and 22) were significantly enriched for histone modifications in both decreased and increased islands. In the same chromosomes in HIV-1 latently infected cells, 38 decreased and 41 increased islands from common islands of H3K4me3 and H3K9ac were selected for functional annotation. In Gene Ontology analysis, the 38 genes associated with decreased islands were involved in the regulation of biological process, regulation of cellular process, biological regulation, and purinergic receptor signaling pathway, while the 41 genes associated with increased islands were involved in nucleic acid binding, calcium-activated cation channel activity, DNA binding, and zinc ion binding. In Pathway Commons analysis, the 38 genes were strongly involved in the p63 transcription factor network, while the 41 genes were involved in the RNA polymerase III transcription termination pathway. Several genes such as Nuclear factor I X (NFIX) and TNF receptor association factor 4 (TRAF4) were selected as candidate genes for HIV latency. Especially, NFIX was highly expressed in HIV-1 latently infected cell lines and showed a dramatic reduction in expression after phorbol-13-myristate-12-acetate (PMA) treatment. CONCLUSIONS: These results show that the unique enrichment of histone modifications and its linked genes in specific chromosomes might play a critical role in the establishment and maintenance of HIV-1 latency.

p53-derived host restriction of HIV-1 replication by protein kinase R-mediated Tat phosphorylation and inactivation.

UNLABELLED: Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53(-/-) and eIF2α(CA) cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat-Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. IMPORTANCE: HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which was accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1.

Highly activated p53 contributes to selectively increased apoptosis of latently HIV-1 infected cells upon treatment of anticancer drugs.

BACKGROUND: Despite the successful inhibition of human immunodeficiency virus type 1 (HIV-1) replication by combination antiretroviral therapy, cells latently infected with HIV-1 remaining in patients are a major obstacle for eradication of HIV-1 infection. The tumor suppressor factor p53 is activated by HIV-1 infection, and restricts HIV-1 replication. However, a therapeutic strategy based on p53 activity has not been considered for elimination of latently infected cells. METHODS: Apoptotic cells were analyzed using flow cytometry with anti-annexin A5-FITC Ab and PI staining upon treatment of anticancer drugs. The expression and activation of p53 and apoptotic molecules in latently HIV-1-infected T cells were compared using Western blot analysis. The role of p53 in the anticancer drug treatment-induced apoptosis of cells latently infected with HIV-1 was determined by knock-down experiment using siRNA against p53. RESULTS: Upon treatment with 5-fluorouracil (5-FU), apoptosis was increased in latently infected ACH2 cells encoding competent p53 compared with uninfected parent A3.01 cells, while the apoptosis of latently infected p53 null J1.1 cells was less than that of uninfected cells. Treatment with 5-FU increased the levels of cleaved caspase-3 and PARP in ACH2 cells compared with uninfected and latently infected p53 null J1.1 cells. The levels of expression and activation of p53 were higher in both latently infected ACH2 and NCHA2 cells than in uninfected cells. Furthermore, the activation levels of p53 in both cells were further increased upon 5-FU treatment. Consistent with p53 status, apoptosis was markedly increased in ACH2 and NCHA2 cells compared with uninfected and latently infected J1.1 cells upon treatment with other anticancer drugs such as doxorubicin and etoposide. Inhibition of p53 in cells with latent HIV-1 infection diminished apoptosis upon 5-FU treatment. CONCLUSION: Evidence described here indicate that when treated with anticancer drugs, apoptosis of cells with latent HIV-1 infection was increased via the p53 activation pathway and may provide information for application of anticancer drugs to selectively eliminate HIV-1 reservoirs.

Functional characteristics of the natural polymorphisms of HIV-1 gp41 in HIV-1 isolates from enfuvirtide-naïve Korean patients.

HIV-1 gp41 plays a key role in viral entry. The insertion of Thr at position 4 and Met/Val/Phe substitutions at position 7 are frequently observed in the fusion peptide (FP) motif of gp41 without major enfuvirtide resistance associated with mutation in heptad repeats 1/2 (HR1/2) of HIV-1 isolates from Korean patients. Here, the influence of these mutations on their biological function was evaluated by employing HIV-1 variants with mutant FPs as shown previously and with recombinant HIV-1 using the env genes of 20 HIV-1 isolates from Korean patients. In an infectivity assay, all FP mutants showed lower infectivity than the wild-type NL4-3. In particular, the substitutions at position 7 led to much greater reductions in infectivity than the insertions at position 4. Nevertheless, the replication kinetics of most mutants were similar to those of the wild type, except that the FP mutants with an Ile insertion at position 4 and a Phe substitution at position 7 showed reduced replication. Moreover, most point mutants showed lower IC50 values for enfuvirtide than the wild type, whereas the L7M substitution resulted in a slightly increased IC50 value. The infectivity using the HIV-1 env recombinant viruses decreased in 14 cases but increased slightly in six cases compared with the wild type. Most recombinants were more susceptible to enfuvirtide than the wild type, except for three recombinants that showed slight resistance. Our findings may help to explain the potential mechanisms corresponding to the natural polymorphism of gp41 and to predict the efficiency of enfuvirtide in treatment of HIV-1-infected patients in Korea.

Impaired IL-2 expression in latent HIV-1 infection.

Regarding the T cell function in HIV-1 infection, activation of T cells is enhanced in acutely HIV-1-infected T cells upon stimuli. However, T cell immune responses underlying the activation of T cell receptor (TCR) signaling molecules and interleukin (IL)-2 production in latently HIV-1-infected cells are poorly understood. The expression and activation of TCR components and its downstream molecules in acutely and latently HIV-1-infected T cells were compared using quantitative reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression and enzyme-linked immunosorbent assay (ELISA) for levels of IL-2 in phytohemagglutinin M (PHA-M). The levels of T cell surface molecules and TCR signaling molecules in latently HIV-1-infected cells were greatly decreased without changes in their mRNA levels. In addition, downstream TCR-signaling molecules in latently HIV-1-infected cells were not activated even in the presence of PHA-M. The phosphorylation of mitogen-activated protein kinases (MAPKs) in the presence of PHA-M was weakly induced in latently HIV-1-infected cells but was greater in acutely HIVNL4-3-infected cells. Finally, the production of IL-2 was significantly decreased in latently HIV-1-infected cells compared with uninfected parent cells. Thus, IL-2-related immunological functions in latently HIV-1-infected T cells were markedly impaired even in the presence of stimuli.

Interferon-inducible protein 10 (IP-10) is associated with viremia of early HIV-1 infection in Korean patients.

Cytokines/chemokines play key roles in modulating disease progression in human immunodeficiency virus (HIV) infection. Although it is known that early HIV-1 infection is associated with increased production of proinflammatory cytokines, the relationship between cytokine levels and HIV-1 pathogenesis is not clear. The concentrations of 18 cytokines/chemokines in 30 HIV-1 negative and 208 HIV-1 positive plasma samples from Korean patients were measured by the Luminex system. Early HIV-1 infection was classified according to the Fiebig stage (FS) based on the characteristics of the patients infected with HIV-1. Concentrations of interleukin-12 (IL-12), interferon-inducible protein-10 (IP-10), macrophage inflammatory protein-1α (MIP-1α) and regulated upon activation, normal T cells expressed and secreted (RANTES) were increased significantly during the early stage of HIV-1 infection (FS II-IV) compared with the HIV-1-negative group. Of these cytokines, an elevated level of IP-10 was the only factor to be correlated positively with a higher viral load during the early stages of HIV-1 infection (FS II-IV) in Koreans (R = 0.52, P < 0.0005). Therefore, these results suggest that IP-10 may be an indicator for HIV-1 viremia and associated closely with viral replication in patients with early HIV-1 infection.

NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter.

BACKGROUND: Human immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication. Transcriptional activity of Tat is modulated by several host factors, but the mechanism responsible for Tat regulation by host factors is not understood fully. RESULTS: Using a yeast two-hybrid screening system, we identified Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) as a novel Tat-interacting partner. Here, we report its function as a positive regulator of Tat. In a coimmunoprecipitation assay, HIV-1 Tat interacted sufficiently with both endogenous and ectopically expressed NUCKS1. In a reporter assay, ectopic expression of NUCKS1 significantly increased Tat-mediated transcription of the HIV-1 LTR, whereas knockdown of NUCKS1 by small interfering RNA diminished Tat-mediated transcription of the HIV-1 LTR. We also investigated which mechanism contributes to NUCKS1-mediated Tat activation. In a chromatin immunoprecipitation assay (ChIP), knockdown of NUCKS1 interrupted the accumulation of Tat in the transactivation-responsive (TAR) region on the LTR, which then led to suppression of viral replication. However, NUCKS1 expression did not increase Tat nuclear localization and interaction with Cyclin T1. Interestingly, the NUCKS1 expression level was lower in latently HIV-1-infected cells than in uninfected parent cells. Besides, expression level of NUCKS1 was markedly induced, which then facilitated HIV-1 reactivation in latently infected cells. CONCLUSION: Taken together, our data demonstrate clearly that NUCKS1 is a novel Tat coactivator that is required for Tat-mediated HIV-1 transcription and replication, and that it may contribute to HIV-1 reactivation in latently HIV-1 infected cells.

Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity.

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development.

Disruption of polycomb repressor complex-mediated gene silencing reactivates HIV-1 provirus in latently infected cells.

OBJECTIVES: Persistent HIV-1 infections are characterized by a long silent infection period in resting CD4+ T cells, which allows them to escape the host immune response. Several HIV-1 latency mechanisms have been reported, but the molecular mechanism underlying polycomb repressor complex (PRC)-mediated HIV-1 latency remains poorly understood. METHODS: Expression of PRC proteins in latent cells was measured by Western blot. Knockdowns of PRC genes were conducted by the specific siRNA and methylations at H3K27 on the proviral LTR were investigated by ChIP assay. RESULTS: PRC proteins (EED, BMI-1, and RNF2) were dramatically downregulated in latent cells after PMA treatment. The downregulation of PRC proteins was followed by a decrease in the methylation of H3K27 and ubiquitination of H2AK119 in the PMA-treated latent cells. siRNA knockdowns of EED and BMI-1 also enhanced HIV-1 reactivation significantly in latently infected cells. By contrast, proteasomal inhibitor MG132 successfully abrogated the PMA-induced downregulation of PRCs. In particular, di-/tri-methylations of histone-3 in the proviral LTR was absent from latent cells after PMA treatment. CONCLUSIONS: This study shows that PRC is strongly related to the control of HIV-1 latency and that PRC-breaking agents may be helpful for purging HIV-1 from latent reservoirs.

Characterization of Gp41 polymorphisms in the fusion peptide domain and T-20 (Enfuvirtide) resistance-associated regions in Korean HIV-1 isolates.

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naïve Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

CISH is induced during DC development and regulates DC-mediated CTL activation.

The cytokine inducible SH2-domain protein (CISH) is a well-known STAT5 target gene, but its role in the immune system remains uncertain. In this study, we found that CISH is predominantly induced during dendritic cell (DC) development from mouse bone marrow (BM) cells and plays a crucial role in type 1 DC development and DC-mediated CTL activation. CISH knockdown reduced the expression of MHC class I, co-stimulatory molecules and pro-inflammatory cytokines in BMDCs. Meanwhile, the DC yield was markedly enhanced by CISH knockdown via cell-cycle activation and reduction of cell apoptosis. Down-regulation of cell proliferation at the later stage of DC development was found to be associated with CISH-mediated negative feedback regulation of STAT5 activation. In T-cell immunity, OT-1 T-cell proliferation was significantly reduced by CISH knockdown in DCs, whereas OT-2 T-cell proliferation was not affected by CISH knockdown. CTLs generated by DC vaccination were also markedly reduced by CISH knockdown, followed by significant impairment of DC-based tumor immunotherapy. Taken together, our data suggest that CISH expression at the later stage of DC development triggers the shutdown of DC progenitor cell proliferation and facilitates DC differentiation into a potent stimulator of CTLs.

The efficient synthesis and biological evaluation of justicidin B.

A facile new synthetic method for the preparation of a Type-A 1-arylnaphthalene lactone skeleton was developed and used to synthesise justicidin B and several derivatives. Key synthesis steps included Hauser-Kraus annulation of a phthalide intermediate and Suzuki-Miyaura cross coupling between a triflated naphthalene lactone intermediate and various potassium organotrifluoroborates. With two exceptions, the derivatives showed significant inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse macrophages. Moreover, several compounds, including justicidin B, had marked cytotoxicity towards six human tumour cell lines.

New Cdc2 Tyr 4 phosphorylation by dsRNA-activated protein kinase triggers Cdc2 polyubiquitination and G2 arrest under genotoxic stresses.

Cell division cycle 2 (Cdc2) protein is an essential subunit of M-phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double-stranded RNA-activated protein kinase (PKR) was involved in the process. The PKR-mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho-mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR-mediated Tyr 4-phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.

PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53.

Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2alpha) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.