Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides

The transactivator responsive region (TAR) present in the 5′-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA–protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNA_TAR) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418–428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNA_TAR–peptide conjugates where uptake of the PNA_TAR–penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC₅₀ values in the range of 28–37 nM while IC₅₀ for inhibition of HIV-1 replication was lowest with PNA_TAR–transportan-27 (0.4 μM) followed by PNA_TAR–tat (0.72 μM) and PNA_TAR–penetratin (0.8 μM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

Synthesis, structure-activity relationships, and mechanism of action of anti-HIV-1 lamellarin α 20-sulfate analogues

Lamellarin α and six different types of lamellarin α 20-sulfate analogues were synthesized and their structure–activity relationships were investigated using a single round HIV-1 vector infection assay. All lamellarin sulfates having pentacyclic lamellarin core exhibited anti-HIV-1 activity at a 10 μM concentration range regardless of the number and position of the sulfate group. On the other hand, non-sulfated lamellarin α and ring-opened lamellarin sulfate analogues did not affect HIV-1 vector infection in similar concentrations. The lamellarin sulfates utilized in this study did not exhibit unfavorable cytotoxic effect under the concentrations tested (IC₅₀ > 100 μM). Confocal laser scanning microscopic analysis indicated that hydrophilic lamellarin sulfates were hardly incorporated in the cell. HIV-1 Env-mediated cell–cell fusion was suppressed by lamellarin sulfates. These results suggested that lamellarin sulfates have a novel anti-HIV-1 activity besides the previously reported integrase activity inhibition, possibly at a viral entry step of HIV-1 replication.     

Native X-DING-CD4 protein secreted by HIV-1 resistant CD4+ T cells blocks activity of IL-8 promoter in human endothelial cells infected with enteric bacteria

Onsets of bacterial infections devastate the compromised immune system in AIDS patients. Damaged gut mucosa permits dissemination of bacterial toxins into deeper layers and hyper-activation of the immune system. We previously reported that the unfractionated supernatants of HIV-resistant CD4(+) T cells impeded the NF-κB/DNA binding in macrophages induced by either HIV-1 or LPS. The active component of this soluble material was identified as X-DING-CD4 (extracellular DING from CD4 T cells). We hypothesized that the anti-inflammatory effect of the X-DING-CD4 protein might extend to non-immune cells, for example endothelial cells, undergoing persistent endotoxin stimulation in the course of advanced HIV disease. To test this proposition, we evaluated the efficiency of NF-κB and Ap-1 binding to the IL-8 promoter in LPS-activated endothelial cells and control human macrophages exposed to native X-DING-CD4 protein. We found a deficiency of NF-κB- but not AP-1-DNA binding in the systems where cells were treated with native soluble X-DING-CD4 protein. The X-DING-CD4-mediated inhibition of the IL-8 promoter also resulted in a reduction of the soluble IL-8 protein in endothelial cells and human macrophages infected with a subset of enteric bacteria frequently causing diarrhea in progressive HIV disease. Bacterial endotoxin did not induce the endogenous X-DING-CD4 mRNA activity in human macrophages and transformed CD4(+)T cells, indicating that the reduction of LPS-mediated IL-8 promoter activation was not related to de novo X-DING-CD4 protein synthesis, but depended on function of the exogenous X-DING-CD4 protein. This study provides evidence that the X-DING-CD4 protein might be developed as a novel biotherapeutic to control LPS-mediated inflammation in advanced HIV disease.     

Anti-HIV-1 Activity of Elafin Depends on Its Nuclear Localization and Altered Innate Immune Activation in Female Genital Epithelial Cells

Elafin (E) and its precursor trappin-2 (Tr) are alarm antiproteases with antimicrobial and immunomodulatory activities. Tr and E (Tr/E) have been associated with HIV-1 resistance. We recently showed that Tr/E reduced IL-8 secretion and NF-κB activation in response to a mimic of viral dsRNA and contributed to anti-HIV activity of cervicovaginal lavage fluid (CVL) of HIV-resistant (HIV-R) commercial sex workers (CSWs). Additionally, Tr, and more so E, were found to inhibit attachment/entry and transcytosis of HIV-1 in human endometrial HEC-1A cells, acting through virus or cells. Given their immunomodulatory activity, we hypothesized that Tr/E could exert anti-HIV-1 activity at multiple levels. Here, using tagged and untagged Tr/E proteins, we comparatively evaluated their protease inhibitory, anti-HIV-1, and immunomodulatory activities, and cellular distribution. E appeared to function as an autocrine/paracrine factor in HEC-1A cells, and anti-HIV-1 activity of E depended on its unmodified N-terminus and altered cellular innate activation, but not its antiprotease activity. Specifically, exogenously added N-terminus-unmodified E was able to enter the nucleus and to reduce viral attachment/entry and transcytosis, preferentially affecting R5-HIV-1_ADA, but not X4-HIV-1_IIIB. Further, anti-HIV-1 activity of E was associated with significantly decreased HIV-1-triggered IL-8 release, attenuated NF-κB/p65 nuclear translocation, and significantly modulated mRNA expression of innate sensors TLR3 and RIG-I in HEC-1A cells. Most importantly, we found that elevated Tr/E in CVLs of HIV-R CSWs were associated with lower mRNA levels of TLRs 2, 3, 4 and RIG-I in the genital ECs from this cohort, suggesting a link between Tr/E, HIV-1 resistance and modulated innate viral recognition in the female genital mucosa. Collectively, our data indicate that unmodified N-terminus is critical for intranuclear localization and anti-HIV-1 activity of E. We also propose that E-mediated altered cellular innate activation most likely contributes to the HIV-R phenotype of these subjects.     

Synthesis of C-2 and C-3 substituted quinolines and their evaluation as anti-HIV-1 agents

A plenty of natural products and synthetic derivatives containing quinoline moiety have been reported to possess various pharmacological activities. Quinolines such as 2-styrylquinolines and 8-hydroxyquinolines are extensively studied for their anti-HIV-1 activity and found to act mainly through HIV-1 integrase enzyme inhibition. In continuation of our efforts to search for newer anti-HIV-1 molecules, thirty-one quinoline derivatives with different linkers to ancillary phenyl ring were synthesized and evaluated for in vitro anti-HIV-1 activity using TZM-bl assays. Compound 31 showed higher activity in TZM-bl cell line against HIV-1_VB59 and HIV-1_UG070 cell associated virus (IC₅₀ 3.35 ± 0.87 and 2.57 ± 0.71 μM) as compared to other derivatives. Compound 31 was further tested against cell free virus HIV-1_VB59 and HIV-1_UG070 (IC₅₀ 1.27 ± 0.31 and 2.88 ± 1.79 μM, TI 42.20 and 18.61, respectively). This lead molecule also showed good activity in viral entry inhibition assay and cell fusion assay defining its mode of action. The activity of compound 31 was confirmed by testing against HIV-1_VB51 in activated peripheral blood mononuclear cells (PBMCs). Binding interactions of 31 were compared with known entry inhibitors.     

TRIM Protein-Mediated Regulation of Inflammatory and Innate Immune Signaling and Its Association with Antiretroviral Activity

Members of the tripartite interaction motif (TRIM) family of E3 ligases are emerging as critical regulators of innate immunity. To identify new regulators, we carried out a screen of 43 human TRIM proteins for the ability to activate NF-κB, AP-1, and interferon, hallmarks of many innate immune signaling pathways. We identified 16 TRIM proteins that induced NF-κB and/or AP-1. We found that one of these, TRIM62, functions in the TRIF branch of the TLR4 signaling pathway. Knockdown of TRIM62 in primary macrophages led to a defect in TRIF-mediated late NF-κB, AP-1, and interferon production after lipopolysaccharide challenge. We also discovered a role for TRIM15 in the RIG-I-mediated interferon pathway upstream of MAVS. Knockdown of TRIM15 limited virus/RIG-I ligand-induced interferon production and enhanced vesicular stomatitis virus replication. In addition, most TRIM proteins previously identified to inhibit murine leukemia virus (MLV) demonstrated an ability to induce NF-κB/AP-1. Interfering with the NF-κB and AP-1 signaling induced by the antiretroviral TRIM1 and TRIM62 proteins rescued MLV release. In contrast, human immunodeficiency virus type 1 (HIV-1) gene expression was increased by TRIM proteins that induce NF-κB. HIV-1 resistance to inflammatory TRIM proteins mapped to the NF-κB sites in the HIV-1 long terminal repeat (LTR) U3 and could be transferred to MLV. Thus, our work identifies new TRIM proteins involved in innate immune signaling and reinforces the striking ability of HIV-1 to exploit innate immune signaling for the purpose of viral replication.