Wei Pang

Sifuvirtide, a potent HIV fusion inhibitor peptide.

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC(50)), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC(50)) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1(IIIB) were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

Current peptide HIV type-1 fusion inhibitors.

There are now 26 antiretroviral drugs and 6 fixed-dose combinations, including reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and fusion (or entry) inhibitors, approved by the US Food and Drug Administration for clinical use. Although they are clinically effective when used in combination, none of the existing drugs are considered ideal because of toxic side effects and the ascendance of inducing drug-resistant mutants. Development of new antiviral agents is essential. In the past decades, there has been great progress in understanding the structure of HIV type-1 (HIV-1) gp41 and the mechanism of HIV-1 entry into host cells. This opened up a promising avenue for rationally designed agents to interfere with this process. A number of fusion inhibitors have been developed to block HIV-1 replication. Enfuvirtide (T20) was one of those approved for clinical use. This signalled a new era in AIDS therapeutics. It is a synthetic polypeptide with potent inhibitory activity against HIV-1 infection. However, it is sensitive to proteolytic digestion and resistant virus strains are easily induced with multiple clinical use. One of the directions in designing new fusion inhibitors is to overcome these shortages. In the past years, large numbers of promising fusion inhibitory peptides have emerged. The antiviral activities are more potent or they can act differently from that of T20. Some of these new compounds have great potential to be further developed as therapeutic agents. This article reviewed some recent developments of these peptides and the possible role in anti-HIV-1 therapy.

Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1(IIIB) entry and replication with EC(50) values of 3.92+/-0.62 and 6.59+/-1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1(KM018) with EC(50) values of 44.44+/-10.20 nM, as well as suppressing HIV-1-induced cytopathic effect with an EC(50) value of 3.04+/-1.20 nM. It also inhibited HIV-2(ROD) and HIV-2(CBL-20) entry and replication in the microM range. Notably, HR212 was highly effective against T20-resistant strains with EC(50) values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/AIDS patients, particularly for those infected by T20-resistant variants.

Expression of syncytin in leukemia and lymphoma cells

Syncytin is a placenta-specific protein and generally believed to play a pivotal role in syncytiotrophoblast morphogenesis. In this study, transcripts of this gene were quantified by real-time RT-PCR and the translated products were measured by an indirect immunofluorescence assay. Results showed that syncytin was found to be expressed in all nine leukemia and lymphoma cell lines studied albeit at different levels and in 43 peripheral blood samples of 57 leukemia or lymphoma patients. Neither the transcripts nor the translated syncytin was detected in blood samples of normal individuals. In conclusion, peripheral blood syncytin may serve as a marker for leukemia and lymphoma.

Design, synthesis and anti-HIV-1 evaluation of hydrazide-based peptidomimetics as selective gelatinase inhibitors.

As our ongoing work on research of gelatinase inhibitors, an array of hydrazide-containing peptidomimetic derivatives bearing quinoxalinone as well as spiro-heterocyclic backbones were designed, synthesized, and assayed for their in vitro enzymatic inhibitory effects. The results demonstrated that both the quinoxalinone (series I and II) and 1,4-dithia-7-azaspiro[4,4]nonane-based hydrazide peptidomimetics (series III) displayed remarkably selectivity towards gelatinase A as compared to APN, with IC50 values in the micromole range. Structure-activity relationships were herein briefly discussed. Given evidences have validated that gelatinase inhibition may be contributable to the therapy of HIV-1 infection, all the target compounds were also submitted to the preliminary in vitro anti-HIV-1 evaluation. It resulted that gelatinase inhibition really has positive correlation with anti-HIV-1 activity, especially compounds 4m and 7h, which gave enhanced gelatinase inhibition in comparison with the positive control LY52, and also decent anti-HIV-1 potencies. The FlexX docking results provided a straightforward insight into the binding pattern between inhibitors and gelatinase, as well as the selective inhibition towards gelatinase over APN. Collectively, our research encouraged potent gelatinase inhibitors might be used in the development of anti-HIV-1 agents. And else, compounds 4m and 7h might be promising candidates to be considered for further chemical optimization.

NHERF1 regulates gp120‐induced internalization and signaling by CCR5, and HIV‐1 production

The scaffolding protein Na+/H+ exchanger regulator factor 1 (NHERF1) plays an important role in the trafficking of G protein‐coupled receptors. We previously demonstrated that NHERF1 is involved in chemokine receptor CCR5 homodimer internalization and signal transduction. Given the importance of CCR5 internalization during HIV‐1 infection, we evaluated NHERF1s contribution in HIV‐1 infection. We challenged human osteosarcoma cells coexpressing CD4 and CCR5 cells expressing either NHERF1 fragment domains or WT NHERF1 with an HIV‐1 strain to examine the effects of NHERF1 on HIV‐1 entry and replication. WT NHERF1 potentiates HIV‐1 envelope gp120‐induced CCR5 internalization, and promotes the replication of HIV‐1. In order to better understand how NHERF1 affects signal transduction, different domains of NHERF1 were overexpressed in cells to analyze their effect on the different signaling pathways. Here, we show that NHERF1 can associate with CCR5, and promote activation of the gp120‐induced MAPK/ERK, focal adhesion kinase and RhoA (Ras homolog gene family member A) signaling pathways. NHERF1 overexpression also increases HIV‐1 host cell migration triggered by gp120 via focal adhesion kinase (FAK) signaling. Finally, NHERF1 enhanced actin filament rearrangement in host cells, an important step in post‐entry HIV‐1 replication events. While postsynaptic density 95/disk‐large/zonula occludens 2 (PDZ2) appears to be the major contributor in those events, other domains also participate in the regulation of gp120‐induced signaling pathways. Altogether, our results suggest a very important role of the scaffold NHERF1 in the regulation of HIV‐1 entry and replication.

The Anti-HIV Actions of 7- and 10-Substituted Camptothecins

Camptothecin (CPT), a traditional anti-tumor drug, has been shown to possess anti-HIV-1 activity. To increase the antiviral potency, the anti-HIV activities of two CPT derivatives, 10-hydroxy-CPT and 7-hydroxymethyl-CPT, were evaluated in vitro. The therapy index (TI) of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-1IIIB in C8166 were 24.2, 4.2 and 198.1, and against clinical isolated strain HIV-1KM018 in PBMC were 10.3, 3.5 and 66.0, respectively. While the TI of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-2CBL-20 were 34.5, 10.7 and 317.0, respectively, and the TI of the three compounds against HIV-2ROD showed the similar values. However, when the antiviral mechanisms were considered, we found there was no inhibition of 7-hydroxymethyl-CPT on viral cell-to-cell transmission, and was no inhibition on reverse transcriptase, protease or integrase in cell-free systems. 7-Hydroxymethyl-CPT showed no selective killing of chronically infected cells after 3 days of incubation. In conclusion, 7-hydroxymethyl-CPT showed more potent anti-HIV activity, while 10-hydroxy-CPT had less efficient activity, compared with the parent CPT. Though the antiviral mechanisms remain to be further elucidated; the modification of -OH residues at C-7 of CPT could enhance the antiviral activity, while of -OH residues at C-10 of CPT had decreased the antiviral activity, which provides the preliminary modification strategy for anti-viral activities enhancement of this compound.

A Novel Enzyme-Linked Immunosorbent Assay for Screening HIV-1 Fusion Inhibitors Targeting HIV-1 Gp41 Core Structure

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein mediates the fusion of viral and host cell membranes. As the HIV-1 enters the host cells, the 2 helical regions, HR1 and HR2, in the ectodomain of gp41 can form a 6-helix bundle, which brings the viral and target cell membranes to close proximity and serves as an attractive target for developing HIV-1 fusion inhibitors. Now, there are several cell- and molecule-based assays to identify potential HIV-1 fusion inhibitors targeting gp41. However, these assays cannot be used universally because they are time-consuming, inconvenient, and expensive. In the present study, the authors expressed and purified GST-HR121 and C43-30a proteins that were derived from the HIV-1 gp41 ectodomain region. GST-HR121 has a function similar to the HR1 peptide of gp41, whereas C43-30a is an HR2-derived peptide that added 50 amino acid residues (aa) in the N-terminal of C43. Further research found they could interact with each other, and a potential HIV-1 fusion inhibitor could inhibit this interaction. On the basis of this fact, a novel, rapid, and economic enzyme-linked immunosorbent assay was established, which can be developed for high-throughput screening of HIV-1 fusion inhibitors.

Northern pig-tailed macaques (Macaca leonina) maintain superior CD4+ T-cell homeostasis during SIVmac239 infection

Gradual depletion of CD4+ Txa0cells is a typical characteristic of pathogenic SIV infection. Intriguingly, we find a spontaneous CD4+ T-cell homeostasis in northern pig-tailed macaques (Macaca leonina) during SIVmac239 infection.