Antiviral Activity of Dual-acting Hydrocarbon-stapled Peptides against HIV-1 Predominantly Circulating in China.

Objective: New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China.

Methods: The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs).

Design of antiviral stapled peptides containing a biphenyl cross-linker.

Here we report the design and synthesis of a panel of stapled peptides containing a distance-matching biphenyl cross-linker based upon a peptide capsid assembly inhibitor reported previously. Compared with the linear peptide, the biphenyl-stapled peptides exhibited significantly enhanced cell penetration and potent antiviral activity in the cell-based infection assays. Isothermal titration calorimetry and surface plasmon resonance experiments revealed that the most active stapled CAI peptide binds to the C-terminal domain of HIV capsid protein as well as envelop glycoprotein gp120 with low micromolar binding affinities, and as a result, inhibits both the HIV-1 virus entry and the virus assembly.

Virtual screening based identification of novel small-molecule inhibitors targeted to the HIV-1 capsid.

The hydrophobic cavity of the C-terminal domain (CTD) of HIV-1 capsid has been recently validated as potential target for antiviral drugs by peptide-based inhibitors; however, there is no report yet of any small molecule compounds that target this hydrophobic cavity. In order to fill this gap and discover new classes of ant-HIV-1 inhibitors, we undertook a docking-based virtual screening and subsequent analog search, and medicinal chemistry approaches to identify small molecule inhibitors against this target. This article reports for the first time, to the best of our knowledge, identification of diverse classes of inhibitors that efficiently inhibited the formation of mature-like viral particles verified under electron microscope (EM) and showed potential as anti-HIV-1 agents in a viral infectivity assay against a wide range of laboratory-adapted as well as primary isolates in MT-2 cells and PBMC.

Evidence for protection against chronic hepatitis C virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus.

Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection.

A cell-penetrating helical peptide as a potential HIV-1 inhibitor.

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer alpha-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.

Attempted therapeutic immunization in a chimpanzee chronic HBV carrier with a high viral load.

Background: We previously reported successful therapeutic immunization in a chimpanzee having a relatively low viral load, which was immunized with recombinant plasmid hepatitis B surface antigen (HBsAg) DNA and boosted with recombinant HBsAg encoding canarypox virus. In the present study, we attempted to confirm these findings in an animal with a high virus load.

Methods And Results: We tested three immunization strategies successively over a 3-year period.

Prospects and strategies for the discovery and development of small-molecule inhibitors of six-helix bundle formation in class 1 viral fusion proteins.

Class I viral fusion proteins have an important role in the fusion of viral membranes with host cell membranes, a critical step in the viral life-cycle. These proteins all have similar structural features and form six-helix bundles in their fusogenic form, a general mechanism of action for virus-cell fusion. The successful discovery of peptide-based inhibitors of fusion proteins, in addition to the US Food and Drug Administration approval of one of these inhibitors as an anti-HIV-1 drug, confirmed that the inhibition of six-helix bundle formation is a viable strategy for identifying antiviral drugs.

Identification of two new members, XPLAC and XTES, of the XK family.

XK, a putative membrane transporter, is a component of the XK/Kell complex of the Kell blood group system. XK's substrate is unknown but absence of the protein, as occurs in the McLeod phenotype, is associated with red cell acanthocytosis and late onset central nervous system and neuromuscular abnormalities known as the McLeod syndrome. We have cloned two cDNAs, XPLAC (GenBank accession no.

Endothelin-3-converting enzyme activity of the KEL1 and KEL6 phenotypes of the Kell blood group system.

The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp(21)-Ile(22) bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype.

Identification of N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamides as a new class of HIV-1 entry inhibitors that prevent gp120 binding to CD4.

We have identified two N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide analogs as a novel class of human immunodeficiency virus type 1 (HIV-1) entry inhibitors that block the gp120-CD4 interaction, using database screening techniques. The lead compounds, NBD-556 and NBD-557, are small molecule organic compounds with drug-like properties. These compounds showed potent cell fusion and virus-cell fusion inhibitory activity at low micromolar levels.

Onset of expression of the components of the Kell blood group complex.

Background: Kell and XK, two distinct red blood cell membrane proteins, are linked by a disulfide bond and form the Kell blood group complex. Kell surface antigens are expressed early during erythropoiesis but the onset of expression of XK which carries the Kx antigen is unknown.

Study Design And Methods: To determine whether Kell and XK are synchronously expressed, sorted human hematopoietic progenitor cells and mouse progenitor cells of defined lineage were studied.

Development of Duffy transgenic mouse: in vivo expression of human Duffy gene with -33T-->C promoter mutation in non-erythroid tissues.

Blood group Duffy gene (FY) promoter in Duffy-negative individuals contains a point mutation in the GATA1 protein-binding motif, which was suggested to be responsible for erythroid suppression of FY. We developed two transgenic mouse lines with FY from both Duffy phenotypes. Transgenic mice with FY from Duffy-positive phenotype expressed Duffy protein both in red blood cells (RBCs) and non-erythroid tissues.

A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5.

HIV-1 infection is initiated by the interaction of the envelope glycoprotein gp120 with the cellular receptor CD4 that triggers conformational changes in gp120 necessary for subsequent interaction with a coreceptor CCR5 (or CXCR4). The CD4-induced (CD4i) conformation of gp120 can be mimicked by a full-length single chain (FLSC) protein consisting of gp120 linked with the D1D2 domains of CD4 by a 20-amino-acid linker. We have used this protein to establish a flow cytometry-based assay and an ELISA-based assay to identify inhibitors that block the binding of gp120 to CCR5.

Hepatitis C virus replication kinetics in chimpanzees with self-limited and chronic infections.

The availability of molecular beacon-based, real time polymerase chain reaction (PCR) and a semi-automated sample extraction procedure have made it possible for us to retrospectively examine HCV replication kinetics in HCV naive chimpanzees infected during the past 20 years. We compared these in 17 animals that developed chronic infection, and in 21 that developed self-limited infection. No differences were found in infecting dose, or replication kinetics in the acute phase between these two types of infection.

Murine bone marrow cells cultured ex vivo in the presence of multiple cytokine combinations lose radioprotective and long-term engraftment potential.

The desire to improve engraftment following transplantation of limited numbers of hematopoietic stem cells (HSC) has spurred the investigation of ex vivo stem cell expansion techniques. While surrogate outcomes, such as an increase in SCID-repopulating cells, suggest successful stem cell expansion in some studies, it is not clear that such assays predict outcomes using a more clinically relevant approach (e.g.

Water dispersible microbicidal cellulose acetate phthalate film.

Background: Cellulose acetate phthalate (CAP) has been used for several decades in the pharmaceutical industry for enteric film coating of oral tablets and capsules. Micronized CAP, available commercially as "Aquateric" and containing additional ingredients required for micronization, used for tablet coating from water dispersions, was shown to adsorb and inactivate the human immunodeficiency virus (HIV-1), herpesviruses (HSV) and other sexually transmitted disease (STD) pathogens. Earlier studies indicate that a gel formulation of micronized CAP has a potential as a topical microbicide for prevention of STDs including the acquired immunodeficiency syndrome (AIDS).

Generation of predictive pharmacophore models for CCR5 antagonists: study with piperidine- and piperazine-based compounds as a new class of HIV-1 entry inhibitors.

Predictive pharmacophore models were developed for a large series of piperidine- and piperazine-based CCR5 antagonists as anti-HIV-1 agents reported by Schering-Plough Research Institute in recent years. The pharmacophore models were generated using a training set consisting of 25 carefully selected antagonists based on well documented criteria. The activity spread, expressed in K(i), of training set molecules was from 0.

Oxysterols suppress constitutive fibrinogen expression.

Elevated levels of both fibrinogen and cholesterol are risk factors in coronary artery disease. Previously we reported a metabolic link between fibrinogen and lipid metabolism in that HepG2 cells that were programmed by transfection of Bbeta-fibrinogen cDNA to overexpress fibrinogen exhibited increased synthesis of cholesterol and increased secretion of apolipoprotein B. In this study we demonstrate that oxysterols, which participate in maintaining cholesterol homeostasis, also down regulate fibrinogen expression.

DNA immunization with hepatitis C virus (HCV) polycistronic genes or immunization by HCV DNA priming-recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1-transgenic mice.

We studied immune responses to hepatitis C virus (HCV) genes delivered as DNA encoding the entire HCV protein coding genome in two polycistronic plasmids encoding HCV capsid-E1-E2-NS2-NS3 and HCV NS3-NS4-NS5 in HLA-A2.1-transgenic mice. Immune responses to HCV DNA prime and recombinant canarypox virus boost were also studied with the above constructs.

Quantitative structure-activity relationship (QSAR) paradigm--Hansch era to new millennium.

The analysis of structure-activity relationships started probably more than hundred years ago but the concept of quantitatively correlating physicochemical properties of molecules with their biological activities, termed as quantitative structure-activity relationship (QSAR), was initiated by Corwin Hansch and his groups in early 1960. Many new methods have emerged since then. The concept evolved from 2D QSAR to 3D QSAR and lately another dimension (4D QSAR) has been added.

Lack of evidence for HIV type 1-related SIVcpz infection in captive and wild chimpanzees (Pan troglodytes verus) in West Africa.

Serum from 387 chimpanzees (Pan troglodytes verus), caught in the wild or bred in captivity, was tested for antibody to HIV-1 and HIV-2, using second- and third-generation enzyme immunoassays. Six samples were repeatedly positive; however, only one of these was Western blot positive. Serial sera drawn before and after the Western blot-positive samples were seronegative, and thus we conclude that this sample represented specimen contamination, or mislabeling.

Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT.

Background: Preservation of the integrity of viral nucleic acids in blood specimens during shipping and handling is crucial for NAT and viral load monitoring. An economical and convenient method is described for nucleic acid stabilization by using an RNA stabilizing solution (RNAlater, Ambion) in plasma that is designed for the shipment of samples to tropical countries.

Study Design And Methods: HCV, HIV, and HBV FFP were compared with RNAlater-treated plasma and dried plasma spots (DPSs) after incubation at 37 degrees C, which was chosen as an upper limit of ambient shipping temperature, for up to 28 days.

Anti-HIV-1 activity of cellulose acetate phthalate: synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles.

Background: Cellulose acetate phthalate (CAP), a promising candidate microbicide for prevention of sexual transmission of the human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted disease (STD) pathogens, was shown to inactivate HIV-1 and to block the coreceptor binding site on the virus envelope glycoprotein gp120. It did not interfere with virus binding to CD4. Since CD4 is the primary cellular receptor for HIV-1, it was of interest to study CAP binding to HIV-1 complexes with soluble CD4 (sCD4) and its consequences, including changes in the conformation of the envelope glycoprotein gp41 within virus particles.

Point mutations in KEL exon 8 determine a high-incidence (RAZ) and a low-incidence (KEL25, VLAN) antigen of the Kell blood group system.

Background And Objectives: The molecular basis of two Kell blood group antigens, RAZ (provisionally KEL27) and VLAN (KEL25), were determined.

Materials And Methods: The DNA sequences of the open reading frames and the flanking intron regions of the 19 KEL exons from RAZ and VLAN probands were compared with that of common KEL. Genotyping assays were designed to confirm and detect RAZ and VLAN phenotypes.