Luis M. Bedoya

Negatively Charged Glyconanoparticles Modulate and Stabilize the Secondary Structures of a gp120 V3 Loop Peptide: Toward Fully Synthetic HIV Vaccine Candidates

The third variable region (V3 peptide) of the HIV-1 gp120 is a major immunogenic domain of HIV-1. Controlling the formation of the immunologically active conformation is a crucial step to the rational design of fully synthetic candidate vaccines. Herein, we present the modulation and stabilization of either the α-helix or β-strand conformation of the V3 peptide by conjugation to negatively charged gold glyconanoparticles (GNPs). The formation of the secondary structure can be triggered by the variation of the buffer concentration and/or pH as indicated by circular dichoism. The peptide on the GNPs shows increased stability toward peptidase degradation as compared to the free peptide. Moreover, only the V3β-GNPs bind to the anti-V3 human broadly neutralizing mAb 447-52D as demonstrated by surface plasmon resonance (SPR). The strong binding of V3β-GNPs to the 447-52D mAb was the starting point to address its study as immunogen. V3β-GNPs elicit antibodies in rabbits that recognize a recombinant gp120 and the serum displayed low but consistent neutralizing activity. These results open up the way for the design of new fully synthetic HIV vaccine candidates.

Multivalent manno-glyconanoparticles inhibit DC-SIGN-mediated HIV-1 trans-infection of human T cells.

Viral entry is a critical step in human immunodeficiency virus (HIV) infection that takes place through a series of sequential interactions between the envelope glycoprotein gp120, cellular receptor CD4, and coreceptors CCR5 or CXCR4 on T cells. Through a mechanism named trans-infection, dendritic cells (DCs) efficiently transfer the virus to T lymphocytes where viral replication occurs. Interference in HIV transmission in the DC–lymphocyte synapse is a preferential but complex target in the development of new compounds displaying anti-HIV activity. HIV–DC interaction is mediated by the glycans of gp120 and the C-type lectin DC-SIGN (dendritic cell-specific ICAM 3grabbing nonintegrin) expressed on DCs. DC-SIGN is tetrameric and specifically recognizes N-linked high-mannose oligosaccharides (Man9GlcNAc2) through multivalent and Ca -dependent protein–carbohydrate interactions. Mimicking the cluster presentation of the oligomannosides on the surface of the virus is a strategy for designing carbohydrate-based antiviral agents. Mannose multivalent systems based on proteins, peptides, liposomes, and dendrimers as scaffolds have recently been prepared for targeting DCs and tested as inhibitors of DC-SIGN binding to gp120 in ELISA and SPR experiments. Nevertheless, none of these systems has so far been tested in cell-based trans-infection models. Glyconanoparticles (GNPs) are polyvalent, biocompatible sugar-functionalized gold nanoclusters. 10] Glyconanoparticle platforms offer the unique potential for simultaneous incorporation of different ligands in variable densities on a single gold cluster. We have prepared a small library of GNPs (mannoGNPs) coated with sets of different structural motifs of the Nlinked high-mannose undecasaccharide Man9ACHTUNGTRENNUNG(GlcNAc)2 of gp120 (or with an unnatural heptasaccharide) and have observed that GNPs that display multiple copies of the disacchaACHTUNGTRENNUNGride Mana1-2Mana block DC-SIGN/gp120 binding at 120 nm in surface plasmon resonance (SPR) experiments. The mannoGNPs were designed to target DC-SIGN receptors present on DCs by mimicking the clustered carbohydrate display of gp120. In this work we present the results obtained with manno-GNPs (Scheme 1) as inhibitors of DC-SIGN-mediated HIV trans-infection of human activated peripheral blood mononuclear cells (PBMCs). We show that manno-GNPs coated with the linear tetrasaccharide Mana1-2Mana1-2Mana1-3Mana inhibit HIV trans-infection of human T cells similarly to GNPs coated with more complex branched pentaand heptaoligomannosides. Hybrid gold manno-GNPs displaying different densities of linear and branched mannose oligosaccharides were prepared (Scheme 1). The manno-conjugates 6–10 were synthesized by conjugation of ethylamino mannosides 1–5, obtained by the protocol of Wong et al. , with isothiocyanate linker 11 and subsequent removal of the acetyl group (Scheme 1 A). A shorter five-carbon atom aliphatic linker was used for the preparation of 5’-mercaptopentyl b-d-glucopyranoside (GlcC5S), and was hidden internally in order to allow correct presentation of the oligomannosides. Manno-GNPs with 100, 50, or 10 % densities of dimannoside (D-100, D-50, and D-10, respectively), trimannoside (T-50 and T-10), tetramannoside (Te-50 and Te-10), pentamannoside (P-50 and P-10), and heptamannoside (H-50) were prepared from mixtures of manno-conjugates 6–10 and gluco-conjugate GlcC5S by using a previously described methodology (Scheme 1 B). GNPs were characterized by transmission electron microscopy (TEM), H NMR, IR, UV/Visible, and elemental analysis as previously reported. The average number of (oligo)mannosides per GNP and their average molecular weights (Table 1) were calculated from elemental analysis and gold cluster size (1–2 nm as determined by TEM; see the Supporting Information). For trans-infection studies, we used Raji DC-SIGN transfected lymphoblastoid B cells, which can capture and transmit HIV with an efficiency similar to that of monocyte-derived DCs. Manno-GNPs are nontoxic to Raji DC-SIGN and to human activated PBMCs at concentrations of 100 mg mL , as determined by the CellTiter cell viability assay (Figure S9 in the Supporting Information). The activities of manno-GNPs against R5 or X4 HIV-1 were evaluated through an original DC-SIGN transfer assay in which inhibition of HIV-1 infection by GNPs was assessed by use of recombinant viruses carrying the Renilla reporter genes in their genomes. In this assay, inhibition of viral replication is proportional to Renilla-luciferase activity in cell lysates. Briefly, Raji DC-SIGN cells were preincubated with GNPs for 1 h and were then pulsed with JR-Renilla (R5) or NL4.3-Renilla (X4) recombinant viruses for 2 h. Afterwards, the cell cultures were extensively washed and co-cultured with activated PBMCs that would be infected through transfer of the virus bound to DC-SIGN in Raji cells. Viral replication was as[a] Dr. O. Mart nezvila, Dr. M. Marradi, Dr. C. Clavel, Prof. Dr. S. Penad s Laboratory of GlycoNanotechnology, CIC biomaGUNE/CIBER-BBN P8 Miram n 182, 20009 San Sebasti n (Spain) Fax: (+ 34) 943-00-53-01 E-mail : spenades@cicbiomagune.es [b] Dr. L. M. Bedoya, Dr. J. Alcam AIDS Immunopathology Unit, National Center of Microbiology Instituto de Salud Carlos III, Ctra.Majadahonda-Pozuelo Km. 2,200 Madrid (Spain) E-mail : ppalcami@isciii.es [c] Dr. C. Clavel Current address : IBMM, UMR 5247CNRS-Universit s Montpellier 2 et 1 ENSCM, Rue de l’Ecole Normale 8, 34296 Montpellier Cedex 5 (France) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900294.

Gold nanoparticles capped with sulfate-ended ligands as anti-HIV agents.

Gold nanoparticles coated with multiple copies of an amphiphilic sulfate-ended ligand are able to bind the HIV envelope glycoprotein gp120 as measured by surface plasmon resonance (SPR) and inhibit in vitro the HIV infection of T-cells at nanomolar concentrations. A 50% density of sulfated ligands on approximately 2 nm nanoparticles (the other ligands being inert glucose derivatives) is enough to achieve high anti-HIV activities. This result opens up the possibility of tailoring both sulfated ligands and other anti-HIV molecules on the same gold cluster, thus contributing to the development of non-cocktail based multifunctional anti-HIV systems.

SJ23B, a jatrophane diterpene activates classical PKCs and displays strong activity against HIV in vitro

Existence of virus reservoirs makes the eradication of HIV infection extremely difficult. Current drug therapies neither eliminate these viral reservoirs nor prevent their formation. Consequently, new strategies are needed to target these reservoirs with the aim of decreasing their size. We analysed a series of jatrophane diterpenes isolated from Euphorbia hyberna and we found that one of them, SJ23B, induces the internalization of the HIV-1 receptors CD4, CXCR4 and CCR5 and prevents R5 and X4 viral infection in human primary T cells at the nanomolar range. Moreover, SJ23B is a potent antagonist of HIV-1 latency. Using Jurkat-LAT-GFP cells, a model for HIV-1 latency, we found that prostratin and SJ23B activate HIV-1 gene expression, with SJ23B being at least 10-fold more potent than prostratin. SJ23B did not elicit transforming foci activity in NIH 3T3 cells but is a potent activator of PKCalpha and delta as measured by in vitro kinase assays and by cellular translocation experiments. By using isoform-specific PKC inhibitors we found that cPKCs are critical for SJ23B-induced HIV-1 reactivation. We also showed that both SJ23B-induced IkappaBalpha degradation and NF-kappaB activation were inhibited by the classical PKC inhibitor, Gö6976. Accordingly, SJ23B synergizes with ionomycin to translocate PKCalpha to the plasma membrane and to activate the NF-kappaB pathway. Moreover, SJ23B activates both NF-kappaB and Sp1-dependent transcriptional activities in primary T cells. We have shown that diterpene jatrophanes represent a new member of anti-AIDS agents that could be developed for mitigating HIV reactivation.

Olean-18-ene triterpenoids from Celastraceae species inhibit HIV replication targeting NF-kB and Sp1 dependent transcription.

In the present study we report the isolation of nine new olean-18-ene triterpenes (1-9), along with three known ones (10-12), from Cassine xylocarpa and Maytenus jelskii. Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques (COSY, ROESY, HSQC and HMBC), and spectrometric methods. The natural compounds and derivatives 13-15 have been tested for their potential as inhibitors of human immunodeficiency virus type 1 replication. Five compounds from this series displayed potent antiviral activity with IC(50)s in the micromolar range (1, 3, 4, 7 and 8) being 1 and 8 the most active compounds. The target of these compounds was different from antiretroviral drugs currently licensed as they act as inhibitors of enhancer-dependent transcription. The structure-activity relationships were established based on the regiosubstitution and oxidation degree of the triterpene scaffold, revealing that these aspects were able to modulate the selectivity and intensity of HIV inhibition.

Structure‐Based Design of an RNA‐Binding p‐Terphenylene Scaffold that Inhibits HIV‐1 Rev Protein Function

Numerous antibiotics bind to ribosomal RNA, and many functional RNA motifs have considerable therapeutic potential. However, the development of small RNA-binding agents has been hampered by the difficulties posed by these structures, which have limited physicochemical diversity and are often flexible. In order for this approach to be successful, it is essential to identify new chemical scaffolds recognizing RNA. The Rev response element (RRE) is a strongly conserved 350-nucleotide structure located in the env gene of human immunodeficiency virus type-1 (HIV-1) RNA. Within subdomain IIB of the RRE, the unusually widened major groove of a 4:6 internal loop forms a high-affinity complex with the arginine-rich a-helix of Rev, a virally encoded 116 amino acid protein that adopts a helix-turn-helix conformation (Figure 1). This interaction between internal loop IIB and the RNA-binding a-helix of Rev (Rev34-50) is essential for virus viability because it triggers a cascade of events that allow the transport of unspliced or incompletely spliced viral RNA molecules into the cytoplasm of the infected cell in the late phase of the viral infectious cycle. These events include the cooperative incorporation of additional Rev molecules into the complex through interactions with further sites on the RRE and protein–protein contacts, and the tethering of the RRE–Rev ribonucleoprotein to the Crm1 host export factor. In addition to RNA nuclear export, Rev has been shown to

Isolation, Structural Modification, and HIV Inhibition of Pentacyclic Lupane-Type Triterpenoids from Cassine xylocarpa and Maytenus cuzcoina

As a part of our investigation into new anti-HIV agents, we report herein the isolation, structure elucidation, and biological activity of six new (1-6) and 20 known (7-26) pentacyclic lupane-type triterpenoids from the stem of Cassine xylocarpa and root bark of Maytenus cuzcoina. Their stereostructures were elucidated on the basis of spectroscopic and spectrometric methods, including 1D and 2D NMR techniques. To gain a more complete understanding of the structural requirements for anti-HIV activity, derivatives 27-48 were prepared by chemical modification of the main secondary metabolites. Sixteen compounds from this series displayed inhibitory effects of human immunodeficiency virus type 1 replication with IC50 values in the micromolar range, highlighting compounds 12, 38, and 42 (IC50 4.08, 4.18, and 1.70 μM, respectively) as the most promising anti-HIV agents.

Environmentally Friendly Procedure Based on Supercritical Fluid Chromatography and Tandem Mass Spectrometry Molecular Networking for the Discovery of Potent Antiviral Compounds from Euphorbia semiperfoliata

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-deoxyphorbol esters (16-18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC50 = 0.45 μM), while compound 15 proved to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC50 = 13 nM). This study showed that a supercritical fluid chromatography-based protocol and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeting molecules of interest, while minimizing the use of toxic solvents.

Preprint: Environmentally-Friendly Workflow Based on Supercritical Fluid Chromatography and Tandem Mass Spectrometry Molecular Networking For the Discovery of Potent Anti-Viral Leads From Plants

A supercritical fluid chromatography-based targeted purification workflow using tandem mass spectrometry and molecular networking was developed to analyze, annotate and isolate secondary metabolites from complex mixture. This approach was applied for targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorboids esters, were present in the samples. The purification procedure using semi-preparative-supercritical fluid chromatography led to the isolation and identification of two jatrophane esters (13 and 14) and four 4-deoxyphorbol esters (15-18). Compound 16 was found to display antiviral activity against chikungunya virus (EC50 = 0.45 µM), while compound 15 was found to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC50 = 13 nM). This study showed that supercritical fluid chromatography-based workflow and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeted molecules of interest, while minimizing the use of toxic solvents.

Neoflavonoids as Inhibitors of HIV-1 Replication by Targeting the Tat and NF-κB Pathways

Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-κB and displayed anti-Tat activity simultaneously. Compounds 10, 14, and 25, inhibited HIV replication in both targets at concentrations <25 μM. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.