Synthesis of 5-substituted 2'-deoxyuridine-5'-phosphonate analogues and evaluation of their antiviral activity

A small series of 5-(hetero)aryl-modified nucleoside phosphonates was synthesized via an 8-step procedure including a Wittig reaction and Suzuki-Miyaura coupling. An unanticipated anomerization during phosphonate deprotection allowed us to isolate both anomers of the 5-substituted 2'-deoxy-uridine phosphonates and assess their antiviral activity against a broad panel of viruses

Mycoplasmas and cancer

The standard of care for patients suffering cancer often includes treatment with nucleoside analogues (NAs). NAs are internalized by cell-specific nucleobase/nucleoside transporters and, after enzymatic activation (often one or more phosphorylation steps), interfere with cellular nucleo(s)(t)ide metabolism and DNA/RNA synthesis. Therefore, their efficacy is highly dependent on the expression and activity of nucleo(s)(t)ide-metabolizing enzymes, and alterations thereof (e.g. by down/upregulated expression or mutations) may change the susceptibility to NA-based therapy and/or confer drug resistance. Apart from host cell factors, several other variables including microbial presence may determine the metabolome (i.e. metabolite concentrations) of human tissues. Studying the diversity of microorganisms that are associated with the human body has already provided new insights in several diseas es (e.g. diabetes and inflammatory bowel disease) and the metabolic exchange between tissues and their specific microbiota was found to affect the bioavailability and toxicity of certain anticancer drugs, including NAs. Several studies report a preferential colonization of tumor tissues with some mycoplasma species (mostly Mycoplasma hyorhinis). These prokaryotes are also a common source of cell culture contamination and alter the cytostatic activity of some NAs in vitro due to the expression of nucleoside-catabolizing enzymes. Mycoplasma infection may therefore bias experimental work with NAs, and their presence in the tumor microenvironment could be of significance when optimizing nucleoside-based cancer treatment

Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy.status: publishe

Resistance of HIV-1 to the broadly HIV-1-neutralizing, anti-carbohydrate antibody 2G12

AbstractThe 2G12 mAb inhibits the infection of HIV-1 laboratory-adapted viruses at 50% inhibitory concentrations (IC50) ranging from 0.02 to 0.2 μg/ml when evaluated in different cell-types. However, isolates from various HIV-1 subtypes (such as clade C, D, A/E, F and group O) were not inhibited by 2G12 mAb (IC50 >20 μg/ml). 2G12 mAb pressure in HIV-1 IIIB- and NL4.3-infected T cell cultures selected for resistant viruses containing only few (1 to 3 N-glycosylation) deletions in gp120. The 2G12-resistant viruses keep their full sensitivity to various mannose-specific lectins and other known HIV entry inhibitors. Moreover, we observed that the NL4.3-2G12-resistant virus, with the N295K mutation in gp120, became significantly more sensitive to several mannose-specific lectins. This is, to our knowledge, the first report showing that a resistant virus generated in vitro against a neutralizing mAb and containing a mutation in gp120, has increased sensitivity to another class of HIV entry inhibitors

Glycan deletions in the HIV-1 gp120 V1/V2 domain compromise viral infectivity, sensitize the mutant virus strains to carbohydrate-binding agents and represent a specific target for therapeutic intervention

AbstractCarbohydrate-binding agents (CBAs), such as the mannose-specific Hippeastrum hybrid agglutinin (HHA) and the GlcNAc-specific Urtica dioica agglutinin (UDA), frequently select for glycan deletions in all different domains of HIV-1 gp120, except in the V1/V2 domain. To reveal the underlying mechanisms, a broad variety of 31 different virus strains containing one or several N-glycan deletions in V1/V2 of the gp120 of the X4-tropic HIV-1NL4.3 were constructed by chimeric virus technology. No co-receptor switch to CCR5 was observed for any of the replication-competent mutant virus strains. With a few exceptions, the more glycans were deleted in the gp120 V1/V2 domain, the more the replication capacity of the mutant viruses became compromised. None of the mutant virus strains showed a markedly decreased sensitivity to the inhibitory activity of HHA and UDA. Instead, an up to 2- to 10-fold higher sensitivity to the inhibitory activity of these CBAs was observed. Our data may provide an explanation why glycan deletions in the gp120 V1/V2 domain rarely occur under CBA pressure and confirm the important functional role of the glycans in the HIV-1 gp120 V1/V2 domain. The gp120 V1/V2 loop glycans of HIV-1 should therefore be considered as a hot spot and novel target for specific therapeutic drug intervention

Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design

BACKGROUND: Development of countermeasures to bioterrorist threats such as those posed by the smallpox virus (variola), include vaccination and drug development. Selective activation of nucleoside analogues by virus-encoded thymidine (dThd) kinases (TK) represents one of the most successful strategies for antiviral chemotherapy as demonstrated for anti-herpes drugs. Vaccinia virus TK is a close orthologue of variola TK but also shares a relatively high sequence identity to human type 2 TK (hTK), thus achieving drug selectivity relative to the host enzyme is challenging. RESULTS: In order to identify any differences compared to hTK that may be exploitable in drug design, we have determined the crystal structure of VVTK, in complex with thymidine 5'-triphosphate (dTTP). Although most of the active site residues are conserved between hTK and VVTK, we observe a difference in conformation of residues Asp-43 and Arg-45. The equivalent residues in hTK hydrogen bond to dTTP, whereas in subunit D of VVTK, Asp-43 and Arg-45 adopt a different conformation preventing interaction with this nucleotide. Asp-43 and Arg-45 are present in a flexible loop, which is disordered in subunits A, B and C. The observed difference in conformation and flexibility may also explain the ability of VVTK to phosphorylate (South)-methanocarbathymine whereas, in contrast, no substrate activity with hTK is reported for this compound. CONCLUSION: The difference in conformation for Asp-43 and Arg-45 could thus be used in drug design to generate VVTK/Variola TK-selective nucleoside analogue substrates and/or inhibitors that have lower affinity for hTK