Pronucleotides of 2′,3′-Dideoxy-2′,3′-Didehydrothymidine as Potent Anti-HIV Compounds

We report on the synthesis and evaluation of three different nucleotide prodrug systems: (i) nucleoside triphosphate analogues in which the γ-phosph(on)ate has two different lipophilic nonbioreversible alkyl residues with d4TDP as the released nucleotide analogue; (ii) nucleoside diphosphate analogues bearing a bioreversible and a stable β-alkyl group; or (iii) nucleoside diphosphate analogues bearing two nonhydrolysable lipophilic alkyl moieties. The delivery of d4TDP (for the triphosphate precursor) and d4TMP (for the diphosphate precursor) was demonstrated in CD4+ T-lymphocyte CEM cell extracts as well as in phosphate buffer saline (PBS). In primer extension assay, we found that γ-dialkylated d4TTP derivatives and d4TDP were accepted as substrates by HIV-RT. Several of these compounds were observed to be extremely active against HIV-1/2 replication in HIV-infected cells. A more than 45,000-fold increase in the anti-HIV activity was detected for compound 18a as compared to the parent d4T which results in a selectivity index value of 37,000

Antiviral Activity of Lipophilic Nucleoside Tetraphosphate Compounds

We report on the synthesis and characterization of three types of nucleoside tetraphosphate derivatives 4–9 acting as potential prodrugs of d4T nucleotides: (i) the δ-phosph­(on)­ate is modified by two hydrolytically stable alkyl residues 4 and 5; (ii) the δ-phosph­(on)­ate is esterified covalently by one biodegradable acyloxybenzyl moiety and a nonbioreversible moiety 6 and 7; or (iii) the δ-phosphate of nucleoside tetraphosphate is masked by two biodegradable prodrug groups 8 and 9. We were able to prove the efficient release of d4T triphosphate (d4TTP, (i)), δ-monoalkylated d4T tetraphosphates (20 and 24, (ii)), and d4T tetraphosphate (d4T4P, (iii)), respectively, by chemical or enzymatic processes. Surprisingly, δ-dialkylated d4T tetraphosphates, δ-monoalkylated d4T tetraphosphates, and d4T4P were substrates for HIV-RT. Remarkably, the antiviral activity of TetraPPPPro-prodrug 7 was improved by 7700-fold (SI 5700) as compared to the parent d4T in CEM/TK– cells, denoting a successful cell membrane passage of these lipophilic prodrugs and an intracellular delivery of the nucleotide metabolites

Antiviral Activity of Lipophilic Nucleoside Tetraphosphate Compounds

We report on the synthesis and characterization of three types of nucleoside tetraphosphate derivatives 4–9 acting as potential prodrugs of d4T nucleotides: (i) the δ-phosph­(on)­ate is modified by two hydrolytically stable alkyl residues 4 and 5; (ii) the δ-phosph­(on)­ate is esterified covalently by one biodegradable acyloxybenzyl moiety and a nonbioreversible moiety 6 and 7; or (iii) the δ-phosphate of nucleoside tetraphosphate is masked by two biodegradable prodrug groups 8 and 9. We were able to prove the efficient release of d4T triphosphate (d4TTP, (i)), δ-monoalkylated d4T tetraphosphates (20 and 24, (ii)), and d4T tetraphosphate (d4T4P, (iii)), respectively, by chemical or enzymatic processes. Surprisingly, δ-dialkylated d4T tetraphosphates, δ-monoalkylated d4T tetraphosphates, and d4T4P were substrates for HIV-RT. Remarkably, the antiviral activity of TetraPPPPro-prodrug 7 was improved by 7700-fold (SI 5700) as compared to the parent d4T in CEM/TK– cells, denoting a successful cell membrane passage of these lipophilic prodrugs and an intracellular delivery of the nucleotide metabolites

Pronucleotides of 2′,3′-Dideoxy-2′,3′-Didehydrothymidine as Potent Anti-HIV Compounds

We report on the synthesis and evaluation of three different nucleotide prodrug systems: (i) nucleoside triphosphate analogues in which the γ-phosph(on)ate has two different lipophilic nonbioreversible alkyl residues with d4TDP as the released nucleotide analogue; (ii) nucleoside diphosphate analogues bearing a bioreversible and a stable β-alkyl group; or (iii) nucleoside diphosphate analogues bearing two nonhydrolysable lipophilic alkyl moieties. The delivery of d4TDP (for the triphosphate precursor) and d4TMP (for the diphosphate precursor) was demonstrated in CD4+ T-lymphocyte CEM cell extracts as well as in phosphate buffer saline (PBS). In primer extension assay, we found that γ-dialkylated d4TTP derivatives and d4TDP were accepted as substrates by HIV-RT. Several of these compounds were observed to be extremely active against HIV-1/2 replication in HIV-infected cells. A more than 45,000-fold increase in the anti-HIV activity was detected for compound 18a as compared to the parent d4T which results in a selectivity index value of 37,000

HIV‑1 X4 Activities of Polycationic “Viologen” Based Dendrimers by Interaction with the Chemokine Receptor CXCR4: Study of Structure–Activity Relationship

A series of “viologen” based dendrimers with polycationic scaffold carrying 10, 18, 26, 42, and 90 charges per molecule were used to determine the structure–activity relationship (SAR) with regard to HIV-1 inhibitory activity. The studies involved five compounds with a high activity against HIV-1 already utilized in our previous study and five new dendrimers. Such dendrimers block HIV-1 entry into the cell, indicating that they bind to HIV-1 surface proteins and/or on the host cell receptors required for entry. The increasing positive character of dendrimers leads to more cytotoxicity. The 10 charges dendrimers (<b>1</b>, <b>6</b>) have less influence on the cell viability but low inhibition of the binding of the CXCR4 mAb clone 1D9. Thus, dendrimers with 18 charges (<b>2</b>, <b>7</b>) are the most promising CXCR4 imaging probes. We report the design, synthesis, and biological activity of new HIV-1 inhibitors that are conceptually distinct from those of the existing HIV-1 inhibitors

Triphosphate Prodrugs of the Anti-HIV-Active Compound 3′-Deoxy-3′-fluorothymidine (FLT)

3′-Fluoro-3′-deoxythymidine (FLT) was identified as one of the most potent inhibitors of human immunodeficiency virus (HIV) replication. However, FLT also showed severe toxicity so that it was abundant as a potential chemotherapeutic agent. Here, we describe various triphosphate prodrugs of FLT aiming for (a) a bypass of all phosphorylation steps needed to convert the nucleoside analogue into its triphosphate (TP) form, (b) an intracellular delivery of hydrolytically and enzymatically stable triphosphate derivatives, and (c) increasing the selectivity for HIV-RT vs three cellular DNA polymerases including the mitochondrial DNA polymerase γ. γ-Alkylated FLTTP compounds fulfill all of these requirements because these compounds proved highly resistant to dephosphorylation and showed strong selectivity for HIV-RT. Moreover, a prodrug form of these compounds proved to be nontoxic in CEM cells

Nucleoside Diphosphate Prodrugs: Nonsymmetric Di<i>PP</i>ro-Nucleotides

Nonsymmetric Di<i>PP</i>ro-nucleotides are described as nucleoside diphosphate (NDP) delivery systems. The concept is to attach different bis­(acyloxybenzyl) moieties at the β-phosphate moiety of a NDP. Di<i>PP</i>ro compounds bearing two alkanoylbenzyl residues and Di<i>PP</i>ro compounds bearing an alkanoylbenzyl or a benzoylbenzyl group as bioreversible prodrug moieties were studied. Compounds bearing short chain alkanoyl esters led to a fast hydrolysis by chemical or enzymatic means. The ester group in the second prodrug group comprised a long lipophilic aliphatic or an aromatic residue. The lipophilicity of this group enabled the prodrug to penetrate the cell membrane. The introduction of two different groups allowed a controlled stepwise removal of the prodrug moieties to achieve a highly selective delivery of the NDP in CEM cell extracts. The compounds were highly active against HIV even in thymidine kinase-deficient CEM cells. Thus, the compounds, although charged at the α-phosphate group, were taken up by the cells and released NDPs

Triphosphate Prodrugs of the Anti-HIV-Active Compound 3′-Deoxy-3′-fluorothymidine (FLT)

3′-Fluoro-3′-deoxythymidine (FLT) was identified as one of the most potent inhibitors of human immunodeficiency virus (HIV) replication. However, FLT also showed severe toxicity so that it was abundant as a potential chemotherapeutic agent. Here, we describe various triphosphate prodrugs of FLT aiming for (a) a bypass of all phosphorylation steps needed to convert the nucleoside analogue into its triphosphate (TP) form, (b) an intracellular delivery of hydrolytically and enzymatically stable triphosphate derivatives, and (c) increasing the selectivity for HIV-RT vs three cellular DNA polymerases including the mitochondrial DNA polymerase γ. γ-Alkylated FLTTP compounds fulfill all of these requirements because these compounds proved highly resistant to dephosphorylation and showed strong selectivity for HIV-RT. Moreover, a prodrug form of these compounds proved to be nontoxic in CEM cells

SARS-CoV-2 Virion Infectivity and Cytokine Production in Primary Human Airway Epithelial Cells

The emergence of new SARS-CoV-2 variants and the replacement of preceding isolates have been observed through B.1.1.7, B.1.351, B.1.617.2, and B.1.1.529 lineages (corresponding to alpha, beta, delta, and omicron variants of concern (VoC), respectively). However, there is still a lack of biological evidence to which extent those VoC differ from the ancestral lineages. By exploiting human airway epithelial cell (HAEC) cultures, which closely resemble the human airway architecture and physiology, we report distinctive SARS-CoV-2 tropism in different respiratory tissues. In general, SARS-CoV-2 VoC predominantly infect and replicate in HAEC better than the progenitor USA-WA1 isolate or the BavPat1 isolate, which contains the D614G mutation, even though there is little to no difference between variants regarding their infectivity (i.e., virion-per-vRNA copy ratio). We also observe differential tissue-specific innate immunity activation between the upper and lower respiratory tissues in the presence of the virus. Our study provides better comprehension of the behavior of the different VoC in this physiologically relevant ex vivo model.</p

Time- and dose-dependent DENV infection of primary microvascular endothelial HMVEC-d cells and the endothelial cell line HMEC-1.

<p>HMEC-1 (A, C) and HMVEC-d cells (E) were infected with DENV-2 at a MOI 1. Viral infectivity was quantified at different times after infection by flow cytometry using an anti-DENV-2 specific antibody (A, E). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells by means of real-time RT-PCR at different times after infection (C). Alternatively, HMEC-1 (B, D) and HMVEC-d cells (F) were treated with medium only (mock) or infected with DENV at a MOI of 1, 2 or 4. Viral infectivity was quantified 24 h post infection by flow cytometry (B, F). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells at 24 h post infection by means of real-time RT-PCR (D). The means and standard deviations of three independent experiments are shown.</p