|Titel:||Membrane-Permeable Nucleoside Triphosphate Prodrugs of Anti-HIV Active Nucleoside Analogues: γ-(Phosphate or Phosphonate)-Modified Nucleotide Analogues||Sonstige Titel:||Membranpermeable Nucleosidtriphosphat-Prodrugs von Anti-HIV-aktiven Nucleosidanaloga: γ- (Phosphat oder Phosphonat) -modifizierte Nucleotidanaloga||Sprache:||Englisch||Autor*in:||Jia, Xiao||Schlagwörter:||nucleoside triphosphate; nucleoside diphosphate; nucleoside monophosphate; nucleoside analogues; prodrugs||GND-Schlagwörter:||Nucleoside||Erscheinungsdatum:||2020||Tag der mündlichen Prüfung:||2020-10-02||Zusammenfassung:||
Nucleoside reverse transcriptase inhibitors (NRTIs) are extensively used as antiviral and anticancer compounds but they require intracellular anabolic phosphorylation into their antivirally active form, the triphosphorylated NRTI metabolites. The development of nucleoside triphosphate prodrugs is still highly interesting and desirable because they bypass all steps of intracellular phosphorylation and can therefore maximize the intracellular concentration of the ultimately bioactive nucleoside analogue triphosphates (NTPs).
This thesis describes the advancement of the nucleoside triphosphate delivery system (TriPPPro-concept), to a system in which the γ-phosphate is covalently modified by two different biodegradable masking units, one alkoxycarbonyloxybenzyl- (ACB) moiety and one acyloxybenzyl- (AB) group, and d4T as nucleoside analogue. This TriPPPro-approach enables the delivery of d4TTP with high selectivity and by enzyme-triggered reactions, as shown in human CD4+ T-lymphocyte CEM cell extracts. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and, more importantly, by enzymes contained in cell extracts. The second part of this work describes the use of this variation of the TriPPPro-concept on a variety of approved, and importantly also on so-far non-active nucleoside analogues to demonstrate the general applicability and the great potential that this approach promises. In antiviral assays, some of the nucleoside triphosphate prodrugs were found to be highly active against HIV-1 and HIV-2 in cultures of infected human T-lymphoblasts (wild-type CEM/0 cells) and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-) as well. The stability, hydrolysis pathway, and antiviral activity were significantly influenced by the acyl chain lengths of the prodrug moieties.
The third part of this work describes the synthesis and evaluation of a new class of nucleoside triphosphate analogues in which the γ-phosphate group has been replaced by a C-alkyl-phosphonate moiety. These compounds were converted into bioreversibly modified lipophilic prodrugs at the γ-phosphonate by covalent attachment of a prodrug group, either an AB or an ACB moiety. The prodrug group was selectively cleaved by chemical hydrolysis and, importantly, by enzyme activity present in pig liver esterase as well as human CD4+ T-lymphocyte CEM cell extracts to give γ-C-(alkyl)-nucleoside triphosphate analogues. In contrast to d4TTP, γ-C-(alkyl)-d4TTPs showed a very high stability in cell extracts towards dephosphorylation. A finding of major importance was that, in primer extension assays, γ-C-(alkyl)-d4TTPs proved to be substrates for HIV-RT but not for cellular DNA-polymerases α, β and γ.
In antiviral assays, the prodrug compounds were found to be highly active against HIV-1 and HIV-2 in cultures of infected wild-type CEM/0 cells. More importantly, the high antiviral activity of the prodrugs was retained in thymidine-deficient CEM cells (TK-) and compared to the parent nucleoside d4T, the anti-HIV activity was improved by 1000-fold. These results demonstrate that these prodrugs efficiently enter cells and deliver the nucleoside triphosphate analogues. Thus, they bypass all steps of the intracellularly needed phosphorylation.
Furthermore, this thesis describes a series of γ-dialkylphosphate-modified-d4TDPs and γ-dialkylphosphonate-modified-d4TDPs. Both γ-modifications are not identical to the previous work; in contrast, they are non-bioreversible, simple alkyl groups. The hydrolysis mechanism proceeded variously to the published cleavage pathway for TriPPPro-NTPs. The delivery of d4TDP rather than d4TTP was observed in CEM/0 cell extracts that was probably due to chemical phosphoanhydride cleavage. Interestingly, γ-(alkyl; alkyl-C18)-phosphate-d4TDPs and γ-(alkyl; alkyl-C18)-phosphonate-d4TDPs were highly active against HIV-1 and HIV-2 in cultures of infected wild-type CEM/0 cells and showed a marked improvement of antiviral activity (1120-fold more active as d4T) in CEM/TK- cells as well, indicating that these compounds were taken-up into cells and delivered a phosphorylated form of d4T.
This thesis describes the advancement of the TriPPPro-prodrug system for intracellular delivery of NTP derivatives. This advanced prodrug offers high potential to be used in antiviral and antitumoral chemotherapies.
|URL:||https://ediss.sub.uni-hamburg.de/handle/ediss/8566||URN:||urn:nbn:de:gbv:18-ediss-86856||Dokumenttyp:||Dissertation||Betreuer*in:||Meier, Chris (Prof. Dr.)|
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|
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