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2',3'-Dideoxynucleoside phosphorylation by deoxycytidine kinase from normal human thymus extracts: activation of
M A Johnson1, D G Johns, A Fridland
1Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Abstract:
As a first step toward improving dideoxynucleoside inhibition of human immunodeficiency virus replication in human lymphocytes, we examined the kinetics of 5'-phosphorylation of a series of 2',3'-dideoxynucleosides, using deoxycytidine kinase purified from human thymus extracts. Nucleosides with the 2'-deoxyribose moiety were activated 30 times faster than were 2',3'-dideoxynucleosides. The adenosine deaminase inhibitor, 2'-deoxycoformycin, showed an unexpected ability to inhibit purine and pyrimidine dideoxynucleoside phosphorylation; such inhibition was not competitive and was not observed when 2'-deoxycytidine was the substrate. 2'-Deoxycytidine, the natural substrate, inhibited dideoxynucleoside phosphorylation in a manner similar to that observed with 2'-deoxycoformycin. Thus, dideoxynucleosides are activated by deoxycytidine kinase through a different catalytic interaction than occurs in 5'-activation of 3'-hydroxynucleosides by this enzyme.
Insights
This study reveals that deoxycytidine kinase activates dideoxynucleosides differently than other nucleosides, impacting human immunodeficiency virus inhibition. Understanding these kinetics is key for developing better antiviral therapies.
Area of Science:
- Biochemistry
- Virology
- Pharmacology
Background:
- Human immunodeficiency virus (HIV) replication in lymphocytes is a target for antiviral drugs.
- Dideoxynucleosides are a class of compounds investigated for their potential to inhibit HIV replication.
- Efficient intracellular phosphorylation is crucial for the activation and efficacy of dideoxynucleoside drugs.
Purpose of the Study:
- To investigate the kinetics of 5'-phosphorylation of 2',3'-dideoxynucleosides by human deoxycytidine kinase.
- To understand the mechanism of dideoxynucleoside activation and identify factors influencing their phosphorylation.
- To explore potential strategies for improving dideoxynucleoside-based HIV inhibition.
Main Methods:
- Purification of deoxycytidine kinase from human thymus extracts.
- Enzymatic assays to measure the kinetics of 5'-phosphorylation of various dideoxynucleosides.
- Investigating the inhibitory effects of 2'-deoxycoformycin and 2'-deoxycytidine on dideoxynucleoside phosphorylation.
Main Results:
- Nucleosides with a 2'-deoxyribose moiety were phosphorylated 30 times faster than 2',3'-dideoxynucleosides.
- 2'-deoxycoformycin, an adenosine deaminase inhibitor, unexpectedly inhibited dideoxynucleoside phosphorylation in a non-competitive manner.
- 2'-deoxycytidine, the natural substrate, also inhibited dideoxynucleoside phosphorylation, similar to 2'-deoxycoformycin's effect.
Conclusions:
- Dideoxynucleosides are activated by deoxycytidine kinase via a distinct catalytic interaction compared to 3'-hydroxynucleosides.
- The findings suggest a different mechanism for the 5'-activation of dideoxynucleosides by deoxycytidine kinase.
- This mechanistic insight could inform the development of more effective dideoxynucleoside analogs for HIV therapy.