Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives -- a screening based on affinity for the
Dimitri Topalis1, Hiroki Kumamoto1, Maria-Fernanda Amaya Velasco1
1Laboratoire d'Enzymologie Moléculaire et Fonctionnelle, FRE 2852 CNRS-Paris 6, Institut Jacques Monod, Paris, France Institut de Chimie Organique et Analytique, UMR CNRS 6005, FR 2708, Université d'Orléans, UFR Sciences, Orléans, France Unité de Biochimie Structurale, URA CNRS 2185, Institut Pasteur, Paris, France Unité de Chimie Organique, URA CNRS 2128, Institut Pasteur, Paris, France Plate-Forme 6- Cristallogénèse et Diffraction des Rayons X, Institut Pasteur, Paris, France Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 2185, Institut Pasteur, Paris, France.
Researchers developed a screening method using human UMP-CMP kinase to identify potential antiviral and anticancer drugs. Novel uracil derivatives showed promise as inhibitors, offering a new avenue for therapeutic development.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Human UMP-CMP kinase is crucial in the nucleoside salvage pathway, utilizing ATP and NMPs.
- Understanding its substrate-binding sites is key for developing targeted therapies.
- Nucleoside salvage pathway kinases play vital roles in cellular metabolism and are targets for antiviral and anticancer drugs.
Purpose of the Study:
- To develop a screening method for identifying antimetabolites for antiviral and anticancer therapies.
- To investigate the binding affinities and specificities of human UMP-CMP kinase.
- To explore novel acyclic phosphonate analogs as potential therapeutic agents.
Main Methods:
- Utilized methylanthraniloyl derivatives of ATP and CDP as fluorescent probes for enzyme binding sites.
- Employed bisubstrate analogs (Ap5X series) to assess donor-site probe displacement and acceptor-site specificity.
- Screened acyclic phosphonate analogs, including uracil vinylphosphonate derivatives, for their affinity to human UMP-CMP kinase.
Main Results:
- Identified broad specificity of the UMP-CMP kinase acceptor site, with analogs displacing the donor-site probe.
- Several uracil vinylphosphonate derivatives exhibited affinities comparable to natural substrates (dUMP, dCMP) and superior to cidofovir.
- These uracil derivatives acted as inhibitors, not substrates, of human UMP-CMP kinase, with reduced inhibition by 5-halogen-substituted analogs on TMP kinase.
Conclusions:
- The developed screening method effectively identifies potential antimetabolites based on UMP-CMP kinase affinity.
- Novel uracil derivatives show significant potential as inhibitors for therapeutic applications.
- The broad specificity of the enzyme's acceptor site, supported by crystal structure data, facilitates the discovery of new drug candidates.
