Nucleotide binding to human UMP‐CMP kinase using fluorescent derivatives − a screening based on affinity for the UMP‐CMP binding site

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor‐binding and acceptor‐binding sites of human UMP‐CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP‐CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in‐line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor‐binding site of the human UMP‐CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP‐CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP‐CMP kinase. Also, the 5‐halogen‐substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor‐binding site is in agreement with a large substrate‐binding pocket, as shown by the 2.1 Å crystal structure.