Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods.

Thymidylate synthase (TS) is a central target for the design of chemotherapeutic agents due to its vital role in DNA synthesis. Structural studies of binary complexes between Escherichia coli TS and various nucleotides suggest the chemotherapeutic agent FdUMP and the natural ligand dUMP bind similarly. We show, however, that FdUMP binding to human TS yields a substantially greater decrease in fluorescence than does dUMP. Because the difference in quenching due to ligand binding was approximately two-fold and this difference was not seen when using ecTS, the intriguing result indicated a significant difference in the mode of FdUMP binding to the human enzyme. We compared the binding affinities of dUMP, FdUMP, and TMP to TS from both species and found no significant differences for the individual ligands. Because binding affinities were not different among the ligands, the method of continuous variation was employed to determine binding stoichiometry. Similar to that found for dUMP binding to human and ecTS, FdUMP displayed single site occupancy with both enzymes. These results show that nucleotide binding differences exist for FdUMP and dUMP binding to the human enzyme. The observed differences are not due to differences in stoichiometry or ligand affinity. Therefore, although the crystal structure of human TS with various nucleotide ligands has not been solved, these results show that the differences observed using fluorescence methods result from as yet unidentified differential interactions between the human enzyme and nucleotide ligands.

Cytokine-mediated disruption of lymphocyte trafficking, hemopoiesis, and induction of lymphopenia, anemia, and thrombocytopenia in anti-CD137-treated mice.

CD137-mediated signals costimulate T cells and protect them from activation-induced apoptosis; they induce curative antitumor immunity and enhance antiviral immune responses in mice. In contrast, anti-CD137 agonistic mAbs can suppress T-dependent humoral immunity and reverse the course of established autoimmune disease. These results have provided a rationale for assessing the therapeutic potential of CD137 ligands in human clinical trials. In this study, we report that a single 200-mug injection of anti-CD137 given to otherwise naive BALB/c or C57BL/6 mice led to the development of a series of immunological anomalies. These included splenomegaly, lymphadenopathy, hepatomegaly, multifocal hepatitis, anemia, altered trafficking of B cells and CD8 T cells, loss of NK cells, and a 10-fold increase in bone marrow (BM) cells bearing the phenotype of hemopoietic stem cells. These events were dependent on CD8 T cells, TNF-alpha, IFN-gamma, and type I IFNs. BM cells up-regulated Fas, and there was a significant increase in the number of CD8+ T cells that correlated with a loss of CD19+ and Ab-secreting cells in the BM. TCR Valphabeta usage was random and polyclonal among liver-infiltrating CD8 T cells, and multifocal CD8+ T cell infiltrates were resolved upon termination of anti-CD137 treatment. Anti-CD137-treated mice developed lymphopenia, thrombocytopenia, and anemia, and had lowered levels of hemoglobin and increased numbers of reticulocytes.