No abstract

Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo through 5-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5-monophosphorylation of ribavirin and viramidine by adenosine kinase. The apparent K m for ribavirin is 540 M, and k cat is 1.8 min ؊1 . Its catalytic efficiency of 3.3 ؋ 10 ؊3 min ؊1 · M ؊1 is 1,200-fold lower than that of adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase, cytosolic 5-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline plus ATP and 2,3-bisphosphoglycerate, the apparent K m for ribavirin is 88 M, and k cat is 4.0 min ؊1 . These findings suggest that cytosolic 5-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10-to 330-fold lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic 5-nucleotidase II could play an important role in phosphorylating ribavirin and contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced hemolytic anemia in erythrocytes.

Ribavirin is an approved broad-spectrum antiviral drug. A liver-targeting prodrug of ribavirin, viramidine, is in clinical trial in an attempt to provide a better therapeutic index. The conversion of viramidine to ribavirin, and of ribavirin to an inactive metabolite through adenosine deaminase, is reported. Kinetic analysis indicates that adenosine deaminase is likely involved in activation of viramidine in vivo, and the process is highly pH sensitive. The differential activities of two consecutive deamination reactions are kinetically studied and interpreted based on adenosine deaminase structural information. A comprehensive understanding of the viramidine and ribavirin deamination mechanism should help in designing better nucleoside therapeutics in the future.

Pyridine derivatives R 0380Identification and Structure-Activity Relationships of Substituted Pyridones as Inhibitors of Pim-1 Kinase. -Chemical library hits along with synthesized derivatives of these compounds act as potent inhibitors of Pim-1 kinase. A complex crystal structure and the mechanism of action are defined for pyridone (IIIa), the most potent Pim-1 inhibitor. Preliminary data further indicate that (IIIa) lacks in vitro activity towards related serine/threonine kinases Pim-2 and MEK1/2. Small molecules similar to (III) may serve as starting scaffolds for the development. -(CHENEY*, I. W.; YAN, S.; APPLEBY, T.; WALKER, H.; VO, T.; YAO, N.; HAMATAKE, R.; HONG, Z.; WU,

Uridine-cytidine nucleoside kinase 2 (UCK2) is the ratelimiting enzyme in the pyrimidine-nucleotide salvage pathway. UCK2 catalyzes the phosphorylation of the natural ribonucleosides cytidine and uridine to cytidine 5 H -monophosphate (CMP) and uridine 5 H -monophosphate (UMP), respectively, and activates several important frontline antimetabolite drugs. The present contribution reports the rapid crystal structure determination of human UCK2 complexed with a magnesium ion and the reaction products adenosine 5 H -diphosphate (ADP) and CMP. Diffraction data were collected on a copper rotating-anode X-ray generator from one native UCK2 crystal and a single samarium-derivative crystal. Utilizing the relatively high anomalous signal from the samarium derivative at the Cu K wavelength, the structure was determined by single isomorphous replacement and single anomalous signal (SIRAS) phasing techniques. Two of the four major samarium sites are located in the active sites of the two UCK2 molecules that form the asymmetric unit and appear to displace the magnesium ions present in the native crystals. The crystal structures of UCK2 alone and in complex with various ligands have recently been determined using traditional multiple isomorphous replacement (MIR) phasing techniques and data from three heavy-atom derivatives. The reported structures validate our independently determined structure. Of more than 1000 kinase crystal structure entries in the Protein Data Bank, less than 1% of them have been determined by SIRAS. For the published kinase crystal structures determined by SIRAS, all data were reportedly collected at various synchrotron-radiation facilities. This study demonstrates that diffraction data collected from a single samarium derivative using Cu K radiation provides suf®cient phasing power to determine a novel macromolecular crystal structure. PDB Reference: human uridine-cytidine kinase 2, 1xrj, r1xrjsf.