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 A crystal structure.     

Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.

Allomyces arbuscula, an aquatic fungus, contains two Ca2+-dependent neutral cysteine proteases (CDP I and CDP II), eluting respectively, at 0.07 and 0.2 M NaCl from DEAE cellulose columns. The purified CDP I has a Mr of 39 kDa whereas CDP II appears as a doublet of 43 and 40 kDa. Both enzymes require free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that despite these similarities the two enzymes are different because affinity-purified CDP II antibodies do not cross-react with CDP I antigen in Western blots. In contrast, there is a strong cross-reaction between the two 43 and 40 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on the cleavage site. This primary specificity is governed by the nature of the amino acids in the P2 and P3 positions. In general either Pro or Gly in P2 is required, with preference for Pro and in P3 position, Gly over Val. CDP II has higher catalytic activity than CDP I. The sulfhydryl reagent NEM is a more potent inhibitor of CDP I than CDP II. Although the function of the phosphorylable site(s) is not clear, both CDP I and CDP II contain phosphorylable serine residue(s).     

CDP840

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-{3-cyclopentyloxy-4-methoxyphenyl}-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC₅₀ 2–30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC₅₀>100 μM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A_330–886, which rolipram inhibited with low affinity (IC₅₀=1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC₅₀=3.9 nM), which was in good agreement with theK _d of 4.8 nM obtained from [³H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [³H]-rolipram binding to PDE-4A, B, C and D with similarK _{d app} (7–19 nM and 3–5 nM, respectively). Thus, the activity of CDP840 at the [³H]-rolipram binding site was in agreement with the inhibitor’s activity at the catalytic site. However, rolipram was ∼100-fold more potent than CDP840 at inhibiting the binding of [³H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).     

Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (β) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-(β,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.