Design and Synthesis of Novel and Selective Phosphodiesterase 2 (PDE2a) Inhibitors for the Treatment of Memory Disorders.

A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2a inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, the increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2a active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2a inhibition.

The phosphodiesterase 3 inhibitor cilostazol does not stimulate growth of colorectal liver metastases after major hepatectomy.

Liver failure after extended hepatectomy represents a major challenge in the surgery of hepatic colorectal metastasis. A previous study has indicated that inhibition of phosphodiesterase type 3 (PDE 3) stimulates liver regeneration. However, little is known whether PDE 3 inhibitors, such as cilostazol, also stimulate the growth of remnant metastases. Therefore, we herein studied the effect of cilostazol on engraftment, vascularization and growth of colorectal liver metastasis after major hepatectomy. WAG-rats underwent either major hepatectomy or sham operation. Metastases were induced by subcapsular implantation of 5 × 10(5) CC531-colorectal cancer cells. Animals were daily treated with cilostazol (5 mg/kg body weight) or glucose solution. Tumor growth was measured by high-resolution ultrasound at days 7 and 14. Tumor vascularization and tumor cell proliferation were determined by immunohistochemistry and western blotting. High-resolution ultrasound analysis in hepatectomized and non-hepatectomized animals showed that cilostazol does not stimulate tumor growth. Accordingly, the number of PCNA-positive tumor cells did not differ between cilostazol-treated animals and sham-treated controls. Interestingly, cilostazol reduced tumor vascularization in both hepatectomized and non-hepatectomized animals. This was indicated by a significantly lower number of platelet-endothelial cell adhesion molecule (PECAM-1)-positive cells in tumors of cilostazol-treated animals compared to sham-treated controls. The PDE 3 inhibitor cilostazol does not stimulate the growth of colorectal metastases during liver regeneration after major hepatectomy.

The effect of divalent cations on the catalytic activity of the human plasma 3'-exonuclease.

The 3'-exonuclease from human plasma is a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9). Here, the possibility of divalent cation influence for the 3'-exonuclease activity was investigated using the phosphorothioate congener of oligonucleotide containing all phosphorothioate internucleotide linkages of the [R(P)]-configuration ([R(P)-PS]-d[T(12)]) as the substrate for this enzyme. It was found that the 3'-exonuclease is a metalloenzyme, i.e. its phosphodiesterase activity was completely abolished at 0.8 mM concentration EDTA and, in turn, it was restored in the presence of Mg(2+) or Mn(2+) ions. In addition, Mg(2+) can be replaced effectively by Ca(2+), Mn(2+), or Co(2+), but not by Ni(2+) and Cd(2+) during the hydrolysis of the phosphorothioate substrate in human plasma. In addition, the mechanism is postulated, by which a single internucleotide phosphorothioate bond of the S(P)-configuration at the 3'-end of unmodified phosphodiesters (PO-oligos), or their phosporothioate analogs (PS-oligos) protects these compounds against degradation in blood.

Sensitizing hormone-refractory prostate cancer cells to drug treatment by targeting 14-3-3sigma.

Advanced and hormone-refractory prostate cancer has long been considered as a chemoresistant disease. Recently, it was found that 14-3-3sigma expression increases as prostate tumor progresses, and that 14-3-3sigma contributes significantly to drug resistance in breast cancers. We, thus, hypothesized that advanced and hormone-refractory prostate cancers may have an increased level of 14-3-3sigma, which in turn may contribute to drug resistance in advanced and hormone-refractory prostate cancers. In this study, we tested this hypothesis and found that, indeed, the expression level of 14-3-3sigma in androgen-independent prostate cancer cell lines DU145, PC3, and CWR22RV are much higher than that in the androgen-dependent cell line LNCaP, and that the androgen-independent cells are more resistant to mitoxantrone and Adriamycin than the androgen-dependent cells. Depleting 14-3-3sigma expression in DU145 and CWR22RV by RNA interference significantly sensitized these cells to mitoxantrone and Adriamycin by abrogating G2-M checkpoint and increasing apoptosis, whereas restoring 14-3-3sigma expression in LNCaP cells enhanced drug resistance. We also showed that 14-3-3sigma deficiency caused nuclear localization of Cdc2 and dephosphorylation of the Tyr15 residue upon DNA damage. Based on these studies, we propose that therapeutic intervention targeting 14-3-3sigma may be useful for sensitizing hormone-refractory prostate cancers to chemotherapy by both G2-M checkpoint abrogation and apoptosis enhancement.

Human hRad1 but not hRad9 protects hHus1 from ubiquitin-proteasomal degradation.

Three of the Rad family proteins, Rad9, Rad1, and Hus1, can interact with each other and form a heterotrimeric complex that is thought to play a role in the sensing step of the DNA integrity checkpoint pathways, but the nature of the Rad9-Rad1-Hus1 complex assembly remains enigmatic. Here, we demonstrate that the human hRad1 protein plays a significant role as molecular chaperone in the process of the hRad9-hRad1-hHus1 heterotrimeric complex formation. In contrast to hRad1, hHus1 is an unstable protein that is actively degraded via the ubiquitin-proteasome pathway. We show that treating cells with proteasome-specific inhibitors stabilizes hHus1 expression. Moreover, hRad1 can associate with hHus1 in the absence of hRad9 and protect hHus1 from ubiquitination and degradation in the cytoplasm. Importantly, genotoxic stress induces hRad1 expression and stabilizes the hHus1 protein. Taken together, these findings suggest a novel role of hRad1 as a potential intrinsic chaperone in the stabilization of hHus1 for the hRad9-hRad1-hHus1 checkpoint complex formation.

3-O-methylquercetin more selectively inhibits phosphodiesterase subtype 3.

Rhamnus nakaharai Hayata (Rhamnaceae), has been used as a folk medicine in Taiwan for treating constipation, inflammation, tumors and asthma. 3-O-methylquercetin (3-MQ), a main constituent of the plant, has been reported to inhibit total cAMP- and cGMP-phosphodiesterase (PDE) of guinea pig trachealis. Therefore we were interested in investigating the inhibitory effect of 3-MQ on various PDE isozymes from guinea pig lungs and hearts. Isolated guinea pig lungs and hearts were homogenized and centrifuged. The supernatant was chromatographed over a column of Q-sepharose, and eluted with various concentrations of NaCl. In the following order, PDE subtypes 1, 5, 2, 4 from lungs, and 3 from hearts were separated. The IC 50 values of 3-MQ on these isozymes were 31.9, 86.9, 18.6, 28.5 and 1.6 microM, respectively. 3-MQ (10-100 microM) non-competitively inhibited PDE2, but competitively inhibited PDE4. 3-MQ (1-10 microM) also competitively inhibited PDE3. However, 3-MQ (10-100 microM) did not competitively inhibit PDE1 and 5, although it had a tendency to competitively inhibit PDE1 at concentrations of 10 - 30 microM. The present results showed that K i value of 3-MQ was similar to that of milrinone in PDE3, and was not significantly different from that of Ro 20 - 1724 in PDE4, respectively. In conclusion, 3-MQ was revealed to be a selective and competitive PDE3/PDE4 inhibitor, although its inhibitory effect on PDE4 was not potent. Therefore, 3-MQ may have a potential in the treatment of asthma beside its antiviral activity.

Characterization of a multi-functional metal-mediated nuclease by MALDI-TOF mass spectrometry.

Mass spectrometric analysis of reaction products allows simultaneous characterization of activities mediated by multifunctional enzymes. By use of MALDI-TOF mass spectrometry, the relative influence of magnesium and manganese promoted exonuclease and phosphatase activities of Esherichia coli exonuclease III have been quantitatively measured, offering a rapid and sensitive alternative to radioactivity quantification and gel electrophoresis procedures for determination of reaction rate constants. Manganese is found to promote higher levels of exonuclease activity, which could be a source of mutagenic effects if this ion were selected as the natural cofactor. Several potential applications of these methods to quantitative studies of DNA repair chemistry are also described.

Cyclic nucleotide-mediated regulation of vascular smooth muscle cell cyclic nucleotide phosphodiesterase activity. Selective effect of cyclic AMP.

Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents.

p53 Protein exhibits 3'-to-5' exonuclease activity.

Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."

Effects of nickel ions on polymerase activity and fidelity during DNA replication in vitro.

Nickel is a genotoxic carcinogen. However, the mechanisms of nickel-induced genotoxicity are not well understood. We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro. The effect of Ni2+ on different DNA polymerases is quite variable. The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity. Some polymerases, such as E. coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication. Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude. T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase. The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner. The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.