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The present study investigated the putative pro-cognitive effects of the novel selective PDE9 inhibitor BAY 73-6691. The effects on basal synaptic transmission and long-term potentiation (LTP) were investigated in rat hippocampal slices. Pro-cognitive effects were assessed in a series of learning and memory tasks using rodents as subjects. BAY 73-6691 had no effect on basal synaptic transmission in hippocampal slices prepared from young adult (7- to 8-week-old) Wistar rats. A dose of 10 microM, but not 30 microM, BAY 73-6691 enhanced early LTP after weak tetanic stimulation. The dose effective in young adult Wistar rats did not affect LTP in hippocampal slices prepared from young (7- to 8-week-old) Fischer 344 X Brown Norway (FBNF1) rats, probably reflecting strain differences. However, it increased basal synaptic transmission and enhanced early LTP after weak tetanic stimulation in hippocampal slices prepared from very old (31- to 35-month-old) FBNF1 rats. BAY 73-6691 enhanced acquisition, consolidation, and retention of long-term memory (LTM) in a social recognition task and tended to enhance LTM in an object recognition task. Bay 73-6691 attenuated the scoplamine-induced retention deficit in a passive avoidance task, and the MK-801-induced short-term memory deficits in a T-maze alternation task. The mechanism of action, possibly through modulation of the NO/cGMP-PKG/CREB pathway, is discussed. Our findings support the notion that PDE9 inhibition may be a novel target for treating memory deficits that are associated with aging and neurodegenerative disorders such as Alzheimer's disease.

We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (BAY 73-6691), the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC50 = 55 nM) and murine (IC50 = 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl)[2-[(4-phenethyl-benzyl)oxy]phenethyl]amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity.

Two gene groups, designated energy converting hydrogenase A (eha) and energy converting hydrogenase B (ehb), each encoding a putative multisubunit membrane-bound [NiFe] hydrogenase, were identified in the genome of Methanobacterium thermoautotrophicum. The length of the transcription units was determined using reverse transcription (RT)-PCR. The eha operon (12.5 kb) and the ehb operon (9.6 kb) were found to be composed of 20 and 17 open reading frames, respectively. Competitive RT-PCR was used to compare the amounts of eha and ehb transcripts with the amounts of transcripts of genes encoding the M. thermoautotrophicum catabolic enzymes cyclohydrolase (mch) and a subunit of heterodisulfide reductase (hdrC). In cells grown under conditions in which H 2 was nonlimiting, the eha transcripts were 250-fold and 125-fold less abundant and the ehb transcripts were approximately sixfold and threefold less abundant than the hdrC and mch transcripts, respectively. In cells grown under H 2 limitation, the amounts of eha and ehb transcripts were about threefold higher than in cells grown with sufficient H 2 when compared to the amounts of hdrC and mch transcripts. Sequence analysis of the deduced proteins indicated that the eha and ehb operons each encode a [NiFe] hydrogenase large subunit, a [NiFe] hydrogenase small subunit, and two conserved integral membrane proteins. These proteins show high sequence similarity to subunits of the Ech hydrogenase from Methanosarcina barkeri, Escherichia coli hydrogenases 3 and 4, and CO-induced hydrogenase from Rhodospirillum rubrum, all of which form a distinct group of multisubunit membrane-bound [NiFe] hydrogenases and show high sequence similarity to the energy-conserving NADH:quinone oxidoreductase (complex I) from various organisms. In addition to these four subunits, the eha operon encodes a 6[4Fe±4S] polyferredoxin, a 10[4F±4S] polyferredoxin, four nonconserved hydrophilic subunits, and 10 nonconserved integral membrane proteins; the ehb operon encodes a 2[4Fe±4S] ferredoxin, a 14[4Fe±4S] polyferredoxin, two nonconserved hydrophilic subunits, and nine nonconserved integral membrane proteins. A function of these putative membrane-bound [NiFe] hydrogenases as proton pumps involved in endergonic reactions, such as the synthesis of formylmethanofuran from CO 2 , H 2 and methanofuran, is discussed.Keywords: complex I; energy conservation; hydrogenase; iron±sulfur protein; Methanobacterium thermoautotrophicum.A small group of multisubunit membrane-bound [NiFe] hydrogenases has been identified recently. The subunits of these enzymes were found to be more closely related to subunits of the proton-pumping NADH:quinone oxidoreductase (complex I) from various organisms than to subunits of other These enzymes are composed of at least six subunits, five of which are highly conserved. Two of the conserved subunits show sequence similarity to the hydrogenase large and small subunit of`standard' [NiFe] hydrogenases. From the crystal structure of the Desulfovibrio gigas and D. vulgaris [NiFe] ...

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.