1 Celltransmissions The Newsletter for Cell Signaling and Neuroscience Research Vol 20, No 1 • March 2004 In this Issue... New Products pp. 12-14 Functions and Pharmacological Inhibitors of Anti-mTOR: marker for translation initiation Cyclic Nucleotide Phosphodiesterases activation Valeria Vasta and Joe Beavo p. 9 XK469: selective topoisomerase II-β inhibitor he discovery of the cyclic nucleotides 3',5'- p. 10 cyclic monophosphate (cAMP, Prod. No. T ANPs A 9501, A 4137) and guanosine 3',5'-cyclic a Guanylin Y-27632: selective inhibitor STa monophosphate (cGMP, Prod. No. G 7504, NO GTP pGC of Rho associated protein sGC kinase p160ROCK G 6129) led to the first formulation of the second cGMP PDEs NO p. 11 messenger concept. These cyclic nucleotides are 5'GMP now known to be ubiquitous intracellular second SB-431542: potent, messengers that mediate the response of cells to PDEs selective inhibitor of activin a variety of extracellular stimuli through the receptor-like kinase (ALK) activation of cyclic nucleotide-dependent protein PKG receptors kinases, ion channels, GTP-exchange factors and CNG Channels p. 15 their downstream effector systems [1]. The ampli- Protein tude and duration of cAMP and cGMP signals are Phosphorylation SCH-28080: potent Na+, Ca2+ + + controlled by their rates of synthesis by adenylyl inhibitor of gastric H /K - ATPase and guanylyl cyclases, respectively, and their p. 15 degradation by 3’,5'-cyclic nucleotide phospho- a large superfamily of enzymes that hydrolyze the diesterases (PDEs) (Figure 1). PDEs, identified 3' phosphodiester bond in cAMP or cGMP to Peptidomimetic isoform shortly after the discovery of cAMP and cGMP, are form the corresponding 5'-nucleotide selective nNOS inhibitors p. 16 continued on page 3 L-685,458: potent, selective γ-secretase Application Note: inhibitor p. 17 ™ The New Panorama Antibody (Ab) Anti-ILK: marker for integrin-mediated Microarray Cell Signaling Kit: A Unique Tool processes for Protein Expression Analysis p. 18 Eliezer Kopf, Dalia Shnitzer and Dorit Zharhary SB-228357: potent, selective 5-HT2C/2B serotonin receptor Introduction provided valuable information antagonist in many biological areas. p. 18 here is a growing need for However, since there is not technologies that allow Anti-Survivin: marker T always a direct correlation global molecular characteriza- for blocking apoptosis between the mRNA level and tion of biological samples. The p. 19 the expression of the protein, a ability to identify multiple pro- method that can directly assay teins simultaneously has many Compound Libraries proteins is required. Whereas pp. 22-23 applications in basic biological DNA/RNA/oligo arrays give research as well as in disease information on the genetic diagnosis and treatment. The defects that may cause disease, use of DNA arrays for profiling protein microarrays provide mRNA expression in cells has sigma-aldrich.com/cellsignaling continued on page 20 3 Functions and Pharmacological Inhibitors of Cyclic Nucleotide Phosphodiesterases Valeria Vasta and Joe Beavo (continued from cover) monophosphate. This overview focuses on the functions and expressed in multiple isoforms due to different transcription pharmacological inhibitors of mammalian PDEs; for more initiation sites or alternative splicing. A large number of extensive reviews see references [2-4]. transcript variants have been reported in the literature or can be deduced from the EST database, but the physiological significance of this phenomenon is only beginning to be PDE Superfamily elucidated. The PDE families can be differentiated gene- The PDEs are now recognized to form a superfamily of 11 tically, on the basis of sequence homology, and functionally, different, but homologous gene-families that all contain a on the basis of substrate specificity, kinetic properties and conserved catalytic domain of around 300 amino acids near sensitivity to endogenous or pharmacological regulators. The their carboxy terminus [3]. The amino acid sequence identity first functional difference identified between the various in the catalytic domain is usually 35-40% between different PDEs was substrate specificity. Thus, some PDEs hydrolyze families, while it is generally at least 70% between members only cAMP, some hydrolyze only cGMP, while others of the same gene family. Twenty-one residues, in particular, hydrolyze both. Table 1 depicts the 11 gene families, are absolutely conserved across all PDE families and two PDE referred to as PDE1-PDE11, together with their substrate signature motifs (H-N-XX-H\H-D-XX-H) in the active site specificity, tissue localization, their basic mode of regulation appear to form a single pocket, binding two metal ions and some of their selective inhibitors. involved in catalysis [5]. The crystal structures of several PDE catalytic domains have recently been determined [6-9]. The Different PDE isozymes can have specific tissue, cellular and amino terminal region of the PDEs is distinguished by a subcellular distributions and more than one type of PDE is domain and motif organization characteristic of each PDE Cyclic Nucleotide usually present in any given cell. However, the pattern of gene family [3]. This region is involved in subunit and Phosphodiesterases PDE expression can exhibit some degree of variability protein-protein interactions, localization, and allosteric and between different species. The types of PDEs expressed in a covalent modifications affecting catalytic activity. The cell, together with their relative proportions and subcellular nomenclature employed to distinguish each PDE isozyme localization, clearly control the cyclic nucleotide phenotype uses the first two letters to designate the species of origin, of that cell. In addition to literature data on the tissue/cell followed by PDE and the Arabic numeral of the gene family specific expression of the various PDEs, which is not entirely (usually reflecting the order of discovery). The next letter catalogued as yet, information on the expression can be represents the individual gene within the family, followed by obtained from mRNA, ESTs and microarray analysis data- an Arabic numeral identifying the transcript variant. For bases. For example, the sequences (mRNA and ESTs) belong- example, HSPDE8A1 refers to the human PDE8 family, gene ing to each PDE gene family and their tissue of derivation A, transcript variant 1. are listed in a cluster with the name of that PDE in the UniGene database (http://www.ncbi.nlm.nih.gov/entrez/- sigma-aldrich.com/cellsignaling At present, 21 different mammalian PDE genes have been query.fcgi? db=unigene) and in the Geo Microarray analysis identified with each gene generally being highly conserved database (http://www.ncbi.nlm.nih.gov:80/entrez/ between species. In addition, many of the genes are query.fcgi?CMD= search&DB=geo). ANPs Hormone, Neurotransmitter a Guanylin b GPCR NO pGC STa GTP G echnical Service: 1-800-325-5832 sGC S Adenylyl T PDEs NO cGMP 5'-AMP G Cyclase S ATP 5'GMP PDEs cAMP PDEs PKG PKA der: 1-800-325-3010 r CNG Channels Protein Phosphorylation GEFs CNG Channels Protein Phosphorylation Na+, Ca2+ Na+, Ca2+ ol 20, No.1, 2004 O V Figure 1. Cellular pathways of cyclic nucleotide function and regulation. The above schematics show the basic synthetic and regulatory pathways for a) cGMP and b) cAMP metabolism. Various agonists can activate guanylyl cyclases (Panel a) and adenylyl cyclase (Panel b) in different cell types and increase the intracellular levels of cGMP or cAMP, respectively. This ® in turn activates downstream effector systems, such as PKA (protein kinase A), PKG (protein kinase G), cAMP-GEFs (guanine nucleotide exchange factors) and CNG (cyclic nucleotide-gated) channels. By degrading cyclic nucleotides, PDEs control the amplitude, duration and compartmentalization of the cyclic nucleotide signal. Panel a) also illustrates the feedback control of PDE activity by cGMP, ANPs (atrial natriuertic peptides), NO (nitric ® oxide), pGC (particulate guanylyl cyclase), sGC (soluble guanylyl cyclase) and STa (heat-stable enterotoxin). Figure adapted from reference [1]. Celltransmissions 4 Inhibitors of Cyclic Nucleotide Phosphodiesterases...(continued) Table 1 Biochemical and pharmacological characteristics of cyclic nucleotide phosphodiesterase (PDE) isozymes Major Tissue Gene Family Genes Substrate Regulators Inhibitors (Sigma Prod. No.) IC in µM* [Reference] Expression 50 Brain, heart, PDE1A Ca2+-CaM smooth muscle, 8-Methoxymethyl-IBMX (M 2547) 4 [2] PDE1 PDE1B cAMP/cGMP PKA olfactory cilia, Vinpocetine (V 6383) 20 [2] PDE1C CaMKII testis Adrenal cortex, EHNA (E-114) 1 [2] PDE2 PDE2A cGMP/cAMP cGMP brain, heart Trequinsin (T 2057) 0.64-2 [48] Cilostamide (C 7971) 0.005 [2] Cilostazol (C 0737) 0.12 [2] PDE3A Heart, adipose cGMP Enoximone (E 1279) 1 [2] PDE3 PDE3B tissue, pancreas, cAMP>cGMP PKA Imazodan (I 0782) 6 [2] platelets PKB Milrinone (M 4659) 0.30 [2] Trequinsin (T 2057) 0.0003 [2] PDE4A PDE4B PKA Rolipram (R 6520) 2 [2] PDE4 Many tissues cAMP PDE4C PKC YM976 (Y 4877) 0.002 [49] PDE4D Dipyridamole (D 9766) 0.90 [2] DMPPO 0.003 [42] Lung, platelets, Sildenafil (Viagra) 0.004 [50] Cyclic Nucleotide PKA smooth muscle, T-0156 (T 8067) 0.0002 [43] PDE5 PDE5A cGMP PKG Phosphodiesterases corpus T-1032 (T 7692) 0.001 [44] cGMP cavernosum Tadalafil (Cialis) 0.005 [51] Vardenafil (Levitra) 0.0007 [52] Zaprinast (Z 0878) 0.76 [2] Dipyridamole (D 9766) 0.38 [2] PDE6A (α) Rod and cone Transducin Sildenafil 0.074 [51] PDE6 PDE6B (β) photoreceptor cGMP PDE6 γ and δ subunits Tadalafil