sign in sign up
<<
Home , Cilomilast, Cilostamide, EHNA, Enoximone, Furafylline, Milrinone

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 activation Valeria Vasta and Joe Beavo p. 9 XK469: selective topoisomerase II-β inhibitor he discovery of the cyclic 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 -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 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 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 between species.Inaddition,manyofthegenesare identified witheachgenegenerallybeinghighlyconserved At present, 21different mammalianPDEgeneshavebeen A, transcriptvariant1. example, HSPDE8A1refers tothehumanPDE8family, gene an Arabicnumeralidentifyingthetranscriptvariant.For represents theindividualgenewithinfamily, followedby (usually reflecting theorder ofdiscovery).Thenextletter followed byPDEandtheArabicnumeralofgenefamily uses thefirsttwoletterstodesignatespeciesoforigin, nomenclature employedto distinguisheachPDEisozyme covalent modificationsaffecting catalyticactivity. The protein-protein interactions,localization,andallosteric gene family[3].Thisregion isinvolvedinsubunitand domain andmotiforganizationcharacteristicofeachPDE amino terminalregion ofthe PDEsisdistinguishedbya catalytic domainshaverecently beendetermined[6-9].The involved incatalysis[5].Thecrystalstructures ofseveralPDE appear toformasinglepocket,bindingtwometalions signature motifs(H-N-XX-H\H-D-XX-H)intheactivesite are absolutelyconservedacross allPDEfamiliesandtwo PDE of thesamegenefamily. Twenty-one residues, inparticular, families, whileitisgenerallyatleast70%betweenmembers in thecatalyticdomainisusually35-40%betweendifferent their carboxyterminus[3].Theaminoacidsequenceidentity conserved catalyticdomainofaround 300aminoacidsnear different, buthomologousgene-familiesthatallcontain a The PDEsare nowrecognized toformasuperfamily of 11 PDE Superfamily extensive reviews seereferences [2-4]. pharmacological inhibitorsofmammalianPDEs;formore monophosphate. Thisoverviewfocusesonthefunctionsand Phosphodiesterases Functions andPharmacologicalInhibitorsofCyclicNucleotide oxide), pGC(particulate guanylylcyclase),sGC(soluble cyclase)andSTa (heat-stableenterotoxin). Figure adapted fr of thecyclicnucleotide signal.Panela)alsoillustratesthefeedback control ofPDEactivity bycGMP, ANPs(atrialnatriuert factors) andCNG(cyclicnucleotide-gated) channels.Bydegradingcyclicnucleotides,PDEscontrol theamplitude,durationand activates downstreamin turn effector systems,suchasPKA(protein kinaseA),PKG(protein kinaseG),cAMP-GEFs(guanine nucle guanylyl cyclases(Panela)andadenylyl cyclase(Panelb)indifferent celltypesandincrease theintracellularlevelsofcGM The aboveschematicsshowthebasicsynthetic andregulatory pathways fora)cGMPandb)cAMPmetabolism.Various agonistscan Figure 1.Cellularpathwaysofcyclicnucleotidefunctionand regulation. a NO PDEs NO sGC Phosphorylation Protein cGMP GTP PKG pGC PDEs 5'GMP Na STa Guanylin ANPs Valeria Vasta andJoeBeavo + , Ca 2+ CNG Channels b query.fcgi?CMD= search&DB=geo). database (http://www.ncbi.nlm.nih.gov:80/entrez/ query.fcgi? db=unigene) andintheGeoMicroarray analysis UniGene database(http://www.ncbi.nlm.nih.gov/entrez/- are listedinaclusterwith the nameofthatPDEin ing toeachPDEgenefamilyandtheirtissueofderivation bases. Forexample,thesequences(mRNAandESTs) belong- obtained from mRNA,ESTs andmicroarray analysisdata- catalogued asyet,informationontheexpression canbe specific expression ofthevarious PDEs,whichisnotentirely of thatcell.Inadditiontoliterature dataonthetissue/cell localization, clearlycontrol thecyclicnucleotidephenotype cell, togetherwiththeirrelative proportions andsubcellular between different species.ThetypesofPDEsexpressed ina PDE expression canexhibit somedegree ofvariability usually present inanygiven cell.However, of thepattern subcellular distributionsandmore thanonetypeofPDEis Different PDEisozymescanhavespecifictissue,cellularand and someoftheirselectiveinhibitors. specificity, tissuelocalization,theirbasicmodeofregulation referred toasPDE1-PDE11,togetherwiththeirsubstrate hydrolyze both.Table 1depictsthe11genefamilies, only cAMP, somehydrolyze onlycGMP, whileothers PDEs wassubstratespecificity. Thus,somePDEshydrolyze first functionaldifference identifiedbetweenthevarious sensitivity toendogenousorpharmacologicalregulators. The on thebasisofsubstratespecificity, kineticproperties and tically, onthebasisofsequencehomology, andfunctionally, elucidated. ThePDEfamiliescanbedifferentiated gene- significance ofthisphenomenonisonlybeginningtobe be deducedfrom theESTdatabase,butphysiological transcript variantshavebeenreported intheliterature orcan splicing.Alargenumberof initiation sitesoralternative expressed inmultipleisoformsduetodifferent transcription GPCR 5'-AMP Phosphorylation Protein PKA PDEs

(continued fromcover)

G

S

Hormone cAMP GEFs

, Neurotrans ,

ic peptides),NO(nitric P orcAMP, respectively. This

G

S

om reference [1].

Na mitter compartmentalization +

, Ca otide exchange ATP 2+ activate Cyclase Adenylyl CNG Channels

Celltransmissions Vol 20, No.1, 2004 Order: 1-800-325-3010 Technical Service: 1-800-325-5832 sigma-aldrich.com/cellsignaling

¨ Cyclic Nucleotide 3 ¨ Phosphodiesterases 4 ¨ Cyclic Nucleotide ¨ Phosphodiesterases

Celltransmissions Vol 20, No.1, 2004 Order: 1-800-325-3010 Technical Service: 1-800-325-5832 sigma-aldrich.com/cellsignaling cGMP-gated cation channelopeningthattransmit thevisual late rapidoscillations inlight-inducedcGMPand thuscontrol best exampleofthisisthephotoreceptor PDEsthatmodu- but alsoitsdurationandcompartmentalization. Perhapsthe ulating notonlytheamplitude ofacyclicnucleotidesignal, and locallyhasemphasizedthe importanceofPDEsformod- that, inmanycells,cyclicnucleotide levelsoscillaterapidly utilize cAMPorcGMPassecond messengers.Therealization these steady-statelevelsfollowingstimulatoryeventsthat control theresting levelofcyclicnucleotidesandtorestore It isassumedthatthemajorfunctionofPDEsincellsto Function andRegulationofPDEs Inhibitors ofCyclicNucleotidePhosphodiesterases... when usingtheseinhibitorsinintactcells.NotethatIBMXinhibitsallPDEswith theexceptionofPDE8andPDE9,although the nextnearest familymember. However, theseinhibitorsare notabsolutelyselectiveandthecaveatsmentionedintextsh inhibitors listed,exceptdipyrimadoleandzaprinast,exhibitsubstantialselectivity forthePDEfamilylisted;inmostcases brackets). Differences ininhibitorsensitivitymayoccurbetweendifferent membersofeachPDEfamilyandbetweentranscriptv *IC 1-50 µMdependingontheisozymeandspeciesexamined. Table 1Biochemicalandpharmacologicalcharacteristicsofcyclicnucleotidephosphodiesterase(PDE)isozymes Gene Family 50 PDE11 PDE10 PDE3 PDE2 PDE1 PDE9 PDE8 PDE7 PDE6 PDE5 PDE4 values (µM)were obtainedfrom PDE6A ( PDE6C ( PDE6B ( PDE11A PDE10A PDE9A PDE8A PDE7A PDE5A PDE4D PDE4C PDE4A PDE3A PDE2A PDE1C PDE1A Genes PDE8B PDE7B PDE4B PDE3B PDE1B β α α ) ) ) tissue, pancreas, Skeletal muscle, Skeletal muscle, smooth muscle, smooth muscle, outer segments Lung, platelets, Adrenal cortex, Heart, adipose photoreceptor Major Tissue Rod andcone olfactory cilia, T-cells, B-cells Many tissues Brain, heart, cavernosum Expression Testis, brain brain, heart Testis, liver, platelets prostate thyroid Kidney corpus in vitro testis determination ofPDEactivityatthesubstrateconcentrationreported inthespecificreferences (shownin cAMP>cGMP cAMP/cGMP cAMP/cGMP cGMP/cAMP cAMP/cGMP Substrate cGMP cGMP cGMP cAMP cAMP cAMP PDE6 Regulators γ Ca Unknown Unknown Unknown Unknown CaMKII and cGMP cGMP cGMP 2+ PKG PKA PKA PKA PKC PKA PKA PKB -CaM δ domains hasbeen obtainedincardiac myocytes [12]aswell sients [11,12].Evidence fortheexistenceofcAMP micro- intensity andspatialprogression ofcyclicnucleotidetran- The combinedactionsofthecyclases andPDEscontrol the tion isanimportantaspectof cyclic nucleotidesignaling. It isbecomingincreasingly evidentthatcompartmentaliza- tide-modulated cellprocesses. gation, sensorialtransductionandmanyothercyclicnucleo- of synaptictransmission,cardiac contractility, plateletaggre- that similarfunctionsare exertedbyPDEsinthemodulation signal viachangesinmembranepotential[2,10].Itislikely subunits 8-Methoxymethyl-IBMX (M2547) Inhibitors (SigmaProd. No.) (D9766) Dipyridamole (D9766) Dipyridamole (D9766) Dipyridamole (D9766) Dipyridamole (D9766) Dipyridamole (D9766) (6383) (C7971) (E1279) (M4659) (2057) Trequinsin (2057) (C0737) (Levitra) (Z0878) Zaprinast (Z0878) Zaprinast (Z0878) Zaprinast (Z0878) Imazodan (I0782) (R6520) (Viagra) YM976 (Y4877) T-1032 (7692) T-0156 (8067) (Cialis) EHNA (E-114) Vardenafil Sildenafil Sildenafil Tadalafil DMPPO (continued) at least20-foldascompared to IC ould benoted,especially ariants ofeachgene.All 50 IC values canvaryfrom 50 in µM*[Reference] 0.82-1.8 [57] 0.0007 [52] 0.0002 [43] 0.64-2 [48] 0.005 [51] 0.001 [44] 0.004 [50] 0.003 [42] 0.011 [52] 0.074 [51] 0.002 [49] 0.0003 [2] 5-28 [57] 9-42 [53] 0.005 [2] 1.10 [56] 0.76 [2] 0.90 [2] 4.5 [54] 0.15 [2] 5.1 [51] 0.38 [2] 0.30 [2] 0.12 [2] 29 [55] 7 [55] 20 [2] 2 [2] 1 [2] 4 [2] 6 [2] 1 [2] of thesegenes[33,35-37]. ing onthecAMP-response elementsfoundinthepromoters and likelyinvolvesPKAactivationoftranscriptionfactorsact- tion hasbeenobservedforPDE3,PDE4andPDE7[32-34] cAMP andadapttochronic activation.Thistypeofregula- mechanism thataugmentstheabilityofacelltocatabolize cAMP levelsseemstobeacommoncompensatoryfeedback unknown. Up-regulation of PDEsfollowinganincrease in and cell-specifictranscriptionregulation isstilllargely transcription andtranslation.Thebasisofthedevelopmental Another mechanismofPDEregulation occursatthelevelof and activationofPDE6bytransducin[10]. activation ofPDE1byCa established mechanismsofregulation ofPDEactivityinclude crosstalk betweencGMPand cAMPpathways[30].Other [26,29]. CyclicGMPwillalsoinhibitPDE3thusestablishinga ing tothecGMP-bindingGAFdomainsoftheseproteins allosteric activationofPDE2andPDE5through cGMPbind- cGMP alsoprovides anegative feedbackcontrol bycausing feedback loopcanberapidlyactivated[27,28].Elevationof of thesamescaffold signalingcomplexwhere thenegative some instances,PKAandPDEhavebeenshowntobepart kinases followinganincrease incyclicnucleotides[23-26].In cGMP-dependent protein kinases(PKA,PKG)orbyother to beactivatedbyphosphorylation,eithercAMP-or about theregulation ofPDEactivity. SomePDEsare known event [21,22].Thisschemealsofitswellwithwhatisknown can beactivatedtoreset basallevelsfollowingastimulatory point tonewsitesofcyclicnucleotidesynthesiswhere PDEs cyclase, distributedinvarioussubcellularcompartments, The existenceofsolubleguanylylcyclaseandadenylyl allow real-time measurements ofcyclicnucleotides[18-20]. PDEs hasbeenconfirmedbytheuseofnewindicatorsthat molecular andphysicalbarrierstodiffusion. Thisrole for wave ofcAMPpropagation incombinationwithintracellular the siteofsynthesis,thusdelimitingspatio-temporal [15-17], wouldberesponsible fordegradationofcAMPnear activated receptors, asseenincardiac myocytesand Tcells as othercelltypes[13,14].Therecruitment ofPDEsto Inhibitors ofCyclicNucleotidePhosphodiesterases... hshdetrs niiosAalbefo Sigma-RBI InhibitorsAvailable from I 5879 I 0782 I 0157 H 9006 F-124 E 1896 E 1279 E-114 D 9766 D-108 D 5385 C 0737 C 7971 C 0750 B 8279 A 1755 3-Isobutyl-1-methylxanthine Imazodan 7-( hydrochloride Enoximone EHNA Dipyridamole 1,3-Dipropyl-7-methylxanthine 1,7-Dimethylxanthine Cilostazol Cilostamide (Ro 20-1724)(PDE4) 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one hydrate β -Hydroxyethyl) (PDE2) (nonselective) (PDE3) (PDE3) (PDE3) (nonselective) (PDE3) (PDE3) 2+ (nonselective) /calmodulin () (nonselective) (nonselective) (SQ20,009) (PDE4) (CaM) binding[31] (IBMX) (nonselective) (nonselective) including sildenafil(Viagra triggered pharmacologically byselectivePDE5inhibitors, penile erection (Figure 2).ThecGMPpathwaycanbe leadingto allows bloodflowintothecorpuscavernosum in vascularsmoothmusclecells.Thiscausesrelaxation and increasesof nitricoxidethatinturn intracellularcGMPlevels sexual stimulation,penilenerveexcitationcausestherelease ment oferectile dysfunction. Itiswellknownthatupon specific expression ofPDE5 hasbeenexploitedforthetreat- cell-specific .Forexample,therelative smoothmuscle- them desirablemoleculartargetsforthedevelopmentof vention, thedifferential tissuedistributionofPDEsmakes signaling. From thepointof viewofpharmacologicalinter- specificity andcompartmentalizationofcyclicnucleotide ofPDEscontributestothe cellular distributionpatterns As discussedearlier, thecomplexityassociatedwith PDE Inhibitors genesis ofatherosclerosis, animportantriskfactorin stroke. expressed inmanycellsthatare importantforthepatho- stroke isstillnotclear, butitisknownthatPDE4D mechanism ofthisdisregulation ofPDE4Dexpression for detected inaffected individuals.Theprecise pathogenic reduced expression ofsomePDE4Dvariantshasbeen cardiogenic andcarotid ischemicstroke [40].Inparticular, as themostlikelycandidategeneforsusceptibilityto ness [38,39].Recently, thePDE4Dgenehasbeenidentified hereditary retinitis pigmentosaandstationarynightblind- units ofPDE6intheretina are thebasisofsomeforms of ed conditionsinhumans.Forexample,mutationssub- Mutations inPDEshavebeenfoundtocausecertaininherit- T-0156 are availableasresearch tools,specificallyDMPPO[42], potent selectivePDE5inhibitorshavebeendevelopedand (Levitra smooth muscle,cautionstheiruse[45-47]. ence ofPDE5inothercelltypes,includinglungplateletsand clinical applicationsfortheabovedrugs.However, thepres- PDE5 inhibitorsare beingdescribed thatcouldleadtoother expressed invariousother celltypessuchthatneweffects of T 7692 Z 3003 Z 0878 Y 4877 V 6383 T 2057 T 1633 T 4500 T 7692 T 8067 R 6520 Q 3504 P 9689 P 1784 P 3510 M 4659 M 2547 ® ) [44].ItisnowrecognizedthatPDE5also (Prod. No. ), thatprevent thebreakdown ofcGMP[41].Other Zaprinast YM976 Vinpocetine Trequinsin hydochloride Theophylline T-1032 T-0156 Rolipram hydrochloride Milrinone (8-Methoxymethyl-IBMX) (PDE1) 8-Methoxymethyl-3-isobutyl-1-methylxanthine (PDE5) (PDE5) (PDE4) T 8067 (PDE4) (continued) (nonselective) (PDE3) (PDE3) (PDE 3/4) (PDE1) (PDE5) (PDE5) (nonselective) (nonselective) ) [43]and ® ), tadalafil(Cialis (PDE3) (nonselective) T-1032 (Prod. No. ® ) andvardanafil

Celltransmissions Vol 20, No.1, 2004 Order: 1-800-325-3010 Technical Service: 1-800-325-5832 sigma-aldrich.com/cellsignaling

¨ Cyclic Nucleotide 5 ¨ Phosphodiesterases 6 ¨ Cyclic Nucleotide ¨ Phosphodiesterases

Celltransmissions Vol 20, No.1, 2004 Order: 1-800-325-3010 Technical Service: 1-800-325-5832 sigma-aldrich.com/cellsignaling icant effect onthephysiological proccess beingstudied. profile asasmallinhibition ofPDEactivitymayhaveasignif- inhibitor effect type beingstudied.Theconcentrationdependenceofan and subcellularlocalizationofthePDEsinspecificcell actual intracellularconcentrationofinhibitorandtheprofile issue ofdifferential cellpermeability, theuncertaintyof take intoconsiderationanumberoffactorsincludingthe The evaluationofPDEinhibitors script variantswithinaPDEfamilyhaveyettobedeveloped. variants [53,60].Specificdrugsfordifferent genesandtran- bers ofeachPDEfamily[53,58,59]andbetweentranscript inhibitor sensitivitycanbeobservedbetweendifferent mem- when takingthiscompound.Moreover, differences in ing thevisualdisturbancesexperiencedbysomepatients trations, itcanalsoinhibitPDE6(IC affinity forPDE5A(IC es theirselectivity. Forexample,sildenafilpossessesahigh competitors, acharacteristicthattosomeextentcompromis- methylxanthines orisoquinolinederivativesandare substrate vitro (i.e. theyexhibitsomedegree ofspecificitywhentestedin more thanonefamily)whileothersare relatively selective inhibitors, someofwhichare non-selective(i.e.theyinhibit discovered PDEfamilies7to11.Table 1listsavarietyofPDE many PDEfamiliesand,inparticular, forthemore recently isoforms. Truly selectivecompoundsare stillnotavailablefor pounds thatdisplayonlypartialselectivityforspecificPDE heavily onalimitednumberofcommercially availablecom- Research onthefunctionofspecificPDEshassofarrelied Inhibitors ofCyclicNucleotidePhosphodiesterases... relaxation bymechanismsthatare stillbeingdefined,butwhichincludeareduction incytosolicCa (sGC), whichresults intheproduction ofcyclicGMP(cGMP)andtheactivationcGMP-dependent (PKG). PKGcaus phosphorylation andcontraction.Thenitric oxide(NO)pathwayleadstorelaxation ofsmoothmusclebystimulatingthesoluble inositol trisphosphate(InsP Figure 2.Mechanismofactionthedrugssildenafil(Viagra ized inerectile tissue,thereby prolonging andenhancingthe effects ofNOandcGMP onbloodflow. Ca myosin lightchains (by activationofMLCphosphataseand/or bysequestrationofMLCKinaphosphorylated formthatisnotread is regulated bycellularCa Penile erection aneffect occurswhenbloodswellsthecorpuscavernosum, facilitatedbyrelaxation ofregional smooth muscle. PDE activityassays).Mostoftheavailableinhibitorsare 2+ /CaM). Sildenafil(Viagra in vivo 50 might alsodiffer from the 10 nM).However, athigherconcen- 2+ 3 ; , whichactivatestheCa 5'-GMP ® inositol 1,4,5-trisphosphate Locally producedNO ), tadalafil(Cialis Levitra Cialis Viagra ® in vivo ® ® PDE5 50 50 nM),likelyexplain- Smooth musclecell or cGMP ® sGC PKG ex vivo ) andvardenafil (Levitra 2+ /calmodulin (CaM)-dependentenzyme myosin lightchainkinase(MLCK),whichleadstoMLC in vitro has to ; Prod.No. MLC ® ), tadalafil(Cialis IP in I 3 R ® 7012 ) specificallyinhibit the breakdown ofcellular cGMP byPDE5thatislocal- Ca Ca Ca 2+ 2+ ) recepto-mediatedCa MLC 2+ (Prod. No. extract orapurifiedenzymeassay. Forexample, complexity ofthe ence foraninhibitor Moreover, adisparitybetweentheconcentrationdepend- Prod. No. selective inhibitor structures ofPDE4DandPDE5A incomplexwiththenon- ture oftheirregulatory domains.Thedefinitionofthecrystal catalytic domain,theirmechanismofcatalysisandthestruc- advantage oftherecent clarification ofthestructure oftheir The developmentofdrugsthattargetPDEswillclearlytake ous cellprocesses tobemore effectively investigated[63,64]. in vivo rolipram PDE4 inhibitors,suchas compromising theirspecificity. Conversely, variousPDE3and been limitedbytherequirement tousehighdoses,thus use toaccessthefunctionoftheseenzymesincellshas and described [58]including type. PotentandpartiallyselectivePDE1inhibitorshavebeen differential expression ofPDEsbyaspecifictissueorcell the functionofeitherPDE2orPDE3,dependingon Therefore, trequinsin canbeusedtoobtaininformationon it isactuallymuchmore potentatinhibitingPDE3[61,62]. act assuch[48].However, and, whentested scoring thelackofselectivitythisinhibitor[7].More ing residues are conservedbetweenPDE4andPDE5,under- mobilization Contraction export 8-methoxymethyl-IBMX ® studies allowingtherole of theseenzymesinnumer- ) andvardenafil (Levitra (Prod. No. I 5879 T 2057 (non-activatable) (inactive) MLCK MLCK Ca 2+ ) showsthatthemajorityofIBMXbind- 2+ ) hasbeendescribedasaPDE2inhibitor 3-isobutyl-1-methylxanthine 2+ nvivo in in vivo (by enhancedCa (continued)

mobilization) anddephosphorylationof P R 6520 nvitro in Ca CaM 2+ milrinone vinpocetine entry MLC situation versusabroken cell in sometissues,hasbeenshownto in vitro (active) MLCK ) ® and P have beenmoreamenableto ). (Prod. No. 2+ in vivo export and/orbyreduced and inmanyothertissues, (Prod. No. (Prod. No. Smooth muscletone guanylyl cyclase can result from the ily activatedby es smooth-muscle M 2547 M 4659 trequinsin V 6383 (IBMX; ), buttheir ) and ) Figure 3.Structures ofselectedphosphodiesteraseinhibitors. http://clinicaltrials.gov). sis, asthmaandchronic obstructivepulmonarydisease(see evaluated asanti-inflammatorydrugstotreat multiplesclero- inhibitors rolipram, andcilomilastare alsobeing and stimulation wasabolished[65].Thisphenotypereinforces the bronchoconstriction response tomuscariniccholinergic an asthmamodel(bymeasuringairwayresistance), the When thephenotypeofPDE4Dnullmousewastestedin potential isillustratedbythefirstPDE4knockoutstudies. provide therationalefordeveloping newtherapies.This and pathophysiologicalprocesses. Suchstudiesshouldalso for understandingtheroles ofindividualPDEsinphysiological which are justbeginningto becomeavailable,willbecrucial It islikelythatthestudyofspecificPDEknockoutmodels, PDE KnockoutModels stroke, andthePDE3inhibitors madole trials forthetreatment ofvariousconditionsincluding pounds are currently approved orbeenevaluatedinclinical pharmacological potentialofPDEinhibition,severalcom- clearly needtobedevelopedinorder tofullyexplore the ing affinity andselectivity[8].Whilemore selectivedrugs will ent inhibitorshassuggested tivity. Similarly, comparison ofPDE5Acomplexedwithdiffer- residues makingcontact,mayplayarole ininhibitor selec- bility oftheloops,aswellchemicalnature ofthe ing oftheinhibitorsuggestingthatconformationalflexi- involved intheformationofactivesiteandbind- interestingly, thesemodelsshowthatloopregions are also Inhibitors ofCyclicNucleotidePhosphodiesterases... milrinone HOCH HOCH N (Aggrenox; Pod.No. 2 N CH CH N 2 (Prod. No.T8067) 2 3 CH (Prod. O O N CH (Prod. No. 2 Dipyridamole N T-0156 3 O O

N N No. D N OCH COOCH N N N

9766) N M 4659 3 CH CH N modifications toimprove bind- 3 HCl 3 D 9766 CH enoximone H 2 CH 3 C 2 CH 2 ) inheartfailure.ThePDE4 O H 2 N O ) forthepreventionof H N O Vardenafil (Prod. No. S (Prod. No.Z0878) O H Zaprinast 3 H OCH C O N See Table 1forisozymeselectivities. E 1279 O H N N dipyri- O N CH 2 CH N 3 H N N N 2 O CH CH N CH Sildenafil ) N S 3 3 3 O ed by role forthisisoforminthe innateimmuneresponse mediat- Similarly, targetingthePDE4Bgenehasrevealed anessential tion isnotalsoresponsible forthesideeffects ofthis [68]. effects associatedwithrolipram, provided thatPDE4Dinhibi- may possesstherapeuticpotentialthatisdevoidoftheside [67]. Itseemspossible,therefore, thatPDE4D-specific drugs likely mediatestheantidepressant-like effects ofrolipram knockout micehaverevealed thatthisspecificvariantmost Behavioral andpharmacologicaltestsperformedonPDE4D promising newtargetsinthetreatment ofdepression [66]. ation, PDEinhibitionisconsidered tobeoneofthemore this agenthavelimiteditsusetodate.However, inthissitu- tive antidepressant, althoughtheemetogenicside-effects of PDE4-familyinhibitor, rolipram, isknowntobeaneffec- The pulmonary disease. contraction, suchasasthmaandchronic obstructive treatment ofairwaydiseasesassociatedwithsmoothmuscle pharmacological potentialofPDE4D-specificinhibitorsinthe isoform tailored drugdevelopment. Itisexpectedthata different conditions,perhapsopeninganeweraforPDE- also pointtospecificvariantsofPDE4asdrugtargetsfor on theinvolvementofPDE4invariousdiseasestates,they While thesestudiesconfirmprevious pharmacologicaldata rheumatoid arthritis,Crohn's diseaseandsepticshock. for thetreatment ofTNF- PDE4B mayrepresent afavorablepharmacologicalstrategy T 5944,6674 HN O O N tumor necrosisfactor CH CH 3 (Prod. (Prod. No.I5879) N 3 H H CH O O 3 3 N CH O C C Rolipram 3 No. CH N IBMX 3 ) [69].Therefoe,selectiveinhibitorsof O N 3

R

6520) (continued) H N N N H α O -mediated diseasessuchas N α (TNF- O CH 3 (Prod. O α; H N CH O Tadalafil Prod. Nos. 3 T-1032 O O No. O N

H N T OCH COOCH

7692) O O N 3 T 7539, CH H 3 NH 2 3 SO 2 4

Celltransmissions Vol 20, No.1, 2004 Order: 1-800-325-3010 Technical Service: 1-800-325-5832 sigma-aldrich.com/cellsignaling

¨ Cyclic Nucleotide 7 ¨ Phosphodiesterases 8 ¨ Cyclic Nucleotide ¨ Phosphodiesterases

Celltransmissions Vol 20, No.1, 2004 Order: 1-800-325-3010 Technical Service: 1-800-325-5832 sigma-aldrich.com/cellsignaling 13. Cooper, D.M., 12. Brunton, L.L., 1 Tasken, K.andAandahl,E.M., 11. 10. Baylor, Patel,S.B.,etal., D., 9. Sung,B.J.,etal., 8. Huai,Q.,etal., 7. . Beavo,J.A.andBrunton,L.L., 1. References physiological processes. PDEs ofspecificcelltypesbelievedtobeinvolvedinpatho- ment ofmore potentand selective drugstotargetthemajor PDE catalyticandallostericsitesshouldpermitthedevelop- substrate competitive,betterstructuralknowledgeofthe so farare basedonthecyclic nucleotidestructure andare ment oftargeteddrugs.Whilemostthedrugsdeveloped addition, thesenewapproaches willhelpguidethedevelop- which noPDEinhibitoriscurrently commercially available.In function ofindividualPDEs,especiallyforthoseisoforms interference, willprovide toolswithwhichtodissectthe These transgenicmodelsystems,andpossiblyRNA specific conditionalknockoutanimalsbecomeavailable. played bydifferent PDEisozymes,particularlywhentissue- knockout micewillleadtoanewunderstandingoftherole likely, therefore, thatfunctionaltestingofspecificPDE part tothelackofavailabilityselectiveinhibitors.Itseems although theirspecificfunctionsare oftenstillunclearduein Several newPDEshavebeendiscovered inthepast10years, Conclusions ing locomotorhyperactivity[72]. whilealsoexhibit- out miceshowadefectinspatiallearning of PDE3Binmodulatinginsulinaction.Finally, PDE1Bknock- in theregulation ofglucosemetabolismandconfirmtherole observations suggestedanewphysiologicalrole forPDE7A mice possessdefectsinglucosehomeostasis[70,71].These metabolic testsshowedthatPDE7AandPDE3Bknockout can bethelimitingstep.Forexample,onlysophisticated experimental approach thatwillreveal analtered phenotype is thecaseinmanyknockoutmodels,determining simply duetotheintrinsicspecificrole ofthatPDE.Also,as phenotypes, possiblyduetocompensationbyotherPDEsor out modelsinvestigatedtodatehavegeneratedmore subtle have yettobefunctionallycharacterized.OtherPDEknock- similar approach willprovide cluestotherole ofPDEsthat . Xu,R.X.,etal., 6. .Huai,Q.,etal., 5. Mehats,C.,etal., 4. Soderling,S.H.andBeavo,J.A., 3. 2. Beavo, J.A., diesterases. work focusesonthefunctionalcharacterization ofnovelphospho- Pharmacology attheUniversityof Washington, Seattle.Hercurrent position asaresearcher withDr. JoeBeavo intheDepartmentof lation ofphosphodiesterase3Bactivity, shetookupher current Maryland, withDr. Manganiellowhere Vince shestudiedtheregu- a visitingscientistattheNationalInstitutesofHealth,Bethesda, University ofFlorence inItaly. Followingaperiod spentworkingas Valeria Vasta Inhibitors ofCyclicNucleotidePhosphodiesterases... About theAuthors Proc. Natl.Acad.Sci.USA, Physiol. Rev., received herPh.D.inBiologicalSciencesfrom the Sci STKE Biochem. J. Science J. Biol.Chem., Biochemistry Nature, Acta Crystallogr. D. Biol.Crystallogr. Trends Endocrinol.Metab 2003, PE44(2003). 288 75 425 , 375 , 1822-1825(2000). , 725-748(1995). , , 98-102(2003). 42 npes(2004). in press , 517-529(2003). Nat. Rev. Mol.CellBiol Physiol. Rev., , 13220-13226(2003). Curr. Opin.CellBiol., 93 , 560-565(1996). ., 84 13 , 137-167(2004). , 29-35(2002). , ., 60 12 3 , 169-171(2004). , 710-718(2002). , 174-179(2000). 3 Hetman,J.M.,etal., 53. SaenzdeTejada, I.,etal., 52. Daugan,A.,etal., 51. Boolell,M.,etal., 50. Aoki,M.,etal., 49. Whalin,M.E.,etal., 48. 5 Soderling,S.H.,etal., 55. Soderling,S.H.,etal., 54. 7 Kruse,C.,etal., 47. Manganiello,V., 46. Raffelmann, T. andKloner, R.A., 45. 6 Soderling,S.H.,etal., 56. 4 Conti,M.,etal., 64. Manganiello,V.C. andDegerman,E., 63. Haynes,J.Jr., etal., 62. Ruppert,D.andWeithmann, K.U., 61. Bolger, G.E.,etal., 60. Wang, P., etal., 59. 0 Gretarsdottir, S.,etal., 40. Gal,A.,etal., 39. Dryja,T.P., etal., 38. Torras-Llort, M.andAzorin,F., 37. 4 Ukita,T., etal., 44. 7 Hetman,J.M.,etal., 57. 8 Yan, C.,etal., 58. 5 Mehats,C.,etal., 65. 7 Zhang,H.T., etal., 67. Holden,C., 66. 9 Jin,S.L.andConti,M., 69. 7 Tasken, K.A.,etal., 27. Rybalkin,S.D.,etal., 26. 25. Murthy, K.S., Houslay, M.D.andAdams,D.R., 24. Smith,C.J.,etal., 23. Zippin,J.H.,etal., 22. Xie,F. andConti,M., 21. Zaccolo,M.,etal., 20. Honda,A.,etal., 19. 8 Dodge,K.L.,etal., 28. 6 LeJeune,I.R.,etal., 36. 3 Ukita,T., etal., 43. Coste,H.andGrondin,P., 42. Francis,S.H.andCorbin,J.D., 41. 8 Robichaud,A.,etal., 68. 2Reed,T.M., etal., 72 Choi,Y.H., et.al., 71. Michaeli,T, et.al., 70. 8 Rich,T.C., etal., 18. 5 Niiya,T., etal., 35. Lee,R.,etal., 34. Sonnenburg,W.K., etal., 31. Maurice,D.H.,etal., 30. Martinez,S.E.,etal., 29. 7 Baillie,G.S.,etal., 17. Arp,J.,etal., 16. Bolger, G.B.,etal., 15. Karpen,J.W. andRich,T.C., 14. 3 D'Sa,C.,etal., 33. Seybold,J.,etal., 32. characterization of newphosphodiesterases. Seattle where hisresearch interests includetheidentification and Department ofPharmacology attheUniversityofWashington in dependent protein kinase.Heiscurrently aProfessor inthe worked ontheisolationandcharacterization ofcyclicAMP- Edwin Krebs atDaviswhere attheUniversityofCalifornia he diesterases. Hethenmovedtoapost-doctoral positionwithDr. studies onthecharacterizationofcyclic nucleotidephospho- cyclic GMP. Duringthistime,hecontributedtomany oftheearly Sutherland andJoelHardman, theco-discoverers ofcyclicAMPand Nashville, Tennessee, duringwhichtimeheworkedwithDrs.Earl Joe Beavo received hisPh.D.from Vanderbilt Universityin Science, Biochem. J. Mol. CellBiol. Cell Signal, Nat. Genet. J. Biol.Chem J. Neurochem. FEBS Lett., J. Med.Chem. Bioorg. Med.Chem.Lett. J. Pharmacol.Exp.Ther. ice.Bohs e.Commun Biochem. Biophys.Res. Brain Proc. Natl.Acad.Sci.USA J. Biol.Chem. Mol. Pharmacol., Invest. Ophthalmol.Vis. Sci. Proc. Natl.Acad.Sci.USA FASEB J., J. Biol.Chem. J. Neurosci FASEB J., J. Biol.Chem. Int. J.Impot.Res. FASEB J., Proc. Natl.Acad.Sci.USA, J. Med.Chem Diabetologia Neuropsychopharmacology Curr. Opin.CellBiol., Biochem. J EMBO J. J. Biol.Chem 302 J. Biol.Chem. Am. J.Physiol., Mol. Pharmacol. J. Biol.Chem. , Mol. Pharmacol EMBO J. Proc. Natl.Acad.Sci.USA Dev. Biol. Proc. Natl.Acad.Sci.USA Proc. Natl.Acad.Sci.USA J. Clin.Invest. J. Biol.Chem. Proc. Natl.Acad.Sci.USA, Proc. Natl.Acad.Sci.USA 126 (continued) Proc. Natl.Acad.Sci.USA Nat. Genet. , 810-813(2003) , 14 , 360 505 J. Biol.Chem. , 241-247(2003). Int. J.Impot.Res 7 Biochem. Pharmacol. , 17 , , 17 , 277-284(2002). , 551(1994). 17 271 ., Science, 23 20 , , 199-208(2001). , ., , A593Part1Suppl(2003). , , 136-140(2001). , , 1831-1841(2003). 81 22 Curr. UrolRep , , 82-84(2003). 22 , 8042-8057(2003). 44 , , 1921-1930(2001). 278 , 328 Biochem. J. 43 ., 265 , 25699-25706(1996). ., 266 273 , 745-757(2002). , 5188-5197(2002). Biochem. J. , , 469-478(2003). Circulation , 2204-2218(2001). 278 63 , 46 , 138Suppl.1(2000). 276 , , , 5492-5496(2003). , , 539-48(1997). 277 261 , , 196-206(2004). Life Sci 35 , 13385-13390(1991). 110 , , 20575-20588(1998). 8 , 1209-12311(2003). ., , 4533-4542(2003). 293 273 , 49230-49238(2003). 39 , 47-52(1996). , 21999-22002(2001). 64 Thomb. Haemost , 131-138(2003). , , 35980-35989(2002). , H487-492(1991). , 14 , 1045-1052(2002). , 711-717(1991). 295 , , 2204-2205(2001). 270 , 15553-15558(1998). , 533-546(2003). , 13 , 160-166(2002). , , ., 98 ., , , 255-260(2000). 98 , 373 ., , , 2341-2345(2003). , 30989-31000(1995). 31 13 108 100 , , 13049-13054(2001). 370 40 4 , 2437-2442(2001). 27 , , , , , 2037-2043(1982). , 457-465(2003). , 282-290(2001). , , 835-843(2003). 99 , 1859-1865(1999). ., 97 97 , 50 95 96 , 239-244(2003). , 940-945(2003). , 587-595(2002). , 1-18(2003). 9 234 , 13260-13265(2002). , 472-476(2000). , 472-476(2000). , 7628-7633(2003). , 1577-1585(1995). , 8991-8996(1998). , 7071-7076(1999). , 320-324(1997). ., 82 , 407-411(1999).