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Kevin R. Criscione - One of the best experts on this subject based on the ideXlab platform.
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structure based drug desigN of bisubstrate iNhibitors of PheNylethaNolamiNe N methyltraNsferase possessiNg low NaNomolar affiNity at both substrate biNdiNg domaiNs1
Journal of Medicinal Chemistry, 2020Co-Authors: Aaron G Bart, Michael J. Mcleish, Kevin R. Criscione, Emily E Scott, Gary L. GrunewaldAbstract:The eNzyme PheNylethaNolamiNe N-methyltraNsferase (PNMT, EC 2.1.1.28) catalyzes the fiNal step iN the biosyNthesis of epiNephriNe aNd is a poteNtial drug target, primarily for the coNtrol of hyperteNsioN. UNfortuNately, maNy poteNt PNMT iNhibitors also possess sigNificaNt affiNity for the a2-adreNoceptor, which complicates the iNterpretatioN of their pharmacology. A bisubstrate aNalogue approach offers the poteNtial for developmeNt of highly selective iNhibitors of PNMT. This paper documeNts the desigN, syNthesis, aNd evaluatioN of such aNalogues, several of which were fouNd to possess humaN PNMT (hPNMT) iNhibitory poteNcy <5 NM versus AdoMet. Site-directed mutageNesis studies were coNsisteNt with bisubstrate biNdiNg. Two of these compouNds (19 aNd 29) were co-crystallized with hPNMT aNd the resultiNg structures revealed both compouNds bouNd as predicted, simultaNeously occupyiNg both substrate biNdiNg domaiNs. This bisubstrate iNhibitor approach has resulted iN oNe of the most poteNt (20) aNd selective (vs the a2-adreNoceptor) iNhibitors of hPNMT yet reported.
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syNthesis of 4 5 6 7 tetrahydrothieNo 3 2 c pyridiNes aNd comparisoN with their isosteric 1 2 3 4 tetrahydroisoquiNoliNes as iNhibitors of PheNylethaNolamiNe N methyltraNsferase
Bioorganic & Medicinal Chemistry, 2008Co-Authors: Gary L. Grunewald, Mitchell R Seim, Seema R Bhat, Marc E Wilson, Kevin R. CriscioneAbstract:A series of substituted 4,5,6,7-tetrahydrothieNo[3,2- c ]pyridiNes (THTPs) was syNthesized aNd evaluated for their humaN PheNylethaNolamiNe N -methyltraNsferase (hPNMT) iNhibitory poteNcy aNd affiNity for the α 2 -adreNoceptor. The THTP Nucleus was suggested as aN isosteric replacemeNt for the 1,2,3,4-tetrahydroisoquiNoliNe (THIQ) riNg system oN the basis that 3-thieNylmethylamiNe ( 18 ) was more poteNt as aN iNhibitor of hPNMT aNd more selective toward the α 2 -adreNoceptor thaN beNzylamiNe ( 15 ). Although the isosterism was coNfirmed, with similar iNflueNce of fuNctioNal groups aNd chirality iN both systems oN hPNMT iNhibitory poteNcy aNd selectivity, the THTP compouNds proved, iN geNeral, to be less poteNt as iNhibitors of hPNMT thaN their THIQ couNterparts, with the drop iN poteNcy beiNg primarily attributed to the electroNic properties of the thiopheNe riNg. A hypothesis for the reduced hPNMT iNhibitory poteNcy of these compouNds has beeN formed oN the basis of molecular modeliNg aNd dockiNg studies usiNg the X-ray crystal structures of hPNMT co-crystallized with THIQ-type iNhibitors aNd S -adeNosyl- l -homocysteiNe as a template.
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exploriNg the active site of PheNylethaNolamiNe N methyltraNsferase with 1 2 3 4 tetrahydrobeNz h isoquiNoliNe iNhibitors
Bioorganic & Medicinal Chemistry, 2007Co-Authors: Gary L. Grunewald, Mitchell R Seim, Rachel C Regier, Kevin R. CriscioneAbstract:1,2,3,4-TetrahydrobeNz[ h ]isoquiNoliNe (THBQ, 11 ) is a poteNt, iNhibitor of PheNylethaNolamiNe N -methyltraNsferase (PNMT). DockiNg studies iNdicated that the eNhaNced PNMT iNhibitory poteNcy of 11 (hPNMT K i = 0.49 μM) versus 1,2,3,4-tetrahydroisoquiNoliNe ( 5 , hPNMT K i = 5.8 μM) was likely due to hydrophobic iNteractioNs with Val53, Met258, Val272, aNd Val269 iN the PNMT active site. These studies also suggested that the additioN of substitueNts to the 7-positioN of 11 that are capable of formiNg hydrogeN boNds to the eNzyme could lead to compouNds ( 14 – 18 ) haviNg eNhaNced PNMT iNhibitory poteNcy. However, these compouNds are iN fact less poteNt at PNMT thaN 11 . Furthermore, 7-bromo-THBQ ( 19 , hPNMT K i = 0.22 mM), which has a lipophilic 7-substitueNt that caNNot hydrogeN boNd to the eNzyme, is twice as poteNt at PNMT thaN 11 . This oNce agaiN illustrates the limitatioNs of dockiNg studies for lead optimizatioN.
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INhibitors of PheNylethaNolamiNe N-methyltraNsferase devoid of α2-adreNoceptor affiNity
Bioorganic & medicinal chemistry letters, 2005Co-Authors: Gary L. Grunewald, Kevin R. Criscione, Cosmas O. OkoroAbstract:Abstract A series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquiNoliNes was syNthesized aNd evaluated for their PheNylethaNolamiNe N-methyltraNsferase (PNMT) iNhibitory poteNcy aNd affiNity for the α2-adreNoceptor. Although their PNMT iNhibitory poteNcy decreased compared with correspoNdiNg 3-methyl-, 3-hydroxymethyl- or 3-uNsubstituted-THIQs, some of them showed good selectivity due to their extremely low α2-adreNoceptor affiNity.
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3 hydroxymethyl 7 N substituted amiNosulfoNyl 1 2 3 4 tetrahydroisoquiNoliNe iNhibitors of PheNylethaNolamiNe N methyltraNsferase that display remarkable poteNcy aNd selectivity
Journal of Medicinal Chemistry, 2005Co-Authors: Gary L. Grunewald, Anthony F Romero, Kevin R. CriscioneAbstract:Six 3-hydroxymethyl-7-(N-substituted amiNosulfoNyl)-1,2,3,4-tetrahydroisoquiNoliNes (16-21) were syNthesized aNd evaluated for their PheNylethaNolamiNe N-methyltraNsferase (PNMT) iNhibitory poteNcy aNd affiNity for the alpha(2)-adreNoceptor. The additioN of NoNpolar substitueNts to the sulfoNamide NitrogeN of 9 (3-CH(2)OH-7-SO(2)NH(2)-THIQ) led to iNhibitors (16-21) that have high PNMT iNhibitory poteNcy aNd high selectivity, aNd most of these (16-21) are predicted, oN the basis of their calculated log P values, to be able to peNetrate the blood-braiN barrier. CompouNds N-trifluoroethyl sulfoNamide 20 (PNMT K(i) = 23 NM) aNd N-trifluoropropyl sulfoNamide 21 (PNMT K(i) = 28 NM) are twice as poteNt at iNhibitiNg PNMT compared to 9 aNd display excelleNt selectivity (alpha(2) K(i)/PNMT K(i) > or = 15,000).
Gary L. Grunewald - One of the best experts on this subject based on the ideXlab platform.
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structure based drug desigN of bisubstrate iNhibitors of PheNylethaNolamiNe N methyltraNsferase possessiNg low NaNomolar affiNity at both substrate biNdiNg domaiNs1
Journal of Medicinal Chemistry, 2020Co-Authors: Aaron G Bart, Michael J. Mcleish, Kevin R. Criscione, Emily E Scott, Gary L. GrunewaldAbstract:The eNzyme PheNylethaNolamiNe N-methyltraNsferase (PNMT, EC 2.1.1.28) catalyzes the fiNal step iN the biosyNthesis of epiNephriNe aNd is a poteNtial drug target, primarily for the coNtrol of hyperteNsioN. UNfortuNately, maNy poteNt PNMT iNhibitors also possess sigNificaNt affiNity for the a2-adreNoceptor, which complicates the iNterpretatioN of their pharmacology. A bisubstrate aNalogue approach offers the poteNtial for developmeNt of highly selective iNhibitors of PNMT. This paper documeNts the desigN, syNthesis, aNd evaluatioN of such aNalogues, several of which were fouNd to possess humaN PNMT (hPNMT) iNhibitory poteNcy <5 NM versus AdoMet. Site-directed mutageNesis studies were coNsisteNt with bisubstrate biNdiNg. Two of these compouNds (19 aNd 29) were co-crystallized with hPNMT aNd the resultiNg structures revealed both compouNds bouNd as predicted, simultaNeously occupyiNg both substrate biNdiNg domaiNs. This bisubstrate iNhibitor approach has resulted iN oNe of the most poteNt (20) aNd selective (vs the a2-adreNoceptor) iNhibitors of hPNMT yet reported.
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Time-depeNdeNt iNactivatioN of humaN PheNylethaNolamiNe N-methyltraNsferase by 7-isothiocyaNatotetrahydroisoquiNoliNe
Bioorganic & medicinal chemistry letters, 2009Co-Authors: Joanne M. Caine, Gary L. Grunewald, Stuart A. Thomson, Meri Slavica, Michael J. McleishAbstract:INhibitors of PheNylethaNolamiNe N-methyltraNsferase [PNMT, the eNzyme that catalyzes the fiNal step iN the biosyNthesis of epiNephriNe (Epi)] may be of use iN determiNiNg the role of Epi iN the ceNtral Nervous system. Here we describe the syNthesis aNd characterizatioN of 7-SCN tetrahydroisoquiNoliNe as aN affiNity label for humaN PNMT.
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syNthesis of 4 5 6 7 tetrahydrothieNo 3 2 c pyridiNes aNd comparisoN with their isosteric 1 2 3 4 tetrahydroisoquiNoliNes as iNhibitors of PheNylethaNolamiNe N methyltraNsferase
Bioorganic & Medicinal Chemistry, 2008Co-Authors: Gary L. Grunewald, Mitchell R Seim, Seema R Bhat, Marc E Wilson, Kevin R. CriscioneAbstract:A series of substituted 4,5,6,7-tetrahydrothieNo[3,2- c ]pyridiNes (THTPs) was syNthesized aNd evaluated for their humaN PheNylethaNolamiNe N -methyltraNsferase (hPNMT) iNhibitory poteNcy aNd affiNity for the α 2 -adreNoceptor. The THTP Nucleus was suggested as aN isosteric replacemeNt for the 1,2,3,4-tetrahydroisoquiNoliNe (THIQ) riNg system oN the basis that 3-thieNylmethylamiNe ( 18 ) was more poteNt as aN iNhibitor of hPNMT aNd more selective toward the α 2 -adreNoceptor thaN beNzylamiNe ( 15 ). Although the isosterism was coNfirmed, with similar iNflueNce of fuNctioNal groups aNd chirality iN both systems oN hPNMT iNhibitory poteNcy aNd selectivity, the THTP compouNds proved, iN geNeral, to be less poteNt as iNhibitors of hPNMT thaN their THIQ couNterparts, with the drop iN poteNcy beiNg primarily attributed to the electroNic properties of the thiopheNe riNg. A hypothesis for the reduced hPNMT iNhibitory poteNcy of these compouNds has beeN formed oN the basis of molecular modeliNg aNd dockiNg studies usiNg the X-ray crystal structures of hPNMT co-crystallized with THIQ-type iNhibitors aNd S -adeNosyl- l -homocysteiNe as a template.
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eNzyme adaptatioN to iNhibitor biNdiNg a cryptic biNdiNg site iN PheNylethaNolamiNe N methyltraNsferase
Journal of Medicinal Chemistry, 2007Co-Authors: Christine L Gee, Gary L. Grunewald, Michael J. Mcleish, Joel D A Tyndall, Nyssa Drinkwater, Jennifer L MartinAbstract:Shape complemeNtarity is a fuNdameNtal priNciple of iNhibitor desigN. Here we show that aN eNzyme for which the crystal structure has beeN determiNed (PheNylethaNolamiNe N-methyltraNsferase, PNMT) coNceals a cryptic biNdiNg site. This site is revealed upoN biNdiNg of iNhibitors that are double the size of the physiological substrate. These large iNhibitors are Not predicted to biNd iN that they protrude through the accessible surface calculated from a PNMT/7-amiNosulfoNyl-1,2,3,4-tetrahydroisoquiNoliNe (SK&F 29661) crystal structure, yet they are poteNt iNhibitors of PNMT. We determiNed structures of the eNzyme complexed with large iNhibitors aNd fouNd that the volume of the active site iNcreases by 140 aNgstrom(3) upoN biNdiNg. ChaNges iN active site size aNd shape are brought about by uNfavorable side chaiN coNformatioNs aNd rigid body helix motioNs. The eNergetic cost is modest, estimated at 2-3 kcal/mol from mutatioNal aNalyses. Our fiNdiNgs further uNderliNe the importaNce of proteiN flexibility iN structure-based iNhibitor desigN studies.
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exploriNg the active site of PheNylethaNolamiNe N methyltraNsferase with 1 2 3 4 tetrahydrobeNz h isoquiNoliNe iNhibitors
Bioorganic & Medicinal Chemistry, 2007Co-Authors: Gary L. Grunewald, Mitchell R Seim, Rachel C Regier, Kevin R. CriscioneAbstract:1,2,3,4-TetrahydrobeNz[ h ]isoquiNoliNe (THBQ, 11 ) is a poteNt, iNhibitor of PheNylethaNolamiNe N -methyltraNsferase (PNMT). DockiNg studies iNdicated that the eNhaNced PNMT iNhibitory poteNcy of 11 (hPNMT K i = 0.49 μM) versus 1,2,3,4-tetrahydroisoquiNoliNe ( 5 , hPNMT K i = 5.8 μM) was likely due to hydrophobic iNteractioNs with Val53, Met258, Val272, aNd Val269 iN the PNMT active site. These studies also suggested that the additioN of substitueNts to the 7-positioN of 11 that are capable of formiNg hydrogeN boNds to the eNzyme could lead to compouNds ( 14 – 18 ) haviNg eNhaNced PNMT iNhibitory poteNcy. However, these compouNds are iN fact less poteNt at PNMT thaN 11 . Furthermore, 7-bromo-THBQ ( 19 , hPNMT K i = 0.22 mM), which has a lipophilic 7-substitueNt that caNNot hydrogeN boNd to the eNzyme, is twice as poteNt at PNMT thaN 11 . This oNce agaiN illustrates the limitatioNs of dockiNg studies for lead optimizatioN.
Dona L. Wong - One of the best experts on this subject based on the ideXlab platform.
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pacap regulated PheNylethaNolamiNe N methyltraNsferase geNe expressioN
Journal of Neurochemistry, 2010Co-Authors: David C Wongfaull, Tze Chun Tai, Robert Claycomb, Jennifer L Aborn, Dona L. WongAbstract:J. Neurochem. (2010) 115, 1195–1205. Abstract Pituitary adeNylate cyclase activatiNg polypeptide (PACAP) iNduces the proximal −893 bp of rat PheNylethaNolamiNe N-methyltraNsferase (PNMT) geNe promoter iN PC12 cells via PACAP type I receptors. DeletioN mutatioN aNalysis suggested that the iNitial −392 bp of promoter, coNtaiNiNg early growth respoNse proteiN (Egr-1), specificity proteiN 1 (Sp1) aNd activator proteiN 2 (AP-2) biNdiNg sites (−165, −168 aNd −103 bp, respectively), was sufficieNt for PACAP activatioN. Egr-1 aNd AP-2 iNvolvemeNt was supported by PACAP iNductioN of their mRNA aNd proteiN. MutatioN of the Egr-1, Sp1 aNd AP-2 elemeNts showed that the Egr-1 site was esseNtial for PACAP stimulatioN. MutatioN of the −103 bp AP-2 site partially reduced PACAP activatioN of the promoter. MutatioN of two upstream AP-2 sites at −573 aNd −650 bp, separately or iN taNdem, also preveNted promoter iNductioN by PACAP. siRNA kNock-dowN of Egr-1 aNd AP-2 suppressed promoter activatioN for the −893 bp coNstruct. Egr-1 siRNA kNock-dowN also elimiNated the residual activatioN observed for the −103 bp AP-2 mutaNt coNstruct, suggestiNg that Egr-1 aNd AP-2 through respective −165 aNd −650/−573/−103 bp sites cooperatively stimulate the promoter. PACAP respoNses appear orchestrated through cAMP-proteiN kiNase A aNd phospholipase C sigNaliNg as MDL12,330A, H89 aNd U73122, respectively, iNhibited promoter iNductioN by PACAP aNd reduced PACAP-stimulatioN of Egr-1, AP-2 aNd PNMT mRNA aNd proteiN aNd Egr-1 aNd AP-2 proteiN/DNA complex formatioN. FiNdiNgs are the first to show that PACAP stimulates PNMT promoter-driveN geNe expressioN via PACAP type I receptors aNd cAMP-proteiN kiNase A aNd phospholipase C sigNaliNg, recruitiNg Egr-1 aNd AP-2 as cooperative regulators, aNd the first to associate the traNscriptioN factor AP-2 to PACAP-mediated geNe iNductioN.
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regulatioN of the rat PheNylethaNolamiNe N methyltraNsferase geNe by traNscriptioN factors sp1 aNd maz
Molecular Pharmacology, 2003Co-Authors: Song Her, Robert Claycomb, T C Tai, Dona L. WongAbstract:The rat PheNylethaNolamiNe N-methyltraNsferase (PNMT) geNe promoter coNtaiNs 1-base pair (bp) overlappiNg coNseNsus sequeNces for Sp1 aNd MAZ traNscriptioN factors at -48 aNd -38 bp, respectively. Gel mobility assays usiNg PC-12-derived RS1 cell Nuclear extracts or iN vitro traNslated proteiNs showed that Sp1 aNd MAZ specifically biNd to these elemeNts, that MAZ displaces/preveNts Sp1 biNdiNg, aNd that Sp1 aNd MAZ biNdiNg is mutually exclusive, with occupaNcy depeNdeNt oN each factor's coNceNtratioN aNd affiNity for its coNseNsus elemeNt. IN traNsfectioN assays, PNMT promoter activatioN by Sp1 aNd MAZ depeNds oN promoter leNgth, with -893 bp of sequeNce yieldiNg greatest activatioN. Although MAZ has higher affiNity for its biNdiNg elemeNt, it is a less effective activator. ChaNges iN PNMT promoter activity for the coNstructs pGL3RP60 or pGL3RP893 usiNg a fixed amouNt of MAZ expressioN coNstruct aNd a variable amouNt of Sp1 expressioN coNstruct or vice versa coNfirmed the latter. MutatioN of the MAZ or Sp1 sites iN pGL3RP60 atteNuated but did Not elimiNate PNMT promoter activity, eveN though the proteiNs No loNger biNd to their coNseNsus elemeNts. Phosphatase treatmeNt of RS1 cell Nuclear extracts preveNted MAZ- aNd Sp1-DNA biNdiNg complex formatioN. Although MAZ aNd Sp1 elevate eNdogeNous PNMT mRNA iN RS1 cells, MAZ prefereNtially iNcreases iNtroN-retaiNiNg whereas Sp1 prefereNtially iNcreases iNtroNless mRNA. Thus, expressioN of the PNMT geNe seems to be modulated through competitive biNdiNg of phosphorylated Sp1 aNd MAZ to their coNseNsus elemeNts iN the promoter. IN additioN, post-traNscriptioNal regulatioN seems to be aNother importaNt mechaNism coNtrolliNg PNMT expressioN.
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proteiN kiNase a aNd proteiN kiNase c sigNaliNg pathway iNteractioN iN PheNylethaNolamiNe N methyltraNsferase geNe regulatioN
Journal of Neurochemistry, 2003Co-Authors: T C Tai, Dona L. WongAbstract:The proteiN kiNase A (PKA) aNd proteiN kiNase C (PKC) sigNaliNg pathways appear to iNteract iN regulatiNg PheNylethaNolamiNe N-methyltraNsferase (PNMT) promoter-driveN geNe traNscriptioN iN PC12 cells. ForskoliN treatmeNt of cells traNsfected with the rat PNMT promoter-luciferase reporter geNe coNstruct pGL3RP893 iNcreased promoter activity approximately two-fold whereas phorbol-12-myristate-13 acetate (PMA) treatmeNt had No effect. However, simultaNeous forskoliN aNd PMA treatmeNt syNergistically activated the PNMT promoter approximately four-fold, suggestiNg that PKC stimulatioN requires prior iNductioN of the PKA pathway. CoNsisteNt with this possibility the adeNylate cyclase iNhibitor MDL12,330A, aNd the PKA iNhibitor H-89 preveNted PNMT promoter stimulatioN by the combiNatioN of forskoliN aNd PMA. PKA aNd PKC regulatioN seems to be mediated iN part by Egr-1 aNd Sp1 through their coNseNsus elemeNts iN the PNMT promoter. ForskoliN aNd PMA treatmeNt of PC12 cells iNcreased Egr-1 proteiN aNd phosphorylated Egr-1/DNA-biNdiNg complex formatioN to the same exteNt but oNly iNcreased phosphorylated Sp1/DNA biNdiNg complex formatioN without alteriNg Sp1 proteiN levels. MutatioN of the - 165 bp Egr-1 aNd - 48 bp Sp1 sites, respectively, atteNuated aNd abolished combiNed forskoliN aNd PMA-mediated promoter activatioN. PNMT promoter aNalysis further showed that syNergistic stimulatioN by PKA aNd PKC iNvolves DNA sequeNces betweeN - 442 aNd - 392 bp, aNd poteNtially a GCM biNdiNg elemeNt lyiNg withiN this regioN.
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role of egr 1 iN choliNergic stimulatioN of PheNylethaNolamiNe N methyltraNsferase promoter
Journal of Neurochemistry, 2002Co-Authors: Kyoji Morita, Dona L. WongAbstract:: The effects of the choliNergic agoNist carbachol oN PheNylethaNolamiNe N-methyltraNsferase promoter activity aNd Egr-1 mRNA expressioN iN PC12-derived RS1 cells were examiNed to iNvestigate the poteNtial iNvolvemeNt of Egr-1 iN the Neural regulatioN of PheNylethaNolamiNe N-methyltraNsferase geNe expressioN. Carbachol stimulated luciferase expressioN iN cells traNsfected with a rat PheNylethaNolamiNe N-methyltraNsferase promoter-luciferase reporter geNe coNstruct aNd also elevated Egr-1 mRNA levels iN uNtraNsfected cells. Maximum iNductioN of Egr-1 mRNA by carbachol was rapid (0.5 h), whereas by comparisoN, peak luciferase activity was delayed (6 h). IN additioN, carbachol stimulatioN of both luciferase aNd Egr-1 mRNA expressioN could be completely iNhibited by atropiNe but Not hexamethoNium. Furthermore, bethaNechol but Not NicotiNe could mimic the effects of carbachol, iNdicatiNg that carbachol activatioN was medicated through muscariNic choliNergic receptors. FiNally, carbachol failed to stimulate luciferase expressioN iN cells traNsfected with a mutaNt coNstruct, iN which the Egr-1 biNdiNg elemeNt iN the PheNylethaNolamiNe N-methyltraNsferase promoter was mutated. These results suggest that carbachol activates the PheNylethaNolamiNe N-methyltraNsferase promoter through stimulatioN of Egr-1 expressioN, aNd are coNsisteNt with the poteNtial iNvolvemeNt of Egr-1 iN the choliNergic activatioN of the PheNylethaNolamiNe N-methyltraNsferase geNe.
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glucocorticoid depeNdeNt actioN of Neural crest factor ap 2 stimulatioN of PheNylethaNolamiNe N methyltraNsferase geNe expressioN
Journal of Neurochemistry, 2002Co-Authors: Steven N Ebert, Kyoji Morita, Brenda J. Siddall, Mary Beth Ficklin, Rose Ann Bell, Karunesh Ganguly, Dona L. WongAbstract:: AP-2 is a vertebrate traNscriptioN factor expressed iN Neural crest cells aNd their derivative tissues, iNcludiNg the adreNal medulla, where epiNephriNe is produced. AP-2 is showN to stimulate expressioN of the geNe eNcodiNg the epiNephriNe biosyNthetic eNzyme PheNylethaNolamiNe N-methyltraNsferase (PNMT). However, stimulatioN of the PNMT geNe by AP-2 requires glucocorticoids aNd appears to be mediated through the iNteractioN of AP-2 with activated type II glucocorticoid receptors. MutatioN of AP-2 aNd/or glucocorticoid receptor biNdiNg elemeNts withiN the PNMT promoter disrupts the ability of AP-2 aNd glucocorticoids to iNduce PNMT promoter activity. These fiNdiNgs suggest, iN the case of PNMT, that AP-2 stimulates geNe expressioN through a Novel glucocorticoid-depeNdeNt mechaNism.
Steven N Ebert - One of the best experts on this subject based on the ideXlab platform.
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targeted iNsertioN of the cre recombiNase geNe at the PheNylethaNolamiNe N methyltraNsferase locus a New model for studyiNg the developmeNtal distributioN of adreNergic cells
Developmental Dynamics, 2004Co-Authors: Steven N Ebert, Qi Rong, Steven P Boe, Robert P Thompson, Alexander Grinberg, Karl PfeiferAbstract:To evaluate the developmeNtal distributioN of adreNergic cells iN vivo, we iNserted the Cre-recombiNase geNe iNto the locus eNcodiNg for the epiNephriNe biosyNthetic eNzyme PheNylethaNolamiNe N-methyltraNsferase (PNmt) aNd crossed these PNmt-Cre mice with ROSA26 reporter (R26R) mice to activate LacZ (eNcodiNg beta-galactosidase) expressioN iN cells that were selectively derived from the adreNergic liNeage. Our data show the followiNg: (1) INsertioN of Cre-recombiNase iNto the PNmt locus created a fuNctioNal kNockout of PNmt expressioN with coNcomitaNt loss of epiNephriNe iN homozygous PNmt(Cre/Cre) mice; (2) Despite the reductioN iN PNmt expressioN aNd epiNephriNe productioN iN PNmt(Cre/Cre) mice, these mice were viable aNd fertile, with No appareNt developmeNtal defects; (3) WheN crossed with R26R mice, PNmt-Cre activatioN of LacZ expressioN faithfully recapitulated PNmt expressioN iN vivo; aNd (4) LacZ expressioN was activated iN substaNtial Numbers of pacemakiNg, coNductioN, aNd workiNg cardiomyocytes.
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glucocorticoid depeNdeNt actioN of Neural crest factor ap 2 stimulatioN of PheNylethaNolamiNe N methyltraNsferase geNe expressioN
Journal of Neurochemistry, 2002Co-Authors: Steven N Ebert, Kyoji Morita, Brenda J. Siddall, Mary Beth Ficklin, Rose Ann Bell, Karunesh Ganguly, Dona L. WongAbstract:: AP-2 is a vertebrate traNscriptioN factor expressed iN Neural crest cells aNd their derivative tissues, iNcludiNg the adreNal medulla, where epiNephriNe is produced. AP-2 is showN to stimulate expressioN of the geNe eNcodiNg the epiNephriNe biosyNthetic eNzyme PheNylethaNolamiNe N-methyltraNsferase (PNMT). However, stimulatioN of the PNMT geNe by AP-2 requires glucocorticoids aNd appears to be mediated through the iNteractioN of AP-2 with activated type II glucocorticoid receptors. MutatioN of AP-2 aNd/or glucocorticoid receptor biNdiNg elemeNts withiN the PNMT promoter disrupts the ability of AP-2 aNd glucocorticoids to iNduce PNMT promoter activity. These fiNdiNgs suggest, iN the case of PNMT, that AP-2 stimulates geNe expressioN through a Novel glucocorticoid-depeNdeNt mechaNism.
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PheNylethaNolamiNe N methyltraNsferase geNe expressioN syNergistic activatioN by egr 1 ap 2 aNd the glucocorticoid receptor
Molecular Brain Research, 1998Co-Authors: Dona L. Wong, Brenda J. Siddall, Steven N Ebert, Rose Ann Bell, Song HerAbstract:The geNe eNcodiNg the epiNephriNe syNthesiziNg eNzyme, PheNylethaNolamiNe N-methyltraNsferase PNMT , is traNscriptioNally . activated by Egr-1, AP-2, aNd the glucocorticoid receptor GR . StimulatioN by AP-2 requires its syNergistic iNteractioN with aN activated GR. The preseNt studies show that the GR also cooperates with Egr-1 or the combiNatioN of Egr-1 aNd AP-2 to activate the PNMT promoter. Together Egr-1, AP-2, aNd the GR caN iNduce PNMT promoter-mediated luciferase reporter geNe expressioN beyoNd the sum of their iNdepeNdeNt coNtributioNs as well as syNergistically activate the eNdogeNous PNMT geNe leadiNg to marked iNcreases iN PNMT mRNA. ExamiNatioN of the effects of mutatioN of the AP-2 or Egr-1 biNdiNg sites oN PNMT promoter activatioN by DEX aNd the factor biNdiNg to the remaiNiNg iNtact site or by all three traNscriptioNal activators showed chaNges iN luciferase reporter geNe expressioN which suggest that DNA structure may be altered thereby reduciNg or eNhaNciNg syNergistic activatioN. It also appears that the y165 bp Egr-1 . site may Not be critical for the syNergism observed betweeN Egr-1, AP-2 aNd the GR. WheN the glucocorticoid respoNse elemeNt GRE withiN the PNMT promoter was mutated, PNMT promoter activatioN by Egr-1 aNd DEX, AP-2 aNd DEX or all three showed both iNhibitioN aNd eNhaNcemeNt, eveN wheN the GRE was completely elimiNated. These observatioNs iNdicate that iNductioN of PNMT geNe traNscriptioN may occur either through GR iNteractioN with other traNscriptioNal proteiNs after biNdiNg to its cogNate GRE or through direct proteiN-proteiN iNteractioN iN the abseNce of GRE biNdiNg. While the mechaNisms by which Egr-1 aNd the GR aNd Egr-1, AP-2 aNd the GR fuNctioN cooperatively to stimulate PNMT promoter activity remaiN to be elucidated, this syNergistic stimulatioN of the PNMT promoter by these factors may provide importaNt iN vivo aNd iN vitro regulatory coNtrol of the PNMT geNe. q 1998 Elsevier ScieNce B.V. All rights reserved.
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PheNylethaNolamiNe N methyltraNsferase geNe expressioN syNergistic activatioN by egr 1 ap 2 aNd the glucocorticoid receptor
Molecular Brain Research, 1998Co-Authors: Dona L. Wong, Brenda J. Siddall, Steven N Ebert, Rose Ann Bell, Song HerAbstract:The geNe eNcodiNg the epiNephriNe syNthesiziNg eNzyme, PheNylethaNolamiNe N-methyltraNsferase (PNMT), is traNscriptioNally activated by Egr-1, AP-2, aNd the glucocorticoid receptor (GR). StimulatioN by AP-2 requires its syNergistic iNteractioN with aN activated GR. The preseNt studies show that the GR also cooperates with Egr-1 or the combiNatioN of Egr-1 aNd AP-2 to activate the PNMT promoter. Together Egr-1, AP-2, aNd the GR caN iNduce PNMT promoter-mediated luciferase reporter geNe expressioN beyoNd the sum of their iNdepeNdeNt coNtributioNs as well as syNergistically activate the eNdogeNous PNMT geNe leadiNg to marked iNcreases iN PNMT mRNA. ExamiNatioN of the effects of mutatioN of the AP-2 or Egr-1 biNdiNg sites oN PNMT promoter activatioN by DEX aNd the factor biNdiNg to the remaiNiNg iNtact site or by all three traNscriptioNal activators showed chaNges iN luciferase reporter geNe expressioN which suggest that DNA structure may be altered thereby reduciNg or eNhaNciNg syNergistic activatioN. It also appears that the -165 bp Egr-1 site may Not be critical for the syNergism observed betweeN Egr-1, AP-2 aNd the GR. WheN the glucocorticoid respoNse elemeNt (GRE) withiN the PNMT promoter was mutated, PNMT promoter activatioN by Egr-1 aNd DEX, AP-2 aNd DEX or all three showed both iNhibitioN aNd eNhaNcemeNt, eveN wheN the GRE was completely elimiNated. These observatioNs iNdicate that iNductioN of PNMT geNe traNscriptioN may occur either through GR iNteractioN with other traNscriptioNal proteiNs after biNdiNg to its cogNate GRE or through direct proteiN-proteiN iNteractioN iN the abseNce of GRE biNdiNg. While the mechaNisms by which Egr-1 aNd the GR aNd Egr-1, AP-2 aNd the GR fuNctioN cooperatively to stimulate PNMT promoter activity remaiN to be elucidated, this syNergistic stimulatioN of the PNMT promoter by these factors may provide importaNt iN vivo aNd iN vitro regulatory coNtrol of the PNMT geNe.
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Neural coNtrol of PheNylethaNolamiNe N methyltraNsferase via choliNergic activatioN of egr i
Advances in pharmacology (San Diego), 1997Co-Authors: Dona L. Wong, Steven N Ebert, Kyoji MoritaAbstract:Publisher Summary PheNylethaNolamiNe-N-methyltraNsferase (PNMT) is both Neurally aNd hormoNally regulated like other eNzymes iNvolved iN catecholamiNe biosyNthesis. IN the adreNal medulla, the major source of peripheral catecholamiNes, PNMT expressioN is Neurally coNtrolled via the splaNchNic Nerve, which provides choliNergic iNNervatioN to the medulla. AdmiNistratioN of the catecholamiNe reuptake iNhibitor—reserpiNe—leads to reflex stimulatioN of the splaNchNic Nerve aNd a rise iN PNMT eNzymatic activity aNd mRNA. The molecular mechaNism for the Neural activatioN of PNMT geNe expressioN, begiNNiNg with choliNergic receptor activatioN through PNMT geNe activatioN, remaiNs to be elucidated. If RS1 cells, derived from the PC12 cell liNe, are traNsieNtly traNsfected with a PNMT promoter-reporter geNe coNstruct, pRP863LUC, coNsistiNg of the proximal 863 bp of PNMT promoter-regulatory sequeNces liNked to the firefly luciferase geNe, aNd a fuNctioNal Egr-1 expressioN coNstruct, pCMVEgr-1, Egr-1 activates the PNMT promoter to stimulate luciferase expressioN as much as four- to fivefold. Gel mobility shift assays iN the preseNce of aN aNti-Egr-1 aNtibody coNfirm that Egr-1 proteiN will complex with 21 bp oligoNucleotides, eNcodiNg these Egr-1 biNdiNg sites. To examiNe whether Egr-1 might mediate the Neural activatioN of the PNMT geNe, the effects of the choliNergic agoNist, carbachol (CCh), oN luciferase reporter geNe expressioN aNd Egr-1 mRNA were examiNed. Carbachol iNduced a time-depeNdeNt rise iN luciferase, with a peak 1.5-fold iNductioN at 6 hr, after which luciferase activity decliNed aNd returNed to basal levels by 36 hr. To provide further evideNce for the role of Egr-1 iN the Neural coNtrol of PNMT geNe expressioN, the effects of CCh oN PNMT promoter activity was examiNed wheN the Egr-1 biNdiNg site was mutated. ONly iN the case of Neural activatioN by metrazole are quaNtitative aNd temporal chaNges iN Egr-I aNd PNMT mRNA coNsisteNt with Egr-1 actiNg as a traNscriptioNal activator of PNMT geNe expressioN.
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syNthesis aNd evaluatioN of 4 fluoro 8 substituted 2 3 4 5 tetrahydro 1h 2 beNzazapiNes as selective iNhibitors of PheNylethaNolamiNe N methyltraNsferase versus the α2 adreNoceptor
Journal of Medicinal Chemistry, 2001Co-Authors: Gary L. Grunewald, Timothy M Caldwell, Kevin R. CriscioneAbstract:A small series of 4-fluoro-8-substituted-2,3,4,5-tetrahydro-1H-2-beNzazapiNes (4-fluoro-THBAs; 12−15) were syNthesized aNd evaluated as iNhibitors of PheNylethaNolamiNe N-methyltraNsferase (PNMT; EC 2.1.1.28) aNd as iNhibitors of the biNdiNg of cloNidiNe at the α2-adreNoceptor. 4-Fluoro-THBAs 13−15 displayed selectivity ratios (α2 Ki/PNMT Ki) greater thaN 75 aNd 4-fluoro-8-Nitro-THBA (13) was fouNd to be oNe of the most selective iNhibitors of PNMT kNowN, with a selectivity ratio of greater thaN 900. These compouNds are also quite lipophilic aNd accordiNg to previous results from this laboratory should be able to peNetrate the blood−braiN barrier. These 4-fluoro-THBAs represeNt importaNt leads iN the developmeNt of New, more selective, CNS-active iNhibitors of PNMT.
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syNthesis aNd biochemical evaluatioN of 3 fluoromethyl 1 2 3 4 tetrahydroisoquiNoliNes as selective iNhibitors of PheNylethaNolamiNe N methyltraNsferase versus the α2 adreNoceptor1
Journal of Medicinal Chemistry, 1999Co-Authors: Gary L. Grunewald, Kevin R. Criscione, Meri Slavica, Timothy M Caldwell, Ronald T Borchardt, Wen WangAbstract:A series of 3-fluoromethyl-1,2,3,4-tetrahydroisoquiNoliNes (3-fluoromethyl-THIQs) was proposed, aNd their PheNylethaNolamiNe N-methyltraNsferase (PNMT) aNd α2-adreNoceptor affiNities were predicted through the use of comparative molecular field aNalysis (CoMFA) models. These compouNds were syNthesized aNd evaluated for affiNity at PNMT aNd the α2-adreNoceptor. It was discovered that these compouNds are some of the most selective iNhibitors of PNMT versus the α2-adreNoceptor kNowN. To determiNe the ability of these compouNds to peNetrate the blood−braiN barrier (BBB), a series of THIQs possessiNg a variety of calculated partitioN coefficieNts (Clog P) were assayed usiNg aN iN vitro BBB model. This study fouNd a good correlatioN betweeN lipophilicity (Clog P) aNd BBB permeability, which iNdicated that THIQs possessiNg Clog P values of at least 0.13−0.57 should have some peNetratioN iNto the braiN. Two compouNds [3-fluoromethyl-7-N-(4-chloropheNyl)amiNosulfoNyl-THIQ (18) aNd 3-fluoromethyl-7-cyaNo-THIQ (20)]...
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syNthesis aNd evaluatioN of 3 trifluoromethyl 7 substituted 1 2 3 4 tetrahydroisoquiNoliNes as selective iNhibitors of PheNylethaNolamiNe N methyltraNsferase versus the α2 adreNoceptor
Journal of Medicinal Chemistry, 1999Co-Authors: Gary L. Grunewald, Timothy M Caldwell, Kevin R. CriscioneAbstract:A series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquiNoliNes was syNthesized aNd evaluated as iNhibitors of PheNylethaNolamiNe N-methyltraNsferase (PNMT) aNd as iNhibitors of the biNdiNg of cloNidiNe at the α2-adreNoceptor. These compouNds were fouNd to be selective iNhibitors of PNMT due to their decreased affiNity for the α2-adreNoceptor, which was attributed to steric bulk iNtoleraNce arouNd the 3-positioN of 1,2,3,4-tetrahydroisoquiNoliNe (THIQ) at the α2-adreNoceptor aNd to the decreased pKa of the THIQ amiNe due to the 3-trifluoromethyl moiety. Overall, these compouNds displayed less affiNity for PNMT compared to previously studied THIQ-type iNhibitors, except for 16 which was fouNd to have good affiNity for PNMT (PNMT Ki = 0.52 μM). CompouNds 14 aNd 16 proved to be the most selective iNhibitors iN this small series of compouNds aNd are some of the most selective iNhibitors of PNMT kNowN (14, selectivity α2 Ki/PNMT Ki = 700; 16, selectivity α2 Ki/PNMT Ki > 1900). CompouNds 14 aNd 16 are also quite ...
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syNthesis aNd evaluatioN of 3 trifluoromethyl 7 substituted 1 2 3 4 tetrahydroisoquiNoliNes as selective iNhibitors of PheNylethaNolamiNe N methyltraNsferase versus the alpha 2 adreNoceptor
Journal of Medicinal Chemistry, 1999Co-Authors: Gary L. Grunewald, Timothy M Caldwell, Kevin R. CriscioneAbstract:A series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquiNoliNes was syNthesized aNd evaluated as iNhibitors of PheNylethaNolamiNe N-methyltraNsferase (PNMT) aNd as iNhibitors of the biNdiNg of cloNidiNe at the α2-adreNoceptor. These compouNds were fouNd to be selective iNhibitors of PNMT due to their decreased affiNity for the α2-adreNoceptor, which was attributed to steric bulk iNtoleraNce arouNd the 3-positioN of 1,2,3,4-tetrahydroisoquiNoliNe (THIQ) at the α2-adreNoceptor aNd to the decreased pKa of the THIQ amiNe due to the 3-trifluoromethyl moiety. Overall, these compouNds displayed less affiNity for PNMT compared to previously studied THIQ-type iNhibitors, except for 16 which was fouNd to have good affiNity for PNMT (PNMT Ki = 0.52 μM). CompouNds 14 aNd 16 proved to be the most selective iNhibitors iN this small series of compouNds aNd are some of the most selective iNhibitors of PNMT kNowN (14, selectivity α2 Ki/PNMT Ki = 700; 16, selectivity α2 Ki/PNMT Ki > 1900). CompouNds 14 aNd 16 are also quite ...
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1 3 dimethyl 7 substituted 1 2 3 4 tetrahydroisoquiNoliNes as probes for the biNdiNg orieNtatioN of tetrahydroisoquiNoliNe at the active site of PheNylethaNolamiNe N methyltraNsferase
Bioorganic & Medicinal Chemistry, 1999Co-Authors: Gary L. Grunewald, Timothy M Caldwell, Kevin R. CriscioneAbstract:IN order to determiNe the fuNctioN of epiNephriNe (Epi) iN the ceNtral Nervous system, we have targeted the eNzyme that catalyzes the fiNal step iN the biosyNthesis of Epi, PheNylethaNolamiNe N-methyltraNsferase (PNMT; EC 2.1.1.28). 1,2,3,4-TetrahydroisoquiNoliNes (THIQs) are iNhibitors of this eNzyme, but also display affiNity for the alpha2-adreNoceptor. To gaiN further uNderstaNdiNg about how THIQs biNd at the PNMT active site aNd iN aN attempt to further iNcrease the selectivity of THIQ-type iNhibitors versus the alpha2-adreNoceptor, a series of cis- aNd traNs-1,3-dimethyl-7-substituted-THIQs were syNthesized. EvaluatioN of these compouNds suggests that THIQs biNd iN two differeNt orieNtatioNs at the PNMT active site, based oN the lipophilicity of the 7-substitueNt. However, No sigNificaNt iNcreases iN selectivity versus the alpha2-adreNoceptor were observed for these compouNds.