<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure"^^ . "Impaired cAMP signaling and dysfunctional calcium handling in cardiomyocytes are both \r\ncharacteristics associated with heart failure. So far, therapeutic attempts to improve contractile \r\nfunction by globally increasing cAMP levels failed clinically due to higher arrhythmia \r\nsusceptibility and increased risk of sudden cardiac death. Novel strategies aiming to modulate \r\ndistinct cAMP pools in cardiomyocytes may improve contraction force without detrimental \r\neffects. Nucleoside diphosphate kinase (NDPK), an enzyme with transphosphorylase activity, \r\ncontributes to heterotrimeric G protein activation by GTP formation from ATP and GDP. \r\nMoreover, oligomers consisting of the isoforms NDPK B and C form a complex with \r\nheterotrimeric G proteins and act as protein histidine kinase on the G subunit leading to direct \r\nactivation of the G protein. NDPK C is upregulated in end-stage human heart failure, where it \r\npromotes the complex formation of NDPKs with Gi proteins and enhances their activity. This \r\nlikely contributes to the suppression of cAMP formation. Therefore, this project aims to identify \r\ncompounds that inhibit NDPK B and C and investigate the consequences of NDPK inhibition \r\nin heart muscle.\r\nTo screen for potential inhibitors of NDPK activity, recombinantly expressed human NDPK A, \r\nB, and C were purified. An ATP-dependent, firefly luciferase-based luminescence assay was \r\nused to quantify the amount of ATP formed from GTP+ADP, monitoring the NDPK \r\ntransphosphorylase activity. Out of the tested library, one small molecule compound \r\n(SanWie3) preferentially reduced the activity of NDPK C > NDPK B >> NDPK A. Further in \r\nvitro studies revealed that SanWie3 is an allosteric inhibitor of the transphosphorylase activity \r\nof NDPK C (IC50 ~ 3 μM). Biophysical protein analyses confirmed that binding of SanWie3 \r\nmediates small structural changes in the NDPK C protein. Preliminary studies on the binding \r\nof SanWie3, using HDX-mass spectrometry and in silico protein modeling, identified a putative \r\nbinding pocket near the catalytically active site. The potential off-target binding of SanWie3 on \r\nother enzymes, channels, and receptors was studied using in vitro screens, which did not \r\nsuggest any additional SanWie3 targets so far. Previously, we reported that the knockdown of \r\nNDPK C reduced the isoproterenol-induced cAMP in neonatal rat cardiomyocytes (NRCM). \r\nTherefore, cAMP formation and PKA-dependent protein phosphorylation were analyzed in \r\nNRCM and adult mouse ventricular cardiomyocytes (AMVCM), stimulated with isoproterenol \r\n(ISO). SanWie3 attenuated the ISO-induced cAMP formation in NRCM, consistent with the \r\nreported complex formation of NDPK C with Gs proteins. In accordance, the SanWie3-induced \r\ndeprivation of cAMP reduced the phosphorylation of the cAMP/PKA downstream target \r\nphospholamban (PLN). Surprisingly SanWie3 treatment of AMVCM and measuring cAMP \r\npools by differentially targeted cAMP-Förster resonance energy transfer (FRET) suggested a \r\ndifferent function in AMVCM. Here, SanWie3 caused an increase of ISO-induced cAMP levels \r\nin the PLN/SERCA2a subdomain and the PKA-dependent PLN phosphorylation. The ISOinduced cAMP formation and PKA-dependent phosphorylation of other targets in other cellular \r\ndomains (cytosol, plasma membrane, and ryanodine receptor-2-subdomain) were unchanged. \r\nConsistently, the influence of SanWie3 on calcium handling in AMVCM showed an accelerated \r\nbasal Ca2+ reuptake, an increase in the basal sarcoplasmic reticulum (SR) Ca2+ load, and an \r\nincrease in Ca2+ transients and spontaneous Ca2+ spark frequency. To analyze the effect of \r\nSanWie3 on ISO-induced ventricular contraction, cardiac muscle stripes from rats were \r\nisolated, and force development was measured in an organ bath. This significant increase in \r\nISO-induced contractility was associated with an increase in PLN phosphorylation. As in \r\nAMVCM, the effect of SanWie3 on ISO-induced PLN phosphorylation was mimicked by the \r\ninhibition of Gi activity by pertussis toxin treatment; the results suggest a constitutive activation \r\nof Gi by NDPK C in a signalosome controlling PLN/SERCA2a activity, thereby modulating Ca2+\r\nload and cardiomyocyte contractility.\r\nThe results demonstrate that the identified small molecular inhibitor SanWie3, which \r\npreferentially attenuates the enzymatic activity of NDPK C, modulates cAMP-dependent \r\nsignaling in NRCM and AMVCM preferentially in the PLN/SERCA2a subdomain. Therefore, \r\nSanWie3 may be a lead compound in designing small molecules able to relieve the detrimental \r\nsuppression of cAMP signaling in specific cellular compartments in cardiomyocytes occurring \r\nin heart failure."^^ . "2025" . . . . . . . "Santosh Kumar"^^ . "Lomada"^^ . "Santosh Kumar Lomada"^^ . . . . . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure (PDF)"^^ . . . "Thesis_Lomada.pdf"^^ . . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Investigations on inhibitors of nucleoside diphosphate kinases as \r\nputative novel therapeutics for the treatment of heart failure (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #31257 \n\nInvestigations on inhibitors of nucleoside diphosphate kinases as \nputative novel therapeutics for the treatment of heart failure\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . . . "610 Medizin"@de . "610 Medical sciences Medicine"@en . .