%0 Generic
%A Lomada, Santosh Kumar
%C Heidelberg
%D 2025
%F heidok:31257
%R 10.11588/heidok.00031257
%T Investigations on inhibitors of nucleoside diphosphate kinases as   putative novel therapeutics for the treatment of heart failure
%U https://archiv.ub.uni-heidelberg.de/volltextserver/31257/
%X Impaired cAMP signaling and dysfunctional calcium handling in cardiomyocytes are both   characteristics associated with heart failure. So far, therapeutic attempts to improve contractile   function by globally increasing cAMP levels failed clinically due to higher arrhythmia   susceptibility and increased risk of sudden cardiac death. Novel strategies aiming to modulate   distinct cAMP pools in cardiomyocytes may improve contraction force without detrimental   effects. Nucleoside diphosphate kinase (NDPK), an enzyme with transphosphorylase activity,   contributes to heterotrimeric G protein activation by GTP formation from ATP and GDP.   Moreover, oligomers consisting of the isoforms NDPK B and C form a complex with   heterotrimeric G proteins and act as protein histidine kinase on the G subunit leading to direct   activation of the G protein. NDPK C is upregulated in end-stage human heart failure, where it   promotes the complex formation of NDPKs with Gi proteins and enhances their activity. This   likely contributes to the suppression of cAMP formation. Therefore, this project aims to identify   compounds that inhibit NDPK B and C and investigate the consequences of NDPK inhibition   in heart muscle.  To screen for potential inhibitors of NDPK activity, recombinantly expressed human NDPK A,   B, and C were purified. An ATP-dependent, firefly luciferase-based luminescence assay was   used to quantify the amount of ATP formed from GTP+ADP, monitoring the NDPK   transphosphorylase activity. Out of the tested library, one small molecule compound   (SanWie3) preferentially reduced the activity of NDPK C > NDPK B >> NDPK A. Further in   vitro studies revealed that SanWie3 is an allosteric inhibitor of the transphosphorylase activity   of NDPK C (IC50 ~ 3 μM). Biophysical protein analyses confirmed that binding of SanWie3   mediates small structural changes in the NDPK C protein. Preliminary studies on the binding   of SanWie3, using HDX-mass spectrometry and in silico protein modeling, identified a putative   binding pocket near the catalytically active site. The potential off-target binding of SanWie3 on   other enzymes, channels, and receptors was studied using in vitro screens, which did not   suggest any additional SanWie3 targets so far. Previously, we reported that the knockdown of   NDPK C reduced the isoproterenol-induced cAMP in neonatal rat cardiomyocytes (NRCM).   Therefore, cAMP formation and PKA-dependent protein phosphorylation were analyzed in   NRCM and adult mouse ventricular cardiomyocytes (AMVCM), stimulated with isoproterenol   (ISO). SanWie3 attenuated the ISO-induced cAMP formation in NRCM, consistent with the   reported complex formation of NDPK C with Gs proteins. In accordance, the SanWie3-induced   deprivation of cAMP reduced the phosphorylation of the cAMP/PKA downstream target   phospholamban (PLN). Surprisingly SanWie3 treatment of AMVCM and measuring cAMP   pools by differentially targeted cAMP-Förster resonance energy transfer (FRET) suggested a   different function in AMVCM. Here, SanWie3 caused an increase of ISO-induced cAMP levels   in the PLN/SERCA2a subdomain and the PKA-dependent PLN phosphorylation. The ISOinduced cAMP formation and PKA-dependent phosphorylation of other targets in other cellular   domains (cytosol, plasma membrane, and ryanodine receptor-2-subdomain) were unchanged.   Consistently, the influence of SanWie3 on calcium handling in AMVCM showed an accelerated   basal Ca2+ reuptake, an increase in the basal sarcoplasmic reticulum (SR) Ca2+ load, and an   increase in Ca2+ transients and spontaneous Ca2+ spark frequency. To analyze the effect of   SanWie3 on ISO-induced ventricular contraction, cardiac muscle stripes from rats were   isolated, and force development was measured in an organ bath. This significant increase in   ISO-induced contractility was associated with an increase in PLN phosphorylation. As in   AMVCM, the effect of SanWie3 on ISO-induced PLN phosphorylation was mimicked by the   inhibition of Gi activity by pertussis toxin treatment; the results suggest a constitutive activation   of Gi by NDPK C in a signalosome controlling PLN/SERCA2a activity, thereby modulating Ca2+  load and cardiomyocyte contractility.  The results demonstrate that the identified small molecular inhibitor SanWie3, which   preferentially attenuates the enzymatic activity of NDPK C, modulates cAMP-dependent   signaling in NRCM and AMVCM preferentially in the PLN/SERCA2a subdomain. Therefore,   SanWie3 may be a lead compound in designing small molecules able to relieve the detrimental   suppression of cAMP signaling in specific cellular compartments in cardiomyocytes occurring   in heart failure.