scholarly journals Discovery of imidazole-based inhibitors of P. falciparum cGMP-dependent protein kinase

2021 ◽  
Author(s):  
Rammohan R Yadav ◽  
Mariana Laureano de Souza ◽  
Mariana Lozano Gonzalez ◽  
Shams Ul Mahmood ◽  
Tyler Eck ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Plasmodium Species ◽  
Initial Structure ◽  
Erythrocytic Stage ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Structure Activity ◽  
Dependent Protein ◽  
Parasitic Activity

The discovery of new targets for treatment of malaria and in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this manuscript presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization and cell-based anti-parasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG poten-cy, low hERG activity and cell-based anti-parasitic activity against multiple Plasmodium species that appears to correlate with in vitro potency.

scholarly journals Discovery of novel potent, cell-permeable inhibitors of P. falciparum cGMP-dependent protein kinase.

2021 ◽  
Author(s):  
Rammohan R. Yadav Bheemanaboina ◽  
Mariana Lozano Gonzalez ◽  
Shams Ul Mahmood ◽  
Tyler Eck ◽  
Tamara Kreiss ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Murine Model ◽  
Dependent Protein Kinase ◽  
Antiparasitic Activity ◽  
Cgmp Dependent Protein Kinase ◽  
Structure Activity ◽  
Dependent Protein ◽  
Parasitic Activity

The discovery of new targets for treatment of malaria advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusi-asm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, we recently reported the discovery and optimization of novel, potent isoxazole-based PfPKG inhibitors that lacked any obvious safety warnings. This manuscript presents representative in vitro ADME, hERG charac-terization and cell-based antiparasitic activity of these PfPKG inhibitors. We also report the discovery and structure-activity relationships of a new series with good potency, low hERG activity and cell-based anti-parasitic activity comparable to a literature standard.

Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro

2003 ◽  
Vol 284 (4) ◽  
pp. H1388-H1397 ◽  
Author(s):  
Hyun Kook ◽  
Hiroshi Itoh ◽  
Bong Seok Choi ◽  
Naoki Sawada ◽  
Kentaro Doi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Physiological Concentration ◽  
Cell Numbers ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Regeneration In Vitro

Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiological concentration (10−11mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations.

scholarly journals Increasing cGMP-dependent protein kinase I activity attenuates cisplatin-induced kidney injury through protection of mitochondria function

2013 ◽  
Vol 305 (6) ◽  
pp. F881-F890 ◽  
Author(s):  
Hasiyeti Maimaitiyiming ◽  
Yanzhang Li ◽  
Wenpeng Cui ◽  
Xiaopeng Tong ◽  
Heather Norman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tubular Damage ◽  
Dependent Protein Kinase ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Renal Tubular

Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear, and the renoprotective agents during cisplatin treatment are still lacking. Here, we demonstrated that the expression and activity of cGMP-dependent protein kinase-I (PKG-I) were reduced in cisplatin-treated renal tubular cells in vitro as well as in the kidney tissues from cisplatin-treated mice in vivo. Increasing PKG activity by both pharmacological and genetic approaches attenuated cisplatin-induced kidney cell apoptosis in vitro. This was accompanied by decreased Bax/Bcl2 ratio, caspase 3 activity, and cytochrome c release. Cisplatin-induced mitochondria membrane potential loss in the tubular cells was also prevented by increased PKG activity. All of these data suggest a protective effect of PKG on mitochondria function in renal tubular cells. Importantly, increasing PKG activity pharmacologically or genetically diminished cisplatin-induced tubular damage and preserved renal function during cisplatin treatment in vivo. Mitochondria structural and functional damage in the kidney from cisplatin-treated mice was inhibited by increased PKG activity. In addition, increasing PKG activity enhanced ciaplatin-induced cell death in several cancer cell lines. Taken together, these results suggest that increasing PKG activity may be a novel option for renoprotection during cisplatin-based chemotherapy.

In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium

1993 ◽  
Vol 106 (4) ◽  
pp. 1369-1376 ◽  
Author(s):  
C.E. Walczak ◽  
D.L. Nelson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Sucrose Gradient ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Ciliary Protein ◽  
Dependent Protein ◽  
Camp And Cgmp

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.

scholarly journals Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase

2018 ◽  
Vol 293 (43) ◽  
pp. 16791-16802 ◽  
Author(s):  
Jessica L. Sheehe ◽  
Adrian D. Bonev ◽  
Anna M. Schmoker ◽  
Bryan A. Ballif ◽  
Mark T. Nelson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Specific Substrate ◽  
Type I ◽  
Amino Acid Residues ◽  
Dependent Protein Kinase ◽  
Constitutive Activation ◽  
Channel Characteristics ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2. In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα–specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

cGMP-dependent ADP depolymerization of actin mediates estrogen increase in cervical epithelial permeability

2000 ◽  
Vol 279 (6) ◽  
pp. C2028-C2036 ◽  
Author(s):  
George I. Gorodeski
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Epithelial Permeability ◽  
Estrogen Action ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
17Β Estradiol ◽  
Ly 83583

Estrogen increases secretion of cervical mucus in women, and the effect depends on fragmentation of the cytoskeleton. The objective of the present study was to understand the molecular mechanism of estrogen action. Treatment of human cervical epithelial cells with 17β-estradiol, sodium nitroprusside (SNP), or 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased cellular monomeric G-actin and decreased polymerized F-actin. The effects of estradiol were blocked by tamoxifen, by the guanylate cyclase inhibitor LY-83583, and by the cGMP-dependent protein kinase inhibitor KT-5823. The effects of SNP were blocked by LY-83583 and KT-5823, while the effects of 8-Br-cGMP were blocked only by KT-5823. Treatment with phalloidin decreased paracellular permeability and G-actin. Treatment with 17β-estradiol, SNP, or 8-Br-cGMP attenuated SNP-induced phosphorylation of [32P]adenylate NAD in vitro: tamoxifen blocked the effect of estrogen; LY-83583 blocked the effect of SNP but not that of 8-Br-cGMP, while KT-5823 blocked effects of both SNP and 8-Br-cGMP. These results indicate that estrogen, nitric oxide (NO), and cGMP stimulate actin depolymerization. A possible mechanism is NO-induced, cGMP-dependent protein kinase augmentation of ADP-ribosylation of monomeric actin.

scholarly journals Ο ρόλος της πρωτεϊνικής κινάσης G στην αγγειογένεση

2010 ◽  
Author(s):  
Βασιλική Κόικα
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Ο αγγειακός ενδοθηλιακός παράγοντας (VEGF) επάγει την παραγωγή του μονοξειδίου του αζώτου(ΝΟ), το οποίο διαμεσολαβεί πολλές από τις αγγειογενετικές δράσεις του. Μολονότι, γνωρίζουμε ότι ο «υποδοχέας του ΝΟ» διαλυτή γουανυλική κυκλάση (sGC) συμμετέχει στην αγγειογένεση που επάγεται από τον VEGF, ελάχιστα είναι χαρακτηρισμένα τα καθοδικά μόρια- εκτελεστές μέσω των οποίων το cGMP που προέρχεται από την sGC κατευθύνει την αγγειογενετική απάντηση. Για να προσδιορίσουμε την συμμετοχή της PKG (cGMP-dependent protein kinase) στην αγγειογένεση που επάγεται από τον VEGF, χρησιμοποιήσαμε τα πεπτίδια DT2 και DT3, δύο επιλεκτικούς αναστολείς της PKGIα. Έχοντας την απάντηση αυτού του ερωτήματος ως στόχο, πραγματοποιήσαμε in vivo (CAM, μοντέλο του κερατοειδή του ματιού κουνελιού, τροποποιημένη δοκιμασία Miles assay) και in vitro (πολλαπλασιασμός και μετανάστευση ενδοθηλιακών κυττάρων, εκβλάστηση σε δακτυλίους αορτής) μελέτες. Επιπλέον εκτιμήθηκε η ικανότητα του DT2 να παρεμβάλλεται στην μεταγωγή σήματος του VEGF. Επώαση CAM μεμβρανών με τους πεπτιδικούς αναστολείς της PKGIα είχε σαν αποτέλεσμα την μείωση του μήκους των αγγείων με δοσο-εξαρτώμενο τρόπο, με το DT3 να είναι πιο αποδοτικό από το DT2. Επιπρόσθετα παρατηρήσαμε, ότι το DT3 καταργεί την αγγεογενετική απάντηση που προέρχεται από τον VEGF στον κερατοειδή χιτώνα του ματιού κουνελιού. Η αναστολή της PKGI εμποδίζει επίσης την αγγειακή διαρροή που επάγεται από τον VEGF. In vitro, χορήγηση VEGF σε ενδοθηλιακά κύτταρα επάγει την φωσφορυλίωση της VASP στην Ser239 (επιλεκτικό υπόστρωμα για την PKGΙ) μέσω της ενεργοποίησης του VEGFR2 ενώ η συνχορήγηση του DT2 έχει σαν αποτέλεσμα μειωμένα επίπεδα φωσφορυλιωμένης VASP πρωτεΐνης αποδεικνύοντας ότι σε άθικτα κύτταρα διέγερση του VEGFR2 οδήγησε σε ενεργοποίηση της PKGI. Επιπλέον παρατηρήθηκε ότι επώαση των ενδοθηλιακών κυττάρων με DT2 ή DT3 αναστέλλει την διαμεσολαβούμενη από τις ΜΑΡΚ κινάσες ERK1/2 και p38 μετανάστευση, πολλαπλασιασμό και εκβλάστηση τους που επάγονται από τον VEGF. Εν κατακλείδι, παρέχουμε αποδείξεις ότι η PKGI είναι μέρος του μεταγωγικού μονοπατιού που διαμεσολαβεί τις αγγειογενετικές δράσεις του VEGF και ότι οι πεπτιδικοί αναστολείς της PKGI θα μπορούσαν να δοκιμαστούν σε ασθένειες που σχετίζονται με ενισχυμένη αγγειογένεση.

scholarly journals Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals

2004 ◽  
Vol 381 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Jing XUE ◽  
Peter J. MILBURN ◽  
Bernadette T. HANNA ◽  
Mark E. GRAHAM ◽  
John A. P. ROSTAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Nerve Terminals ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Phosphoamino Acid Analysis ◽  
The Brain ◽  
Major Phosphorylation Site

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.

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