ABSTRACT
Acute nociceptive pain in mice caused by subcutaneous (intraplantar) injection of TRPV1 ion channel agonist capsaicin (1.6 µg/mouse) and the effects of protein kinase A inhibitor H-89 (0.05 mg/mouse, intraplantar injection) and NMDA receptor channel antagonists MK-801 (7.5 and 15 µg/mouse, topical application) and hemantane (0.5 mg/mouse, topical application) on the pain were assessed. MK-801 and hemantane were found to reduce the duration of the pain response. H-89 did not significantly affect the pain in animals, but preliminary administration of this drug abolished the antinociceptive effect of MK-801 (7.5 µg/mouse) and weakens the effect of hemantane (0.5 mg/mouse).
Subject(s)
Analgesics , Capsaicin , Dizocilpine Maleate , Receptors, N-Methyl-D-Aspartate , Animals , Capsaicin/pharmacology , Mice , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Male , Dizocilpine Maleate/pharmacology , Analgesics/pharmacology , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Nociceptive Pain/drug therapy , Nociceptive Pain/chemically induced , Pain Measurement/drug effects , Pain Measurement/methodsABSTRACT
Ferroptosis is a type of regulated cell death characterized by iron accumulation and lipid peroxidation. The molecular mechanisms underlying ferroptosis regulation in non-small cell lung cancer (NSCLC) are poorly understood. In this study, we found that protein kinase A (PKA) inhibition enhanced ferroptosis susceptibility in NSCLC cells, as evidenced by reduced cell viability and increased lipid peroxidation. We further identified cAMP-responsive element protein 1 (CREB1), a transcription factor and a substrate of PKA, as a key regulator of ferroptosis. Knockdown of CREB1 sensitized NSCLC cells to ferroptosis inducers (FINs) and abolished the effects of PKA inhibitor and agonist, revealing the pivotal role of CREB1 in ferroptosis regulation. Using a high-throughput screening approach and subsequent validation by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, we discovered that CREB1 transcriptionally activated stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids. SCD conferred ferroptosis resistance by decreasing the availability of polyunsaturated fatty acids for lipid peroxidation, and its overexpression rescued the effect of CREB1 knockdown on ferroptosis in vitro. Besides, CREB1 knockdown suppressed xenograft tumor growth in the presence of Imidazole Ketone Erastin (IKE), a potent FIN, and this effect was reversed by SCD. Finally, we showed that high expression of CREB1 was associated with poor prognosis in NSCLC patients from public datasets and our institution. Collectively, this study illustrates the effect of PKA/CREB1/SCD axis in regulating ferroptosis of NSCLC, targeting this pathway may provide new strategies for treating NSCLC patients.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Ferroptosis , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Ferroptosis/genetics , Lipid Peroxidation , Lung Neoplasms/geneticsABSTRACT
Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.
Subject(s)
Chondrocytes/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Natriuretic Peptide, C-Type/pharmacology , Osteogenesis/physiology , Animals , Cell Differentiation , Cell Line , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP/metabolism , Enzyme Activation/drug effects , Fluorescence Resonance Energy Transfer , Growth Plate/growth & development , Mice , Mice, Transgenic , Osteogenesis/drug effects , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.
Subject(s)
C9orf72 Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dipeptides/metabolism , Proteolysis , Small Molecule Libraries/pharmacology , Animals , Cell Line , Codon, Initiator/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Repeat Expansion/genetics , Disease Models, Animal , Drosophila/drug effects , Frontotemporal Dementia/pathology , HEK293 Cells , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells/pathology , Isoquinolines/pharmacology , Longevity/drug effects , Motor Neurons/drug effects , Motor Neurons/pathology , Protein Biosynthesis/drug effects , Proteolysis/drug effects , RNA Interference , Sulfonamides/pharmacologyABSTRACT
Indole derivatives from various plants are known to have health benefits because of their anti-cancer, anti-oxidant, anti-inflammatory, and anti-tubercular effects. However, their effects on adipogenesis have not been fully elucidated yet. Herein, we show that a newly synthesized indole derivative, CF3-allylated indole, [(E)-1-(pyrimidin- 2-yl)-2-(4,4,4- trifluorobut-2-enyl)-1H-indole], effectively inhibits adipogenesis. We found that CF3-allylated indole inhibited lipid accumulation and suppressed the expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator activated receptor γ (PPARγ) in 3T3-L1 cells. The inhibitory effect of CF3-allylated indole primarily occurred at the early phase of adipocyte differentiation by increasing intracellular cyclic adenosine monophosphate (cAMP) levels and enhancing protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) signaling. Conversely, depletion of PKA or treatment with a protein kinase A inhibitor (H89) reversed such inhibitory effects of CF3-allylated indole on adipogenesis and PPARγ expression. These results suggest that CF3-allylated indole inhibits early stages of adipogenesis by increasing phosphorylation of PKA/AMPK, leading to decreased expression of adipogenic genes in 3T3-L1 cells. These results indicate that CF3-allylated indole has potential for controlling initial adipocyte differentiation in metabolic disorders such as obesity.
Subject(s)
Adipogenesis/drug effects , Indoles/pharmacology , 3T3-L1 Cells , AMP-Activated Protein Kinases/metabolism , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Adipogenesis/genetics , Adipogenesis/physiology , Animals , Anti-Obesity Agents/chemistry , Anti-Obesity Agents/pharmacology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression/drug effects , Indoles/chemistry , Lipid Metabolism/drug effects , Mice , Obesity/drug therapy , Phosphorylation , Signal Transduction/drug effectsABSTRACT
Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , Drug Development/methods , Fusaric Acid , Mycotoxins , Cell Line, Tumor , Cell Survival/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Fusaric Acid/chemistry , Fusaric Acid/metabolism , Fusaric Acid/toxicity , Fusarium/metabolism , Humans , Molecular Dynamics Simulation , Mycotoxins/chemistry , Mycotoxins/metabolism , Mycotoxins/toxicityABSTRACT
Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Serine-Arginine Splicing Factors/metabolism , Tretinoin/pharmacology , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Humans , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacologyABSTRACT
Protein kinase A (PKA) is a central regulator of cardiac performance and morphology. Myocardial PKA activation is induced by a variety of hormones, neurotransmitters, and stress signals, most notably catecholamines secreted by the sympathetic nervous system. Catecholamines bind ß-adrenergic receptors to stimulate cAMP-dependent PKA activation in cardiomyocytes. Elevated PKA activity enhances Ca2+ cycling and increases cardiac muscle contractility. Dynamic control of PKA is essential for cardiac homeostasis, as dysregulation of PKA signalling is associated with a broad range of heart diseases. Specifically, abnormal PKA activation or inactivation contributes to the pathogenesis of myocardial ischaemia, hypertrophy, heart failure, as well as diabetic, takotsubo, or anthracycline cardiomyopathies. PKA may also determine sex-dependent differences in contractile function and heart disease predisposition. Here, we describe the recent advances regarding the roles of PKA in cardiac physiology and pathology, highlighting previous study limitations and future research directions. Moreover, we discuss the therapeutic strategies and molecular mechanisms associated with cardiac PKA biology. In summary, PKA could serve as a promising drug target for cardioprotection. Depending on disease types and mechanisms, therapeutic intervention may require either inhibition or activation of PKA. Therefore, specific PKA inhibitors or activators may represent valuable drug candidates for the treatment of heart diseases.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Heart Diseases/enzymology , Myocardial Contraction , Myocardium/enzymology , Animals , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Activation , Heart Diseases/drug therapy , Heart Diseases/pathology , Heart Diseases/physiopathology , Humans , Molecular Targeted Therapy , Myocardium/pathology , Phosphorylation , Protein Kinase Inhibitors/therapeutic use , Signal TransductionABSTRACT
Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the ß-agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to ß-adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation).
Subject(s)
Atrial Fibrillation/metabolism , Calcium Signaling , Cyclic AMP/metabolism , Adrenergic beta-Antagonists/pharmacology , Aged , Animals , Calcium/metabolism , Calcium Channels, L-Type/metabolism , Calcium Signaling/drug effects , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Carvedilol/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytosol/drug effects , Cytosol/metabolism , Female , Humans , Male , Middle Aged , Receptors, Adrenergic, beta/metabolism , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolismABSTRACT
Mammalian cell cytoskeletal reorganization for efficient directional movement requires tight coordination of actomyosin and microtubule networks. In this study, we show that LRAP35a potentiates microtubule stabilization by promoting CLASP2/EB1 interaction besides its complex formation with MRCK/MYO18A for retrograde actin flow. The alternate regulation of these two networks by LRAP35a is tightly regulated by a series of phosphorylation events that dictated its specificity. Sequential phosphorylation of LRAP35a by Protein Kinase A (PKA) and Glycogen Synthase Kinase-3ß (GSK3ß) initiates the association of LRAP35a with CLASP2, while subsequent binding and further phosphorylation by Casein Kinase 1δ (CK1δ) induce their dissociation, which facilitates LRAP35a/MRCK association in driving lamellar actomyosin flow. Importantly, microtubule dynamics is directly moderated by CK1δ activity on CLASP2 to regulate GSK3ß phosphorylation of the SxIP motifs that blocks EB1 binding, an event countered by LRAP35a interaction and its competition for CK1δ activity. Overall this study reveals an essential role for LRAP35a in coordinating lamellar contractility and microtubule polarization in cell migration.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Casein Kinase Idelta/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Tumor Suppressor Proteins/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Casein Kinase Idelta/antagonists & inhibitors , Casein Kinase Idelta/genetics , Cell Line, Tumor , Cell Movement , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Microtubule-Associated Proteins/chemistry , Mutagenesis, Site-Directed , Phosphorylation , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/geneticsABSTRACT
Two series of novel amino acid Schiff base ligands containing heterocyclic moieties, such as quinazolinone 3-11 and indole 12-20 were successfully synthesized and confirmed by spectroscopic techniques and elemental analysis. Furthermore, all compounds were investigated in silico for their ability to inhibit mitochondrial NADH: ubiquinone oxidoreductase (complex I) by targeting the AMPK/mTOR signaling pathway and inhibiting hexokinase, a key glycolytic enzyme to prevent the Warburg effect in cancer cells. This inhibitory pathway may be an effective strategy to cause cancer cell death due to an insufficient amount of ATP. Our results revealed that, out of 18 compounds, two (11 and 20) were top-ranked as they exhibited the highest binding energies of -8.8, -13.0, -7.9, and -10.0 kcal/mol in the docking analysis, so they were then selected for in vitro assessment. Compound 11 promoted the best cytotoxic effect on MCF-7 with IC50 = 64.05 ± 0.14 µg/mL (0.135 mM) while compound 20 exhibited the best cytotoxic effect on MDA-231 with IC50 = 46.29 ± 0.09 µg/mL (0.166 mM) Compounds 11 and 20 showed significant activation of AMPK protein and oxidative stress, which led to elevated expression of p53 and Bax, reduced Bcl-2 expression, and caused cell cycle arrest at the sub-G0/G1 phase. Moreover, compounds 11 and 20 showed significant inhibition of the mTOR protein, which led to the activation of aerobic glycolysis for survival. This alternative pathway was also blocked as compounds 11 and 20 showed significant inhibitory effects on the hexokinase enzyme. These findings demonstrate that compounds 11 and 20 obeyed Lipinski's rule of five and could be used as privileged scaffolds for cancer therapy via their potential inhibition of mitochondrial complex I-associated hexokinase.
Subject(s)
Amino Acids/pharmacology , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Mitochondria/drug effects , Amino Acids/chemical synthesis , Amino Acids/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Drug Screening Assays, Antitumor , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hexokinase/antagonists & inhibitors , Hexokinase/metabolism , Humans , Mitochondria/metabolism , Schiff Bases/chemical synthesis , Schiff Bases/chemistry , Schiff Bases/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Tumor Cells, CulturedABSTRACT
In 1984, Japanese researchers led by the biochemist Hiroyoshi Hidaka described the first synthetic protein kinase inhibitors based on an isoquinoline sulfonamide structure (Hidaka et al. Biochemistry, 1984 Oct 9; 23(21): 5036-41. doi: 10.1021/bi00316a032). These led to the first protein kinase inhibitor approved for medical use (fasudil), an inhibitor of the AGC subfamily Rho kinase. With potencies strong enough to compete against endogenous ATP, the isoquinoline compounds established the druggability of the ATP binding site. Crystal structures of their protein kinase complexes, including with cAMP-dependent protein kinase (PKA), showed interactions that, on the one hand, could mimic ATP but, on the other hand, could be optimized for high potency binding, kinase selectivity, and diversification away from adenosine. They also showed the flexibility of the glycine-rich loop, and PKA became a major prototype for crystallographic and nuclear magnetic resonance (NMR) studies of protein kinase mechanism and dynamic activity control. Since fasudil, more than 70 kinase inhibitors have been approved for clinical use, involving efforts that progressively have introduced new paradigms of data-driven drug discovery. Publicly available data alone comprise over 5000 protein kinase crystal structures and hundreds of thousands of binding data. Now, new methods, including artificial intelligence techniques and expansion of protein kinase targeting approaches, together with the expiration of patent protection for optimized inhibitor scaffolds, promise even greater advances in drug discovery. Looking back to the time of the first isoquinoline hinge binders brings the current state-of-the-art into stark contrast. Appropriately for this Perspective article, many of the milestone papers during this time were published in Biochemistry (now ACS Biochemistry).
Subject(s)
Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Drug Design/history , Protein Kinase Inhibitors/pharmacology , Adenosine Triphosphate/metabolism , Artificial Intelligence , Binding Sites/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/ultrastructure , Data Science/history , Data Science/trends , Drug Design/methods , Drug Design/trends , Drug Discovery/history , Drug Discovery/methods , Drug Discovery/trends , History, 20th Century , Isoquinolines/chemistry , Isoquinolines/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Protein Kinase Inhibitors/chemistryABSTRACT
The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC-EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies.
Subject(s)
Adenoviridae/physiology , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Metapneumovirus/physiology , Respiratory Syncytial Virus, Human/physiology , Virus Replication , A549 Cells , Cell Line , Chemokines/immunology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , HEK293 Cells , Humans , Quinolines/pharmacologyABSTRACT
The present study investigated hyperalgesia during sickness syndrome in female rats. Hyperalgesia was induced by an intraperitoneal injection of lipopolysaccharide (LPS) or an intracerebroventricular injection of prostaglandin E2 (PGE2). No differences were found in basal mechanical and thermal thresholds or in LPS-induced hyperalgesia in sham-operated animals in the diestrus or proestrus phase or in ovariectomized (OVX) animals. However, higher levels of PGE2 where found in the cerebrospinal fluid of OVX animals compared to sham-operated females. Intracerebroventricular injection of PGE2 produced rapid mechanical hyperalgesia in sham-operated rats while these responses were observed at later times in OVX animals. The protein kinase A (PKA) inhibitor H-89 reduced mechanical PGE2-induced hyperalgesia in OVX female rats, whereas no effect was observed in sham-operated animals. In contrast, the exchange protein activated by cyclic adenosine monophosphate (cAMP; Epac) inhibitor ESI-09 reduced mechanical PGE2-induced hyperalgesia, whereas no effect was observed in OVX animals. PGE2 also induced thermal hyperalgesia in sham-operated and OVX female rats and a similar effect of ESI-09 was observed. These results suggest that PGE2-induced hyperalgesia that is observed during sickness syndrome has different signaling mechanisms in cycling and OVX female rats involving the activation of the cAMP-Epac or cAMP-PKA pathways, respectively.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dinoprostone/pharmacology , Estrous Cycle/drug effects , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Illness Behavior/drug effects , Animals , Cyclic AMP/antagonists & inhibitors , Dinoprostone/administration & dosage , Disease Models, Animal , Female , Hydrazones/pharmacology , Isoquinolines/pharmacology , Isoxazoles/pharmacology , Lipopolysaccharides/pharmacology , Ovariectomy , Rats , Rats, Wistar , Signal Transduction/drug effects , Sulfonamides/pharmacologyABSTRACT
DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr11 phosphorylation (H3-pThr11 ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr11 both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Damage , E2F1 Transcription Factor/genetics , Endoribonucleases/metabolism , Histones/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 1/metabolism , RNA-Binding Proteins/metabolism , CRISPR-Cas Systems , Cell Proliferation , Cells, Cultured , Checkpoint Kinase 1/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Endoribonucleases/deficiency , Endoribonucleases/isolation & purification , Epigenetic Repression , Gene Expression Regulation , Humans , Phosphoprotein Phosphatases/deficiency , Phosphoprotein Phosphatases/isolation & purification , Phosphorylation , Promoter Regions, Genetic , Protein Processing, Post-Translational , RNA, Messenger/metabolism , RNA-Binding Proteins/isolation & purification , Receptors, Neuropeptide Y/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Ultraviolet RaysABSTRACT
Organic anion transporter 3 (OAT3) plays an important role in the disposition of various anionic drugs which impacts the pharmacokinetics and pharmacodynamics of the therapeutics, thus influencing the pharmacological effects and toxicity of the drugs. In this study, we investigated the effect of insulin on the regulation of OAT3 function, expression, and SUMOylation. We demonstrated that insulin induced an increase in OAT3 transport activity through a dose- and time-dependent manner in COS-7 cells. The insulin-induced elevation in OAT3 function was blocked by PKA inhibitor H89, which correlated well with OAT3 protein expression. Moreover, both PKA activator Bt2-cAMP-induced increase and insulin-induced increase in OAT3 function were blocked by PKB inhibitor AKTi1/2. To further investigate the involvement of SUMOylation, we treated OAT3-expressing cells with insulin in presence or absence of H89 or AKTi1/2 followed by examining OAT3 SUMOylation. We showed that insulin enhanced OAT3 SUMOylation, and such enhancement was abrogated by H89 and AKTi1/2. Lastly, insulin increased OAT3 function and SUMOylation in rat kidney slice. In conclusion, our investigations demonstrated that insulin regulated OAT3 function, expression, and SUMOylation through PKA/PKB signaling pathway. Graphical abstract.
Subject(s)
Insulin, Regular, Human/pharmacology , Organic Anion Transporters, Sodium-Independent/metabolism , Animals , COS Cells , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Diabetes Mellitus, Type 2/drug therapy , Estrone/analogs & derivatives , Estrone/metabolism , Humans , Insulin, Regular, Human/therapeutic use , Isoquinolines , Kidney/drug effects , Kidney/metabolism , Models, Animal , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Rats , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Signal Transduction/drug effects , Sulfonamides , Sumoylation/drug effectsABSTRACT
Previously, we demonstrated that one single physical exercise session could positively modulate recognition memory persistence by D1/D5 activation. Here, we aim to investigate whether the effect of physical exercise on memory occurs due to the activation of both receptors, D1 and D5, or only one of them. Adult male Wistar rats were habituated on a treadmill one week before experiments. After learning session in the object recognition task, some animals received intrahippocampal infusions of the vehicle or a D1/D5 agonist (SKF 38393, 12.5 µg/µL/side), whereas others performed a single session of physical exercise on a treadmill (30 min at an intensity of 60-70% of indirect VO2 max.). Immediately after physical exercise, some animals received intrahippocampal infusions of vehicle or D1/D5 antagonist (SCH 23390, 1 µg/µL/side). Signaling pathways of D1 and D5 receptors in the hippocampus were evaluated by pharmacological activation or inactivation of protein kinases A (PKA) and C (PKC), respectively. According to previous findings, D1/D5 agonist and a single physical exercise session after learning promoted memory persistence, and D1/D5 block impaired physical exercise effect. Importantly, here we demonstrated for the first time that PKA inhibition, but not PKC, impairs the effect of acute physical exercise on memory persistence. Besides, PKA stimulation can promote its effects on memory. Therefore, we provide evidence that corroborates the idea that D1-like dopaminergic receptors, by activation of the PKA pathway, are involved in the effects of acute physical exercise on memory.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Hippocampus/metabolism , Memory/physiology , Physical Conditioning, Animal/physiology , Receptors, Dopamine D1/metabolism , Signal Transduction/physiology , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology , Animals , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dopamine Agonists/pharmacology , Exercise Test/methods , Hippocampus/drug effects , Male , Memory/drug effects , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Wistar , Receptors, Dopamine D1/agonists , Signal Transduction/drug effectsABSTRACT
Identifying a pharmacological agent that targets only one of more than 500 kinases present in humans is an important challenge. One potential solution to this problem is the development of bivalent kinase inhibitors, which consist of two connected fragments, each bind to a dissimilar binding site of the bisubstrate enzyme. The main advantage of bivalent (type V) kinase inhibitors is generating more interactions with target enzymes that can enhance the molecules' selectivity and affinity compared to single-site inhibitors. Earlier type V inhibitors were not suitable for the cellular environment and were mostly used in in vitro studies. However, recently developed bivalent compounds have high kinase affinity, high biological and chemical stability in vivo. This review summarized the hetero-bivalent kinase inhibitors described in the literature from 2014 to the present. We attempted to classify the molecules by serine/threonine and tyrosine kinase inhibitors, and then each target kinase and its hetero-bivalent inhibitor was assessed in depth. In addition, we discussed the analysis of advantages, limitations, and perspectives of bivalent kinase inhibitors compared with the monovalent kinase inhibitors.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Receptor, EphA1/antagonists & inhibitors , Receptor, EphA1/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolismABSTRACT
Prostaglandin E2 (PGE2)-induced coughs in vivo and vagal nerve depolarization in vitro are inhibited by systemic and local administration of prostaglandin EP3 receptor (L-798106) and TRPV1 antagonists (JNJ 17203212). These results indicate a modulating effect of TRPV1 on the EP3 receptor-mediated cough responses to PGE2 likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA, mainly stimulating TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically are co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) there was a cross-effect of TRPV1 and EP3 receptor on these neural excitations. To this end, aerosolized CA or PGE2 was inhaled by unanesthetized guinea pigs pretreated without or with each antagonist given in aerosol form. Immunofluorescence was applied to identify the co-expression of TRPV1 and EP3 receptor in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell voltage patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in individual vagal pulmonary C-neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the evoked currents. We found that PGE2-induced cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. Approximately 1/4 of vagal pulmonary C-neurons co-expressed EP3 with a cell size < 20 µm. Both CAP- and PGE2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the cross-effect of both antagonists on cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the cough response to PGE2.
Subject(s)
Bronchi/metabolism , Cough/metabolism , Nerve Fibers, Unmyelinated/metabolism , Receptors, Prostaglandin E, EP3 Subtype/metabolism , TRPV Cation Channels/metabolism , Aminopyridines/pharmacology , Animals , Capsaicin , Citric Acid/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Dinoprostone , Guinea Pigs , Ion Channel Gating/drug effects , Male , Models, Biological , Nerve Fibers, Unmyelinated/drug effects , Nodose Ganglion/drug effects , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pulmonary Ventilation/drug effects , Receptors, Prostaglandin E, EP3 Subtype/antagonists & inhibitors , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , TRPV Cation Channels/antagonists & inhibitors , Vagus Nerve/drug effects , Vagus Nerve/metabolismABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive decline. In hippocampal neurons, the pathological features of AD include the accumulation of extracellular amyloid-beta peptide (Aß) accompanied by oxidative stress, mitochondrial dysfunction, and neuron loss. A decrease in neuroprotective Protein Kinase A (PKA) signaling contributes to mitochondrial fragmentation and neurodegeneration in AD. By associating with the protein scaffold Dual-Specificity Anchoring Protein 1 (D-AKAP1), PKA is targeted to mitochondria to promote mitochondrial fusion by phosphorylating the fission modulator dynamin-related protein 1 (Drp1). We hypothesized that (1) a decrease in the endogenous level of endogenous D-AKAP1 contributes to decreased PKA signaling in mitochondria and that (2) restoring PKA signaling in mitochondria can reverse neurodegeneration and mitochondrial fragmentation in neurons in AD models. Through immunohistochemistry, we showed that endogenous D-AKAP1, but not other mitochondrial proteins, is significantly reduced in primary neurons treated with Aß42 peptide (10µM, 24 h), and in the hippocampus and cortex from asymptomatic and symptomatic AD mice (5X-FAD). Transiently expressing wild-type, but not a PKA-binding deficient mutant of D-AKAP1, was able to reduce mitochondrial fission, dendrite retraction, and apoptosis in primary neurons treated with Aß42. Mechanistically, the protective effects of D-AKAP1/PKA are moderated through PKA-mediated phosphorylation of Drp1, as transiently expressing a PKA phosphomimetic mutant of Drp1 (Drp1-S656D) phenocopies D-AKAP1's ability to reduce Aß42-mediated apoptosis and mitochondrial fission. Overall, our data suggest that a loss of D-AKAP1/PKA contributes to mitochondrial pathology and neurodegeneration in an in vitro cell culture model of AD.