ABSTRACT
BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for the treatment of various malignancies; however, its therapeutic potential is limited because of the frequent occurrence of tumor cell resistance. In this study, we determined whether TRAIL resistance acquired by repeated administration could be overcome by HDAC inhibition in human colorectal cancer cells. METHODS: TRAIL-resistant HCT116 human colorectal cancer cells (HCT116-TR) were generated by repeated treatment with 10 and 25 ng/mL TRAIL twice weekly for 28 days. RESULTS: The resulting TRAIL-resistant cells were noncross-resistant to other chemotherapeutic agents. The levels of histone acetylation-related proteins, such as ac-histone H4 and HDAC1, were altered in HCT116-TR cells compared with the parental HCT116 cell line. The combined treatment with TRAIL and HDAC inhibitors significantly increased apoptosis in HCT116-TR cells and indicated a synergistic effect. The mechanism by which HDAC inhibition sensitizes HCT116-TR cells to TRAIL is dependent on the intrinsic pathway. In addition, we found that HDAC inhibition enhanced the sensitivity of cells to TRAIL through mitogen-activated protein kinases/CCAAT/enhancer-binding protein homologs of protein-dependent upregulation of death receptor 5. CONCLUSION: These results suggest that histone acetylation is responsible for acquired TRAIL resistance after repeated exposure and acquired resistance to TRAIL may be overcome by combination therapies with HDAC inhibitors.
ABSTRACT
Developing novel therapies that outperform the existing chemotherapeutic treatments is required for treatment-resistant ovarian clear cell carcinoma. We investigated the antitumor effect of metformin on ovarian clear cell carcinoma, enhancement of the antitumor effect by its combination with chemotherapy, and its molecular regulatory mechanism. First, we evaluated the viability of ovarian clear cell carcinoma lines using the water-soluble tetrazolium-1 assay and found that metformin suppressed cell viability. Cell viability was significantly suppressed by co-treatment with cisplatin and metformin. In contrast, co-treatment with paclitaxel and metformin showed no significant difference in viability compared with the group without metformin. Western blot analysis showed increased phosphorylation of AMP-activated protein kinase in some cell lines and suppressed phosphorylation of the mammalian target of rapamycin in a particular cell line. Flow cytometry analysis revealed a significant increase in the rate of apoptosis in the metformin-treated group and rate of cell cycle arrest at the G2/M phase in a particular cell line. These results indicated that metformin may be effective against cultured ovarian clear cell carcinoma cells, particularly in combination with cisplatin.
Subject(s)
Adenocarcinoma, Clear Cell , Antineoplastic Agents , Apoptosis , Cell Survival , Cisplatin , Metformin , Ovarian Neoplasms , Metformin/pharmacology , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Female , Cisplatin/pharmacology , Apoptosis/drug effects , Adenocarcinoma, Clear Cell/drug therapy , Adenocarcinoma, Clear Cell/pathology , Cell Line, Tumor , Cell Survival/drug effects , Antineoplastic Agents/pharmacology , AMP-Activated Protein Kinases/metabolism , Paclitaxel/pharmacology , Phosphorylation/drug effects , TOR Serine-Threonine Kinases/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , G2 Phase Cell Cycle Checkpoints/drug effectsABSTRACT
Alzheimer's disease is a multifaceted neurodegenerative disease. Cholinergic dysfunction, amyloid ß toxicity, tauopathies, oxidative stress, neuroinflammation are among the main pathologies of the disease. Ligands targeting more than one pathology, multi-target directed ligands, attract attention in the recent years to tackle Alzheimer's disease. In this review, we aimed to cover different biochemical pathways, that are revealed in recent years for the pathology of the disease, as druggable targets such as cannabinoid receptors, matrix metalloproteinases, histone deacetylase and various kinases including, glycogen synthase kinase-3, mitogen-activated protein kinase and c-Jun N-terminal kinase, and their ligands for the treatment of Alzheimer's disease in the hope of providing more realistic insights into the field.
ABSTRACT
The dysregulation of kinases has emerged as a major class of targets for anticancer drug discovery given its node roles in the etiology of tumorigenesis, progression, invasion, and metastasis of malignancies, which is validated by the FDA approval of 28 small molecule kinase inhibitor (SMKI) drugs for cancer treatment at the end of 2015. While the preclinical and clinical data of these drugs are widely presented, it is highly essential to give an updated review on the medical indications, design principles and binding modes of these anti-tumor SMKIs approved by the FDA to offer insights for the future development of SMKIs with specific efficacy and safety.
Subject(s)
Antineoplastic Agents , Drug Approval , Neoplasms , Protein Kinase Inhibitors , Small Molecule Libraries , United States Food and Drug Administration , Humans , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Neoplasms/drug therapy , United States , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Molecular Structure , Binding Sites , Structure-Activity RelationshipABSTRACT
Loss-of-function mutants are fundamental resources for gene function studies. However, it is difficult to generate viable and heritable knockout mutants for essential genes. Here, we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1 (OsMPK1) results in weak mutants. This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations. Using the same C-terminal editing approach, we also obtained viable mutants for a wall-associated protein kinase (Os07g0493200) and a leucine-rich repeat receptor-like protein kinase (Os01g0239700), while the null mutations of these genes were lethal. These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus, which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00165-5.
ABSTRACT
OBJECTIVES: Pilocarpine is commonly used clinically to treat dry mouth. The long-term administration of pilocarpine reportedly improves salivary secretion more effectively than short-term administration. Therefore, we hypothesized that pilocarpine alters gene expression in salivary glands via muscarinic receptor stimulation. This study aimed to investigate the effects of pilocarpine use on gene expression mediated by mitogen-activated protein kinase (MAPK) activity. METHODS: The effects of pilocarpine on gene expression were investigated in rats and human salivary gland (HSY) cells using several inhibitors of intracellular signaling pathways. Gene expression in the rat submandibular gland and HSY cells was determined using reverse transcription-quantitative polymerase chain reaction analysis of total RNA. RESULTS: In animal experiments, at 7 days after pilocarpine stimulation, Ctgf and Sgk1 expressions were increased in the submandibular gland. In cell culture experiments, pilocarpine increased Ctgf expression in HSY cells. The mitogen-activated protein kinase kinase inhibitor trametinib, the Src inhibitor PP2, and the muscarinic acetylcholine receptor antagonist atropine suppressed the effect of pilocarpine on gene expression. CONCLUSIONS: Pilocarpine enhances Ctgf and Sgk1 expressions by activating Src-mediated MAPK activity. Although further studies are required to fully understand the roles of Ctgf and Sgk1, changes in gene expression may play an important role in improving salivary secretions.
ABSTRACT
Melatonin (Mel) is recognized as a prominent plant growth regulator. This study investigated the alleviating effect of Mel pretreatment on growth inhibition caused by low-temperature (LT) stress (10 °C/6 °C) in cucumber seedlings and explored the role of the Ca2+/Calcium-dependent protein kinases (CPKs) signaling pathway in Mel-regulated LT tolerance. The main results are as follows: compared to LT treatment alone, 100 µM Mel increased both the content of Ca2+ (highest about 42.01%) and the expression levels of Ca2+ transporter and cyclic nucleotide-gated channel (CNGC) genes under LT. Similarly, Mel enhanced the content of CPKs (highest about 27.49%) and the expression levels of CPKs family genes in cucumber leaves under LT. Additionally, pretreatment with 100 µM Mel for three days strengthened the antioxidant defense and photosynthesis of seedlings under LT. Genes in the ICE-CBF-COR pathway and the MAPK cascade were upregulated by Mel, with maximum upregulations reaching approximately 2.5-fold and 1.9-fold, respectively, thus conferring LT tolerance to cucumber seedlings. However, the above beneficial effects of Mel were weakened by co-treatment with calcium signaling blockers (LaCl3 or EGTA) or CPKs inhibitors (TFP or W-7), suggesting that the Ca2+/CPKs pathway is involved in the Mel-mediated regulation of LT tolerance. In conclusion, this study revealed that Mel can alleviate growth inhibition in cucumber seedlings under LT stress and demonstrated that the Ca2+/CPKs signaling pathway is crucial for the Mel-mediated enhancement of LT tolerance. The findings hold promise for providing theoretical insights into the application of Mel in agricultural production and for investigating its underlying mechanisms of action.
Subject(s)
Cold Temperature , Cucumis sativus , Melatonin , Plant Proteins , Seedlings , Signal Transduction , Cucumis sativus/drug effects , Cucumis sativus/genetics , Cucumis sativus/metabolism , Cucumis sativus/growth & development , Melatonin/pharmacology , Seedlings/drug effects , Seedlings/growth & development , Seedlings/metabolism , Signal Transduction/drug effects , Plant Proteins/metabolism , Plant Proteins/genetics , Calcium/metabolism , Gene Expression Regulation, Plant/drug effects , Protein Kinases/metabolism , Protein Kinases/genetics , Photosynthesis/drug effectsABSTRACT
Protein phosphorylation is an important link in a variety of signaling pathways, and most of the important life processes in cells involve protein phosphorylation. Based on the amino acid residues of phosphorylated proteins, protein kinases can be categorized into the following families: serine/threonine protein kinases, tyrosine-specific protein kinases, histidine-specific protein kinases, tryptophan kinases, and aspartate/glutamyl protein kinases. Of all the protein kinases, most are serine/threonine kinases, where serine/threonine protein kinases are protein kinases that catalyze the phosphorylation of serine or threonine residues on target proteins using ATP as a phosphate donor. The current socially accepted classification of serine/threonine kinases is to divide them into seven major groups: protein kinase A, G, C (AGC), CMGC, Calmodulin-dependent protein kinase (CAMK), Casein kinase (CK1), STE, Tyrosine kinase (TKL) and others. After decades of research, a preliminary understanding of the specific classification and respective functions of serine/threonine kinases has entered a new period of exploration. In this paper, we review the literature of the previous years and introduce the specific signaling pathways and related therapeutic modalities played by each of the small protein kinases in the serine/threonine protein kinase family, respectively, in some common cardiovascular system diseases such as heart failure, myocardial infarction, ischemia-reperfusion injury, and diabetic cardiomyopathy. To a certain extent, the current research results, including molecular mechanisms and therapeutic methods, are fully summarized and a systematic report is made for the prevention and treatment of cardiovascular diseases in the future.
Subject(s)
Cardiovascular Diseases , Protein Serine-Threonine Kinases , Humans , Cardiovascular Diseases/enzymology , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/metabolism , Animals , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Phosphorylation , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacologyABSTRACT
In our previous study, we found that small ubiquitin-related modifier (SUMO)-activating enzyme ubiquitin-associated-2 domain (UBA2) was upregulated in hepatocellular carcinoma (HCC) patients who were insensitive to chemoembolization. In this study, we aimed to investigate the role of UBA2 in HCC progression. Three cohorts were used to evaluate the efficacy of UBA2 as a prognostic factor for HCC. Our results indicated that UBA2 was associated with aggressive clinical behaviors and was a strong indicator of poor prognosis in HCC. In vitro experiments demonstrated that UBA2 accelerated cell growth, invasion, and migration. These results were further supported by in vivo experiments. RNA-sequencing analysis indicated NQO1 as a target of UBA2, with its levels altering following UBA2 manipulation. The results were verified by western blotting (WB) and quantitative PCR. The SUMOplot Analysis Program predicted lysine residue K240 as a modification target of UBA2, which was confirmed by immunoprecipitation (IP) assays. Subsequent mutation of NQO1 at K240 in HCC cell lines and functional assays revealed the significance of this modification. In addition, the oncogenic effect of UBA2 could be reversed by the SUMO inhibitor ML792 in vivo and in vitro. In conclusion, our study elucidated the regulatory mechanism of UBA2 in HCC and suggested that the SUMO inhibitor ML792 may be an effective combinatory treatment for patients with aberrant UBA2 expression.
ABSTRACT
Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 µM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 µM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 µM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.
Subject(s)
Docosahexaenoic Acids , Embryonic Development , Energy Metabolism , In Vitro Oocyte Maturation Techniques , Oocytes , Animals , Docosahexaenoic Acids/pharmacology , Docosahexaenoic Acids/administration & dosage , Oocytes/drug effects , Swine/embryology , In Vitro Oocyte Maturation Techniques/veterinary , In Vitro Oocyte Maturation Techniques/methods , Energy Metabolism/drug effects , Embryonic Development/drug effects , Homeostasis/drug effects , FemaleABSTRACT
The scaffold proteins JIP1 and JIP2 intervene in the c-Jun N-terminal kinase (JNK) pathway to mediate signaling specificity by coordinating the simultaneous assembly of multiple kinases. Using NMR, we demonstrate that JIP1 and JIP2 heterodimerize via their SH3 domains with the affinity of heterodimerization being comparable to homodimerization. We present the high-resolution crystal structure of the JIP2-SH3 homodimer and the JIP1-JIP2-SH3 heterodimeric complex. The JIP2-SH3 structure reveals how charge differences in residues at its dimer interface lead to formation of compensatory hydrogen bonds and salt bridges, distinguishing it from JIP1-SH3. In the JIP1-JIP2-SH3 complex, structural features of each homodimer are employed to stabilize the heterodimer. Building on these insights, we identify key residues crucial for stabilizing the dimer of both JIP1 and JIP2. Through targeted mutations in cellulo, we demonstrate a functional role for the dimerization of the JIP1 and JIP2 scaffold proteins in activation of the JNK signaling pathway.
ABSTRACT
Insulin is a promising neuroprotector. To better understand the mechanism of insulin action, it was important to show its ability to diminish autophagic neuronal death in animals with brain ischemic and reperfusion injury. In forebrain ischemia and reperfusion, the number of live neurons in the hippocampal CA1 region and frontal cortex of rats decreased to a large extent. Intracerebroventricular administration of the autophagy and apoptosis inhibitors to ischemic rats significantly increased the number of live neurons and showed that the main part of neurons died from autophagy and apoptosis. Intranasal administration of 0.5 IU of insulin per rat (before ischemia and daily during reperfusion) increased the number of live neurons in the hippocampal CA1 region and frontal brain cortex. In addition, insulin significantly diminished the level of autophagic marker LC3B-II in these forebrain regions, which markedly increased during ischemia and reperfusion. Our studies demonstrated for the first time the ability of insulin to decrease autophagic neuronal death, caused by brain ischemia and reperfusion. Insulin administered intranasally activated the Akt-kinase (activating the mTORC1 complex, which inhibits autophagy) and inhibited the AMP-activated protein kinase (which activates autophagy) in the hippocampus and frontal cortex of rats with brain ischemia and reperfusion.
ABSTRACT
A recent study in mBio reports the construction and preliminary screening of a library containing mutants of 99 of the 119 predicted protein kinases in Candida albicans (the majority of the remaining 20 are probably essential) (J. Kramara, M.-J. Kim, T. L. Ollinger, L. C. Ristow, et al., mBio e01249-24, 2024, https://doi.org/10.1128/mbio.01249-24). Using a quantitative competition assay in 10 conditions that represent nutritional, osmotic, cell wall, and pH stresses that are considered to model various aspects of the host environment allowed them to phenotypically cluster kinases, which highlight both the integration and specialization of signaling pathways, suggesting novel functions for many kinases. In addition, they tackle two complex and partially overlapping differentiation events, hyphal morphogenesis and biofilm formation. They find that a remarkable 88% of the viable kinase mutants in C. albicans affect hyphal growth, illustrating how integrated morphogenesis is in the overall biology of this organism, and begin to dissect the regulatory relationships that control this key virulence trait.
Subject(s)
Biofilms , Candida albicans , Hyphae , Mutation , Protein Kinases , Candida albicans/genetics , Candida albicans/enzymology , Candida albicans/growth & development , Hyphae/growth & development , Hyphae/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Biofilms/growth & development , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Signal Transduction , Virulence/geneticsABSTRACT
The involvement of inwardly rectifying potassium channel 4.1 (Kir4.1) in neuropathic pain has been established. However, there is limited understanding of the downstream mechanism through which Kir4.1 contributes to orofacial neuropathic pain. The objective of this study was to examine the regulation of Kir4.1 on the expression of pannexin 3 (Panx3) in the trigeminal ganglion (TG) and the underlying mechanism in the context of orofacial neuropathic pain caused by chronic constriction injury of the infraorbital nerve (CCI-ION). The study observed a significant increase in Panx3 expression in the TG of mice with CCI-ION. Inhibition of Panx3 in the TG of CCI-ION mice resulted in alleviation of orofacial mechanical allodynia. Furthermore, conditional knockdown (CKD) of Kir4.1 in the TG of both male and female mice led to mechanical allodynia and upregulation of Panx3 expression. Conversely, overexpression of Kir4.1 decreased Panx3 levels in the TG and relieved mechanical allodynia in CCI-ION mice. In addition, silencing Kir4.1 in satellite glial cells (SGCs) decreased Panx3 expression and increased the phosphorylation of P38 MAPK. Moreover, silencing Kir4.1 in SGCs increased the levels of reactive oxygen species (ROS). The elevated phosphorylation of P38 MAPK resulting from Kir4.1 silencing was inhibited by using a superoxide scavenger known as the tempol. Silencing Panx3 in the TG in vivo attenuated the mechanical allodynia caused by Kir4.1 CKD. In conclusion, these findings suggest that the reduction of Kir4.1 promotes the expression of Panx3 by activating the ROS-P38 MAPK signalling pathway, thus contributing to the development of orofacial neuropathic pain.
Subject(s)
Connexins , Neuralgia , Reactive Oxygen Species , p38 Mitogen-Activated Protein Kinases , Animals , Male , Reactive Oxygen Species/metabolism , Neuralgia/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Mice , Female , Connexins/metabolism , Connexins/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Facial Pain/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Trigeminal Ganglion/metabolism , Hyperalgesia/metabolism , Mice, Inbred C57BL , MAP Kinase Signaling System/physiologyABSTRACT
OBJECTIVES: To observe the effect of electroacupuncture (EA) stimulation of "Zusanli"(ST36) and"Xuehai"(SP10) on the angiogenesis of the local injured skin tissue in mice with psoriasis, so as to explore its mechanisms underlying improvement of psoriasis-induced skin lesions. METHODS: A total of 24 female BALB/c mice aged 6-8 weeks were randomly divided into control, model and EA groups, with 8 mice in each group. The psoriasis-like skin lesion model was established by application of 5% imiquimod (IMQ) cream to the mice's back skin, 62.5 mg/d, for 7 days after local depilation, and the mice of the control group received local application of an equal amount of petroleum jelly once a day for 7 days. EA stimulation (2 Hz/100 Hz) was applied to ST36 and SP10 for 30 min, once daily for 7 consecutive days. Photos of the topical injured skin at the back were taken every day, and the severity of psoriasis lesions (psoriasis area and severity index ï¼»PASIï¼½) was scaled. Following H.E. staining, the morphological changes in the injured skin tissue were observed with epidermal thickness analyzed, and the Masson staining was used to observe the proportion of collagen fibers in the injured skin tissues. Immunohistochemical method was used to detect the expression of microvascular markers CD31 and vascular endothelial growth factor (VEGF) and the microvascular density (MVD) was calculated. Western blot was used to detect the expression levels of CD31, VEGF proteins and mitogen activated protein kinases (MAPK) signaling pathway related proteins p38, phosphorylated p38 (p-p38), extracellular regulated protein kinases (ERK), p-ERK, c-Jun N-terminal kinase (JNK) and p-JNK in the injured skin tissue. RESULTS: Compared with the control group, the mice in the model group showed an evident increase in the erythema score, scales score, skin thickening score and PASI score, epidermal thickness, proportion of the collagen fibers, MVD value of CD31 and VEGF, and expression levels of CD31 and VEGF proteins, and p-p38/p38, p-ERK/ERK and p-JNK/JNK ratios in the injured skin tissue (P<0.001, P<0.01). In contrast to the model group, the EA group had a significant decrease in the levels of all the indexes mentioned above (P<0.05, P<0.01, P<0.001). CONCLUSIONS: EA intervention can improve the psoriasis-like skin lesions induced by IMQ in mice, which may be related with its functions in down-regulating the expression of angiogenic related factors CD31 and VEGF proteins and MAPK signaling pathway related proteins in the topical injured skin tissue.
Subject(s)
Electroacupuncture , Mice, Inbred BALB C , Psoriasis , Vascular Endothelial Growth Factor A , Animals , Psoriasis/therapy , Psoriasis/metabolism , Mice , Female , Humans , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/genetics , Skin/blood supply , Skin/metabolism , Neovascularization, Pathologic/therapy , Neovascularization, Pathologic/metabolism , Disease Models, Animal , Acupuncture Points , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/genetics , AngiogenesisABSTRACT
AIMS: Leishmaniasis, caused by the protozoan parasite poses a significant health burden globally. With a very few specific drugs, increased drug resistance it is important to look for drug repurposing along with the identification of pre-clinical candidates against visceral leishmaniasis. This study aims to identify potential drug candidates against visceral leishmaniasis by targeting leishmanial MAP kinases and screening FDA approved protein kinase inhibitors. MATERIALS AND METHODS: MAP kinases were identified from the Leishmania genome. 12 FDA approved protein kinase inhibitors were screened against Leishmania MAP kinases. Binding affinity, ADME and toxicity of identified drug candidates were profiled. The anti-proliferative effects and mechanism of action were assessed in Leishmania, including changes in cell morphology, flagellar length, cell cycle progression, reactive oxygen species (ROS) generation, and intra-macrophage parasitic burden. KEY FINDINGS: 23 MAP kinases were identified from the Leishmania genome. Sorafenib and imatinib emerged as repurposable drug candidates and demonstrated excellent anti-proliferative effects in Leishmania. Treatment with these inhibitors resulted in significant changes in cell morphology, flagellar length, and cell cycle arrest. Furthermore, sorafenib and imatinib promoted ROS generation and reduced intra-macrophage parasitic burden, and elicited anti-leishmanial activity in in vivo experimental VL models. SIGNIFICANCE: Collectively, these results imply involvement of MAP kinases in infectivity and survival of the parasite and can pave the avenue for repurposing sorafenib and imatinib as anti-leishmanial agents. These findings contribute to the exploration of new treatment options for visceral leishmaniasis, particularly in the context of emerging drug resistance.
Subject(s)
Antiprotozoal Agents , Drug Repositioning , Leishmania , Protein Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Mice , Leishmania/drug effects , Leishmania/enzymology , Antiprotozoal Agents/pharmacology , Reactive Oxygen Species/metabolism , Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , Mice, Inbred BALB C , Humans , Macrophages/parasitology , Macrophages/drug effects , Macrophages/metabolism , Female , Sorafenib/pharmacology , Imatinib Mesylate/pharmacologyABSTRACT
Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-ß4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-ß4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-ß4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.
Subject(s)
Molecular Dynamics Simulation , Allosteric Regulation , Adenosine Triphosphate/metabolism , Catalytic Domain , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Protein Conformation , Protein Binding , Nucleotides/metabolism , Substrate Specificity , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/chemistry , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/geneticsABSTRACT
Traumatic spinal cord injury (SCI) is a life-threatening and life-altering condition that results in debilitating sensorimotor and autonomic impairments. Despite significant advances in the clinical management of traumatic SCI, many patients continue to suffer due to a lack of effective therapies. The initial mechanical injury to the spinal cord results in a series of secondary molecular processes and intracellular signaling cascades in immune, vascular, glial, and neuronal cell populations, which further damage the injured spinal cord. These intracellular cascades present promising translationally relevant targets for therapeutic intervention due to their high ubiquity and conservation across eukaryotic evolution. To date, many therapeutics have shown either direct or indirect involvement of these pathways in improving recovery after SCI. However, the complex, multifaceted, and heterogeneous nature of traumatic SCI requires better elucidation of the underlying secondary intracellular signaling cascades to minimize off-target effects and maximize effectiveness. Recent advances in transcriptional and molecular neuroscience provide a closer characterization of these pathways in the injured spinal cord. This narrative review article aims to survey the MAPK, PI3K-AKT-mTOR, Rho-ROCK, NF-κB, and JAK-STAT signaling cascades, in addition to providing a comprehensive overview of the involvement and therapeutic potential of these secondary intracellular pathways following traumatic SCI.
Subject(s)
Signal Transduction , Spinal Cord Injuries , Spinal Cord Injuries/metabolism , Humans , Animals , TOR Serine-Threonine Kinases/metabolismABSTRACT
A study of two enzymes in the brain reveals new insights into how redox reactions regulate the activity of protein kinases.
Subject(s)
Oxidation-Reduction , Brain/metabolism , Brain/physiology , Humans , Animals , Protein Kinases/metabolismABSTRACT
The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule -1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.