Understanding the pathogenic significance of altered calcium-calmodulin signaling in T cells in autoimmune diseases.

Calcium/calmodulin-dependent protein kinase IV (CaMK4) serves as a pivotal mediator in the regulation of gene expression, influencing the activity of transcription factors within a variety of immune cells, including T cells. Altered CaMK4 signaling is implicated in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and psoriasis, which are characterized by dysregulated immune responses and clinical complexity. These conditions share common disturbances in immune cell functionality, cytokine production, and autoantibody generation, all of which are associated with disrupted calcium-calmodulin signaling. This review underscores the consequences of dysregulated CaMK4 signaling across these diseases, with an emphasis on its impact on Th17 differentiation and T cell metabolism-processes central to maintaining immune homeostasis. A comprehensive understanding of roles of CaMK4 in gene regulation across various autoimmune disorders holds promise for the development of targeted therapies, particularly for diseases driven by Th17 cell dysregulation.

Sphingosylphosphorylcholine alleviates pressure overload-induced myocardial remodeling in mice via inhibiting CaM-JNK/p38 signaling pathway.

Apoptosis plays a critical role in the development of heart failure, and sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid naturally occurring in blood plasma. Some studies have shown that SPC inhibits hypoxia-induced apoptosis in myofibroblasts, the crucial non-muscle cells in the heart. Calmodulin (CaM) is a known SPC receptor. In this study we investigated the role of CaM in cardiomyocyte apoptosis in heart failure and the associated signaling pathways. Pressure overload was induced in mice by trans-aortic constriction (TAC) surgery. TAC mice were administered SPC (10 µM·kg-1·d-1) for 4 weeks post-surgery. We showed that SPC administration significantly improved survival rate and cardiac hypertrophy, and inhibited cardiac fibrosis in TAC mice. In neonatal mouse cardiomyocytes, treatment with SPC (10 µM) significantly inhibited Ang II-induced cardiomyocyte hypertrophy, fibroblast-to-myofibroblast transition and cell apoptosis accompanied by reduced Bax and phosphorylation levels of CaM, JNK and p38, as well as upregulated Bcl-2, a cardiomyocyte-protective protein. Thapsigargin (TG) could enhance CaM functions by increasing Ca2+ levels in cytoplasm. TG (3 µM) annulled the protective effect of SPC against Ang II-induced cardiomyocyte apoptosis. Furthermore, we demonstrated that SPC-mediated inhibition of cardiomyocyte apoptosis involved the regulation of p38 and JNK phosphorylation, which was downstream of CaM. These results offer new evidence for SPC regulation of cardiomyocyte apoptosis, potentially providing a new therapeutic target for cardiac remodeling following stress overload.

Inhibition of Erb-B2 Receptor Tyrosine Kinase 3 and Associated Regulatory Pathways Potently Impairs Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, currently untreatable Schwann cell-derived neoplasms with hyperactive mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. To identify potential therapeutic targets, previous studies used genome-scale shRNA screens that implicated the neuregulin-1 receptor erb-B2 receptor tyrosine kinase 3 (erbB3) in MPNST proliferation and/or survival. The current study shows that erbB3 is commonly expressed in MPNSTs and MPNST cell lines and that erbB3 knockdown inhibits MPNST proliferation and survival. Kinomic and microarray analyses of Schwann and MPNST cells implicate Src- and erbB3-mediated calmodulin-regulated signaling as key pathways. Consistent with this, inhibition of upstream (canertinib, sapitinib, saracatinib, and calmodulin) and parallel (AZD1208) signaling pathways involving mitogen-activated protein kinase and mammalian target of rapamycin reduced MPNST proliferation and survival. ErbB inhibitors (canertinib and sapitinib) or erbB3 knockdown in combination with Src (saracatinib), calmodulin [trifluoperazine (TFP)], or proviral integration site of Moloney murine leukemia kinase (AZD1208) inhibition even more effectively reduces proliferation and survival. Drug inhibition enhances an unstudied calmodulin-dependent protein kinase IIα phosphorylation site in an Src-dependent manner. The Src family kinase inhibitor saracatinib reduces both basal and TFP-induced erbB3 and calmodulin-dependent protein kinase IIα phosphorylation. Src inhibition (saracatinib), like erbB3 knockdown, prevents these phosphorylation events; and when combined with TFP, it even more effectively reduces proliferation and survival compared with monotherapy. These findings implicate erbB3, calmodulin, proviral integration site of Moloney murine leukemia kinases, and Src family members as important therapeutic targets in MPNSTs and demonstrate that combinatorial therapies targeting critical MPNST signaling pathways are more effective.

Knockdown of CALM2 increases the sensitivity to afatinib in HER2-amplified gastric cancer cells by regulating the Akt/FoxO3a/Puma axis.

Gastric cancer (GC) is a global health issue that lacks effective treatment options. Afatinib is a tyrosine kinase inhibitor (TKI) that has shown promising results in the treatment of GC. However, resistance to afatinib is inevitable and hampers its clinical application. To date, there is limited knowledge regarding the mechanisms underlying the resistance of GC cells to afatinib. This study aimed to identify novel factors that may contribute to the resistance of GC cells to afatinib. We found that upregulation of calmodulin 2 (CALM2), a member of the CALM family, confers resistance to afatinib in GC cells. Knockdown of CALM2 can overcome the resistance to afatinib by promoting mitochondrial apoptosis in a caspase-dependent manner. Mechanistically, it was found that the downregulation of CALM2 led to the upregulation of the FoxO3a/Puma axis. Inhibition of either FoxO3a or Puma abrogated the effects of CALM2 downregulation in GC cells. In addition, we revealed that CALM2 knockdown inhibited Akt signaling, which is responsible for blocking the FoxO3a/Puma axis. Altogether, our results indicated that CALM2 could be considered a potential target to overcome the resistance of GC cells to afatinib.

Combined ligand-based and structure-based design of PDE 9A inhibitors against Alzheimer's disease.

PDE9 enzyme hydrolyzes cGMP, which is involved in the regulation of synaptic plasticity through the NMDA pathway (a well-known excitotoxic target for AD) via activation of calcium/calmodulin-dependent neuronal NO synthases in the postsynaptic neurons. The inhibition of PDE9 leads to elevated cGMP levels, causing enhanced NMDA signaling and thus contributing to an increase in synaptic plasticity and stabilization. Therefore, it could be considered a pertinent target for AD drug discovery. PF-04447943 and BI-409306 targeting PDE9 are undergoing clinical trials (Phase II). The present study encompasses a pharmacophoric approach to identify potent PDE9 inhibitors using various computational methods. Pharmacophores generated from the PDB 6A3N yielded 37,554 virtual hits, which underwent drug likeliness and PAINS filtering to arrive at a few virtual leads. The leads were further subjected to extra precision docking, ADMET predictions, and molecular dynamics. The final hits, ZINC000001305675 and ZINC000000377099, showed superior docking scores of - 10.90 and - 10.30 kcal/mol and satisfactory predicted ADMET scores. The hits were subjected to molecular dynamics (MD) studies, wherein they formed stable complexes with PDE9 protein and had ligand RMSDs within acceptable limits. The processes involved in the combined ligand and structure-based strategies.

¿Es el cinacalcet un tratamiento coste-efectivo en el hiperparatiroidismo secundario severo en pacientes en hemodiálisis? / No disponible

Introducción: Un estudio previo mostró que la adición de cinacalcet a la vitamina D conseguía una mejor respuesta del Hiperparatiroidismo secundario (HPTS) en pacientes en hemodiálisis. El objetivo del presente estudio fue conocer el coste adicional que supone la adición de cinacalcet al tratamiento estándar en pacientes con HPTS severo teniendo en cuenta los objetivos terapéuticos obtenidos. Métodos: Estudio prospectivo durante 12 meses en 23 pacientes con HPTS severo, en los que no se podía mantener un tratamiento continuado con vitamina D por presentar hipercalcemia y/o hiperfosforemia. Se analizaron 2 regímenes de tratamiento: tratamiento estándar (m 0) y tratamiento estándar asociado a cinacalcet (m 12). Se analizaron las siguientes variables: iPTH, calcio, fósforo, producto CaxP, dosis de vitamina D, dosis de captores del fósforo y% de cumplimiento de indicadores. Los resultados del análisis se expresan en términos de coste-incremental y coste consecuencia por paciente que consigue el objetivo terapéutico en base a 5 marcadores: PTH < 800 pg/ml, PTH entre 150 y 300 pg/ml, Calcio < 9,5 mg/dl, Fósforo < 5,5mg/dl y Ca x P < 55.Resultados: A los 12 meses de tratamiento con cinacalcet, la proporción de pacientes que alcanzaron los 4 objetivos simultáneamente pasó de 0% a 52,1%. Cinacalcet permitió un ahorro en medicación concomitante (sevelamer, vitamina D e hidróxido de aluminio), que minimizó su coste adicional, suponiendo un incremento global de 149 e/mes. En términos de costes y consecuencias, cinacalcet conseguía una reducción del porcentaje de pacientes con PTH > 800 pg/ml a la mitad de coste que el tratamiento estándar. (651,35 e vs 1.363,68 e). La falta de pacientes con PTH entre 150 y 300 pg/ml en el m0 (sin cinacalcet) no permitió la comparación entre el momento basal y el final del estudio. Cinacalcet permitía una consecución de los objetivos de calcio, fósforo y producto calcio-fósforo en su conjunto más coste-efectiva (2.164,2 e vs 2.684,8 e). Conclusiones: Los pacientes tratados con cinacalcet presentan un coste por éxito terapéutico menor que los pacientes sin cinacalcet (pre-tratamiento), a pesar del mayor coste de adquisición de cinacalcet. La capacidad de cinacalcet de reducir la secreción de PTH, junto a la reducción en el Ca, P, y producto Ca x P, proporciona una alternativa al tratamiento tradicional y debe ser tenida en cuenta en el HPTS sever (AU)

Background: A previous study using cinacalcet, as compared to vitamin D alone, showed a better reduction response of PTH levels and a significant diminution of secondary effects. The objective of present study was to evaluate the additional cost of adding cinacalcet to the standard treatment of patients with severe secondary hyperparathyroidism (SHPT) taking into account the treatment goals achieved. Methods: 12 month prospective study of 23 patients with severe SHPT. Two treatment regimens were considered: standard treatment (m 0) and standard treatment plus cinacalcet (m 12). Four consequences of inadequate control of SHPT were registered: parathiroid hormone (PTH), Calcium (Ca), Phosphorus (P) and the Ca x P product serum levels. Treatment effectiveness was measured as percentage of patients who achieved treatment goal according to each indicator: PTH < 800 pg/mL, PTH between 150 and 300 pg/mL, Calcium < 9.5 mg/dL, Phosphorus < 5.5 mg/dL, and Ca x P product < 55. Annual and monthly costs were calculated for both treatment regimens using Spanish 2007 tariffs, and taking into account the dose reduction in some other treatments. Results are presented as incremental costs and cost per patient who achieved treatment goal. Results: At 12 month it was observed a higher percentage of patients who achieved simultaneously the 4 therapeutic goals with respect to basal moment, from 0% to 52.1%. Cinacalcet allowed to save costs in concomitant drugs, achieving a total saving of 149 e per patient and month. At 12 month, Cinacalcet achieved a reduction of percentage of patients with PTH > 800 pgr/mL with half of costs than standard treatment (651.35 e vs 1,363.68 e). It was not possible to calculate the cost for PTH indicator since at the study onset, there was no patient who achieved a level between 150 and 300 pg/mL. Cinacalcet allowed reaching treatment goals in Calcium, Phosphorus and Ca x P product in a more cost-effective way (2,164.2 e vs 2,684.8 e). Conclusions: Although Cinacalcet is expensive, patients treated with Cinacalcet showed a minor cost per patient who achieved treatment goal than patients without Cinacalcet. The ability of cinacalcet to reduce PTH secretion, along with the reductions in the serum Ca, P, and Ca x P product, provides an alternative to the traditional treatment paradigm, and should be a welcomed addition in the management of SHPT (AU)

Hemoglobin substitutes.

Orthopaedic patients frequently require blood transfusions to treat peri-operative anemia. Research in the area of hemoglobin substitutes has been of great interest since it holds the promise of reducing the reliance on allogeneic blood transfusions. The three categories of hemoglobin substitutes are (1) cell-free, extracellular hemoglobin preparations made from human or bovine hemoglobin (hemoglobin-based oxygen carriers or HBOCs); (2) fluorine-substituted linear or cyclic carbon chains with a high oxygen-carrying capacity (perfluorocarbons); and (3) liposome-encapsulated hemoglobin. Of the three, HBOCs have been the most extensively studied and tested in preclinical and clinical trials that have shown success in diminishing the number of blood transfusions as well as an overall favorable side-effect profile. This has been demonstrated in vascular, cardiothoracic, and orthopaedic patients. HBOC-201, which is a preparation of cell-free bovine hemoglobin, has been approved for clinical use in South Africa. These products may well become an important tool for physicians treating peri-operative anemia in orthopaedic patients.

DY-9760e, a novel calmodulin antagonist, reduces infarction after permanent focal cerebral ischemia in rats.

DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate), a novel calmodulin antagonist, provides effective protection against Ca(2+) ionophore-induced cytotoxicity and brain injury induced by transient focal ischemia. In this study, we evaluated the effect of DY-9760e on ischemic infarct volume in rats subjected to permanent focal ischemia. DY-9760e (0.5 mg/kg/h for 6 h) significantly reduced the infarct volume when administered immediately after middle cerebral artery occlusion. Furthermore, this neuroprotection was also exerted by treatment with a 3-hour delay, implying that the therapeutic time window for this compound is at least 3 h. In addition, although treatment with 0.1 mg/kg/h for 24 h was ineffective, the combination of a loading dose of 0.3 mg/kg/h for 2 h followed by 0.1 mg/kg/h for 22 h yielded a significant reduction in infarct volume. Thus, prolonged infusion preceded by a loading dose is an efficacious dosing regimen for DY-9760e, especially at a low infusion rate. These data demonstrate the substantial neuroprotective effect of DY-9760e in a permanent focal ischemia model and indicate that this neuroprotectant may be of therapeutic value for the treatment of acute stroke.

Further assessment of the protective effect of calmodulin inhibitors against reperfusion injury after acute coronary occlusion in the dog.

OBJECTIVE: To re-evaluate the suitability of chlorpromazine and trifluoperazine to prevent postischemic reperfusion injury of the myocardium. DESIGN: Acute occlusion (60 mins) and subsequent reperfusion (120 mins) of the left anterior descendent coronary artery with monitoring of hemodynamic, morphological and biochemical variables of the heart. SETTING: Experimental study. ANIMALS: Seventy adult mongrel dogs. INTERVENTIONS: Chlorpromazine (15 mg/kg body weight) or trifluoperazine (2 mg/kg body weight) given intravenously 30 mins after the onset of occlusion. MAIN RESULTS: Reperfusion alone increased the regional bloodflow and left ventricular end-diastolic pressure (P < 0.05 to 0.01), and reduced the size of the occluded area. Reperfusion also decreased the dp/dtmax, Vmax, mean aortic pressure, cardiac index, etc, but failed to improve cardiac ultrastructure and metabolism. Chlorpromazine or trifluoperazine induced a further reduction (P < 0.05 to 0.01) in infarct size, left ventricular end-diastolic pressure and systemic resistance index, and caused an increase in dp/dtmax, Vmax, cardiac index and regional bloodflow in the ischemic and border zones of the left ventricle. Moreover, these drugs preserved, to a certain extent, the metabolism of the myocardium and its ultrastructure. CONCLUSIONS: In spite of their considerable preventive effect, neither chlorpromazine nor trifluoperazine provided a complete prevention of reperfusion injury to the myocardium.

Potentiation of vincristine effect in human and murine gliomas by calcium channel blockers or calmodulin inhibitors.

The effects of calcium channel blockers and calmodulin inhibitors on vincristine cytotoxicity were studied in vitro with five glioma cell lines: three human glioblastomas, one rat glioma, and one mouse ependymoblastoma. One human glioblastoma and the rat glioma were resistant to vincristine in contrast to other glioma cells. The resistance to vincristine was considerably decreased by nontoxic or marginally toxic concentrations of calcium channel blockers or calmodulin inhibitors, although the former was more effective than the latter. In the presence of verapamil, the vincristine cytotoxicity, as measured by cell doubling times, increased 90- and 84-fold in the vincristine-resistant human glioblastoma and rat glioma, respectively. The decrease in the resistance to vincristine was related to a marked increase in the intracellular level of that drug, probably mediated by inhibiting its outward transport. The in vivo studies showed that verapamil or nicardipine administered daily with vincristine for 10 days significantly enhanced the chemotherapeutic effect of vincristine in an intracranially transplanted rat glioma model. An approximately 32% to 118% increase in life span occurred with 15 mg/kg/day of verapamil, depending on the doses of vincristine.

Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth.

Two drugs known to inhibit the action of calmodulin, prochlorperazine offP) and N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide (W7), were investigated for their ability to control cell proliferation in murine B16 melanoma cells in culture. PCP and W7 inhibited [3H]thymidine uptake in these cells, 50% inhibition occurring with 13 microM PCP and 40 microM W7. In the presence of relatively high concentrations of fetal calf serum (FCS), cells withstood high concentrations of both drugs (100 microM PCP and 200 microM W7) and showed increased pigment production. Drug-inhibited DNA synthesis could be reversed by the addition of fresh medium containing FCS or by the addition of exogenous pure calmodulin. Extracellular calmodulin itself stimulated DNA synthesis. FCS was found to contain calmodulin-like activity at concentrations that may be relevant to the stimulation of [3H]thymidine uptake by cells in culture.

[Calcium transport in the erythrocytes of rats with spontaneous hypertension: characteristics of the effect of calmodulin]. / Transport kal-tsiia v éritrotsitakh krys so spontannoi gipertenziei: osobennosti vliianiia kal'modulina.

Transport features of calcium in red cells of rats with spontaneous genetic hypertension (SHR) have been studied. It is shown that in the presence of calmodulin the rate of calcium accumulation by the inside-out vesicles of SHR red cell membranes is roughly twice less than in the control normotensive rats. It is suggested that upset interrelationship between calmodulin and Mg-Ca-ATPase of plasma membrane may be the cause of increase of intracellular calcium recorded in a number of tissues in primary hypertension.