Prototype of a family of at least nine members, c-src tyrosine kinase is a therapeutically interesting target, because its inhibition might be of interest not only in a number of malignancies, but also in a diverse array of conditions, from neurodegenerative pathologies to certain viral infections. Computational methods in drug discovery are considerably cheaper than conventional methods and offer opportunities of screening very large numbers of compounds in conditions that would be simply impossible within the wet lab experimental settings. We have explored the use of global QSAR models and molecular ligand docking in the discovery of new c-src tyrosine kinase inhibitors. Using a data set of 1038 compounds from ChEMBL and 19 blocks of molecular descriptors, we have developed over 200 QSAR classification models, based on six machine learning algorithms and 17 feature selection methods. We have selected 49 with reasonably good performance (positive predictive value and balanced accuracy higher than 70% in nested cross validation) and the models were assembled by stacking with a simple majority vote and used for the virtual screening of over the “named” ZINC data set (over 100,000 compounds). 744 compounds were predicted by at least 50% of the QSAR models as active, 147 compounds were within the applicability domain and predicted by at least 75% of the models to be active. The latter 147 compounds were submitted to molecular ligand docking using Vina and Ledock, and a number of 90 were predicted to be active based on the binding energy. External data from CHEMBL and PUBCHEM confirmed that at least 7.83% (in the case of QSAR) or 6.67% (in the case of integrated QSAR and molecular docking) of the compounds are active on the c-src target.

I have recently reiterated that the cross-bridge is a calcium ATPase that is inhibited by magnesium and this arises because in normal hearts Myosin binding Protein-C prevents the use of MgATP as rate limiting substrate ensuring that Ca²⁺ replaces Mg²⁺ in the excitation pathway. Here I revisit the studies on [Ca²⁺] dependency of ATPase and tension under diastolic stretch with a different conclusion on Hill coefficients. This reveals the underlying mechanisms of the Frank-Starling Law and Hypertrophic myopathy are not the same, the former being kinase controlled.

Aurora kinase A (AURKA) is a normal cell proliferation-inducing enzyme encoded by AURKA gene, with over-expression observed in different types of malignancies. Hence, the goal is to find potential inhibitors against AURKA. In this study, molecular docking, Standard Precision and Extra Precision methods were employed. After the docking study, the ligands showed an extremely low binding score which suggested very high binding affinity of the ligands. Furthermore, Quantum polarized ligand docking (QPLD) was performed to predict the binding status of the molecules. Based on the binding affinity, the top four compounds were chosen for further analysis. The docked complexes were further analyzed in explicit water conditions using 100 ns molecular dynamics simulations and binding free energy calculation. Then, density functional theory (DFT) calculation was used to calculate the molecular properties of the molecules. Finally, systems biology experiments validated the molecular docking and molecular dynamics simulation studies and indicated that quercetin, kaempferol, luteolin and rutin could inhibit the AURKA. The results show that, these four molecules have high binding affinity to the AURKA and significant interactions (LEU139, GLU211and ALA213) were also identified with the hinge region of Aurora kinase A. Thus, LEU139, GLU211, and ALA213 were identified as the crucial protein mechanisms.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm constituting approximately 15% of newly diagnosed leukemia in adult patients. Development of tyrosine kinase inhibitors (TKIs) have dramatically improved outcomes in patients with chronic CML in chronic phase. However, adverse drug events (ADEs) associated with TKI therapy have influenced drug adherence, resulting in adverse clinical outcomes and a decline in the quality of life (QoL). In this study, we carried out a unique questionnaire survey to evaluate ADEs, which comprised 14 adverse events. We compared drug adherence rates between patients using imatinib and those who switched from imatinib to nilotinib, a second-generation TKI. Following the switch, the total number of ADEs decreased considerably in most cases. Simultaneously, better QoL was observed in the nilotinib group than in the imatinib group. Drug adherence was measured using Morisky’s 9-item Medication Adherence Scale (MMAS). MMAS increased significantly after switching to nilotinib in all cases. Drug adherence is a critical factor for achieving molecular response in patients with CML. In fact, our results showed a strong inverse correlation between clinical outcome [international scale (IS)] and adherence (MMAS), with a stronger tendency in the nilotinib group than in the imatinib group. In conclusion, low occurrence of ADEs induced a high level of QoL and a good clinical response with second-generation TKI nilotinib treatment.

Genetic activation of the class I PI3K pathway is very common in cancer. This mostly results from oncogenic mutations in PIK3CA, the gene encoding the ubiquitously expressed PI3Kα catalytic subunit, or from inactivation of the PTEN tumour suppressor, a lipid phosphatase which opposes class I PI3K signalling. The clinical impact of PI3K inhibitors in solid tumours, aimed at dampening cancer-cell-intrinsic PI3K activity, has thus far been limited. Challenges include poor drug tolerance, incomplete pathway inhibition and pre-existing or inhibitor-induced resistance. The principle of pharmacologically targeting cancer-cell-intrinsic PI3K activity also assumes that all cancer-promoting effects of PI3K activation are reversible, which might not be the case. Emerging evidence suggests that genetic PI3K pathway activation can induce and/or allow cells to tolerate chromosomal instability, which – even if occurring in a low fraction of the cell population – might help to facilitate and/or drive tumour evolution. While it is clear that such genomic events cannot be reverted pharmacologically, a role for PI3K in the regulation of chromosomal instability could be exploited by using PI3K pathway inhibitors to prevent those genomic events from happening and/or reduce the pace at which they are occurring, thereby dampening cancer development or progression. Such an impact might be most effective in tumours with clonal PI3K activation and achievable at lower drug doses than the maximum-tolerated doses of PI3K inhibitors currently used in the clinic.

Rett syndrome (RTT) is mainly caused by mutations in methyl CpG-binding protein 2, cyclin-dependent kinase-like 5, or forkhead box protein G1. These RTT-causing proteins harbor an intrinsically disordered region (IDR) whose conformation exhibits spatiotemporal heterogeneity, which not only confer versatility to the protein, but also implicates them in diseases. The IDR generally evolves more rapidly than an ordered structure. In this study, we examined the relationship between pathogenic RTT-associated point mutations in RTT-causing proteins and the evolutionary dynamics of sequence features including structural order–disorder, phosphorylation sites, and evolutionary rates. We also analyzed the molecular properties and evolution of proteins that interact with RTT-causing proteins in terms of phylogenetic profiles, tissue specificity, subcellular localization, expression level, and functions. The results indicate that constrained IDRs may function by forming contacts with other regions in the protein sequence causing pathogenic missense mutations likely to arise in the rapidly evolving IDR and affect molecular networks, leading to disease. The results also provide novel insights into the genetic basis for RTT and the evolution of the neocortex in higher vertebrates.

In most of acute myeloid leukemia patients there is an aberrant tyrosine kinases activity. The Sprouty family proteins were originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. This family proteins are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in AML at diagnosis, suggesting a multistep regulation of RAF–MEK–ERK signaling in human cancers.

Despite the impact of immune checkpoint inhibitors on malignancies treatment is unprecedented, a lack of response to these molecules is observed in several cases. Differently from melanoma and non-small cell lung cancer, where the use of immune checkpoint inhibitors results in a high efficacy, the response rate in other tumors, such as gastrointestinal cancers, breast cancer, sarcomas, and part of genitor-urinary cancers remains low. The first strategy evaluated to improve the response rate to immune checkpoint inhibitors is the use of predictive factors for the response as PD-L1 expression, tumor mutational burden, and clinical features. In addition to the identification of the patients with a high sensibility to immune checkpoint inhibitors, another approach currently under intensive investigation is the use of therapeutics in a combinatory manner with immune checkpoint inhibitors to obtain an enhancement of efficacy through the modification of the tumor immune microenvironment. In addition to the abscopal effect induced by radiotherapy, a lot of studies are evaluating several drugs able to improve response rate to immune checkpoint inhibitors, including microbiota modifiers, drugs targeting co-inhibitors receptors, anti-angiogenic therapeutics, small molecules, and oncolytic viruses. In view of the rapid and extensive development of this research field, we conducted a systematic review of the literature identifying which of these drugs are closer to achieving validation in the clinical practice.

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how MAPK/ERK activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms’ tumor.

Breast Cancer (BC) is the second most common type of cancer worldwide and displays the highest cancer-related mortality among women worldwide. Targeted therapies have revolutionized the way BC has been treated in the last decades improving life expectancies of millions of women. Among the different molecular pathways that have been of interest for the development of targeted therapies are the Cyclin-Dependent Kinases (CDK). CDK inhibitors are a class of molecules that already exist in nature and those belonging to the INK4 protein family specifically inhibit the CDK4/6 proteins. CDK4/6 inhibitors specifically block the transition from the G1 to the S phase of the cell cycle by dephosphorylation of the retinoblastoma tumor suppressor protein. In the past four years CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib received their first FDA approval for the treatment of Hormone Receptor (HR)-positive and Human Epidermal growth factor Receptor 2 (HER2)-negative breast cancer after showing significant improvements in progression-free survival in the PALOMA-1, MONALEESA-2 and the MONARCH-2 randomized clinical trials, respectively. After the encouraging results from these clinical trials, CDK4/6 inhibitors have also been investigated in the other BC subtypes. In HER2-positive BC, combination of CDK4/6 inhibitors with HER2-targeted therapies showed promise in preclinical studies and their clinical evaluation is ongoing. Moreover, in triple-negative BC, CDK4/6 inhibitors efficacy has been investigated in combination with other targeted therapies or immunotherapies. This review summarizes the molecular background and clinical efficacy of CDK4/6 inhibitors as single agents or in combination with other targeted therapies for the treatment of BC. Future directions of ongoing clinical trials and predictive biomarkers will be further debated.

Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis and is highly expressed in various cancer tissues. Although high PKM2 expression is observed in prostate cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells. PKM2 expression was silenced using various PKM2 small interfering RNAs (siRNAs) and then we measured PKM2-related cellular pathways associated with autophagy. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange staining and immunoblotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. To the best of our knowledge, this is the first study to show that PKM2 inhibition alters cancer cell metabolism and induces autophagy. Thus, the present study provides a strategy for the development of PKM2-targeted novel anticancer drugs for the treatment of prostate cancer.

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type–specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR`s actions but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter`s activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

The involvement of astrocytic SN1 (SNAT3) transporter in ammonia-induced L-glutamine retention was recently documented in mouse cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot and confocal microscopy, respectively. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. Ammonia-induced Sp1 regulatory role in SN1-mediated [³H]glutamine transport was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to Snat3 promoter, along with Sp1 dependence of system N-mediated [³H]glutamine transport were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, a treatment likely to dephosphorylate Sp1. Furthermore, silencing of PKCδ isoform abolished the increase of Sp1 level by ammonia. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity.

Identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly targeted strategies designed to improve the outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3, is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD), present in 30%-35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus has encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance is induced by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain at residue Asp835 (D835) to form a ‘dual mutation’ (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades however, currently little is known which divergent signaling pathways are controlled by each of these FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways regulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise for development of more durable and personalized therapeutic approaches targeting mutant FLT3, to improve the treatment of AML.

Hypothesis: Anti-diabetic drugs modulate p-21 activated kinase (PAK) signaling Introduction: Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease associated with increased cancer risk. PAK signaling is implicated in cellular homeostasis when regulated, and cancer when unrestrained. Recent reports provided a role for PAK signaling in glucose homeostasis, but the role of PAKs in the pathogenesis of T2DM is unknown. Here we performed a mini-meta analysis to explore if anti-diabetic drugs modify PAK signaling pathways, and provide insight regarding modulation of these pathways to potentially reduce diabetes-associated cancer risk. Methods: PAK interacting partners in T2DM were identified using online STRING database. Correlation studies were performed via systematic literature review to understand the effect of anti-diabetic drugs on PAK signaling. Mini meta analysis correlated multiple clinical studies and revealed the overall clinical response rate and percentage of adverse events in piogliazone (n=53) and metformin (n=91) treated patients with PAK-associated diseases. Results: A total of 30 PAK interacting partners were identified (10: reduced beta-cell mass; 10: beta-cell dysfunction; 10: obesity-insulin resistance) which were highly associated with Wnt, and G-protein signaling. Anti-diabetic drug metformin activated signaling pathways upstream; whereas pioglitazone inhibited pathways downstream of PAK. Overall clinical response upon pioglitazone treatment was 53%. 79% of pioglitazone and 75% of metformin treated patients had adverse events. Pioglitazone reduced molecular-PAK biomarkers of proliferation (Ki67 and CyclinD1), and metformin had the opposite effect. Conclusions: PAK signaling in T2DM likely involves Wnt and G-protein signaling which may be altered by anti-diabetic drugs metformin and pioglitazone. Apart from the therapeutic limitations of adverse events, pioglitazone may be promising in chemoprevention, however long-term multi-centered studies, which initiate pioglitazone treatment early will be required to fully assess the full potential of these drugs.

Pancreatic ductal adenocarcinoma (PDA) remains the most lethal malignancy due to lack of an effective treatment. P21-activated kinases (PAKs) play key roles in PDA growth, and the PAK inhibitor PF-3758309 synergistically reduced PDA growth with gemcitabine. The aim of this study was to determine the effect of PF-3758309 with multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines. Cells were treated with PF-3758309 plus or minus gemcitabine, 5-fluorouracil (5-FU) or abraxane, and cell proliferation was determined. Protein expression profiles were measured by Western blot. PDA cells were subcutaneously injected into the flanks of SCID mice which were then treated with PF-3758309, gemcitabine, PF-3758309 plus gemcitabine, or gemcitabine plus abraxane. Tumour growth was measured by volume and weight. PF-3758309 enhanced the inhibitory effects of 5-FU, gemcitabine and abraxane on a panel of patient-derived PDA cells, inhibited HIF-1 protein expression and reduced the protein levels of palladin and -SMA in these cells. The combination of PF-3758309 with gemcitabine maximally inhibited PDA growth in vivo, which was comparable to the combination of gemcitabine with abraxane. PF-3758309 enhanced the suppressive effects of multiple chemotherapeutic reagents on the growth of a panel of patient-derived PDA cell lines. The combination of PF-3758309 with gemcitabine provides a potential treatment option with less toxicity than gemcitabine plus abraxane.

The transforming growth factor-β (TGF-β), in which overexpression have been associated with various diseases, has become an attractive molecular target for the treatment of cancers. Three series of 3-substituted-4-(quinoxalin-6-yl) pyrazoles 14a–h, 15a–h, 16a–h, 22a, 22b, 22d, 23a, 23b, 23d, 24b, and 24d were synthesized and evaluated for their activin receptor-like kinase 5 (ALK5) and p38α mitogen activated protein (MAP) kinase inhibitory activity in an enzymatic assays. Among these compounds, the most active compound 16f inhibited ALK5 phosphorylation with an IC₅₀ value of 0.28 µM, with 98% inhibition at 10 µM. Compound 16f also had good selectivity index of >35 against p38α MAP kinase, with 9.0-fold more selective than clinical candidate, compound 3 (LY-2157299). Molecular docking study was performed to identify the mechanism of action of the synthesized compounds and their good binding interactions were observed. ADMET prediction of good active compounds showed that these ones possess good pharmacokinetics and drug-likeness behavior.

Hypothesis: Anti-diabetic drugs modulate p-21 activated kinase (PAK) signaling Introduction: Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease associated with increased cancer risk. PAK signaling is implicated in cellular homeostasis when regulated, and cancer when unrestrained. Recent reports provided a role for PAK signaling in glucose homeostasis, but the role of PAKs in the pathogenesis of T2DM is unknown. Here we explored whether PAK signaling should be targeted via chemoprevention to reduce diabetes-associated cancer risk. Methods: PAK interacting partners in T2DM were identified using online STRING database. Systematic literature review provided the effect of anti-diabetic drugs on PAK signaling. Review of clinical studies revealed the overall clinical response rate and percentage of adverse events in piogliazone (n=53) and metformin (n=91) treated patients with PAK-dependent diseases. Results: A total of 30 PAK interacting partners were identified (10: reduced beta-cell mass; 10: beta-cell dysfunction; 10: obesity-insulin resistance) which were highly associated with Wnt, and G-protein signaling. Anti-diabetic drug metformin activated signaling pathways upstream; whereas pioglitazone inhibited pathways downstream of PAK. Overall clinical response upon pioglitazone treatment was 53%. 79% of pioglitazone and 75% of metformin treated patients had adverse events. Pioglitazone reduced molecular-PAK biomarkers of proliferation (Ki67 and CyclinD1), and metformin had the opposite effect. Conclusions: PAK signaling in T2DM involves Wnt and G-protein signaling which is altered by anti-diabetic drugs metformin and pioglitazone. Apart from the therapeutic limitations of adverse events, pioglitazone is promising in chemoprevention, however long-term multi-centered studies, which initiate pioglitazone treatment early will be required to fully assess the full potential of these drugs.

Neuroinflammatory responses are implicated in the pathogenesis of neurodegenerative diseases. In neurodegenerative diseases, neuroinflammatory reactions to neuronal injury are modulated by microglial cells, which are vital innate immune cells in the central nervous system. Activated microglial cells release proinflammatory cytokines, mediators, and neurotoxic factors that induce fatal neuronal injury. The present study investigated the anti-neuroinflammatory effects of cudratricusxanthone L (1), which was isolated from Cudrania tricuspidata. This compound reduced the levels of lipopolysaccharide-stimulated inflammatory mediators and cytokines, including nitric oxide, prostaglandin E2, interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and IL-12. These effects suggested that cudratricusxanthone L (1) suppressed the nuclear factor-kappa B (NF-κB) signaling pathway. Specifically, cudratricusxanthone L (1) also attenuated the phosphorylation of Jun kinase and inhibited p38 mitogen-activated protein kinase (MAPK) signaling in BV2 and rat primary microglial cells. These results indicated that cudratricusxanthone L (1) effectively repressed neuroinflammatory processes in BV2 and rat primary microglial cells by inhibiting NF-κB and the MAPK signaling pathway.

The search for improved therapeutic approaches to breast cancer are still on going. Breast tumor kinase (BRK, also known as PTK6) is one of the targets, it is highly expressed in breast carcinomas while displaying low or no expression in the normal mammary gland, which hints at the oncogenic role of this enzyme in breast cancer. In these twenty years, an increasing number of studies have focused on understanding the cellular roles of BRK in breast cancers. This review outlines the advances made towards understanding the cellular and physiological role of BRK, the molecular and chemical inhibitors and its therapeutic significance in breast cancer.

Akt1 is a proto-oncogene that is over active in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specifically phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed 4-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

The enzyme-coupled transient receptor potential channel subfamily M member 7, TRPM7, has been associated with immunity and immune cell signalling. Here, we review the role of this remarkable signalling protein in lymphocyte proliferation, differentiation, activation and survival. We also discuss its role in mast cell, neutrophil and macrophage function and highlight the potential of TRPM7 to regulate immune system homeostasis. Further, we shed light on how the cellular signalling cascades involving TRPM7 channel and/or kinase activity culminate in pathologies as diverse as allergic hypersensitivity, arterial thrombosis, and graft versus host disease (GVHD), stressing the need for TRPM7 specific pharmacological modulators.

Purpose: This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. Methods: Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 10⁷ pigment particles/mL for 48 hours before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. Result: Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. Conclusion: RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Objective: The Rho GTPase/Rho kinase pathway is an important target in glaucoma treatment. This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor developed for cancer treatment, in a porcine ex vivo pigmentary glaucoma model. Materials and Methods: Twenty-eight fresh porcine anterior chambers were perfused with pigment medium (1.67 × 10⁷ pigment particles/mL) for 48 hours before being subjected to the RKI-1447 (n = 16) or the vehicle control (n = 12). Another twelve eyes with normal medium perfusion served as the control. The intraocular pressure (IOP) was recorded at two-minute intervals and the outflow facility was calculated. To investigate the intracellular mechanism of the IOP reduction, primary trabecular meshwork cells were exposed to RKI-1447 or the vehicle control and then analyzed for changes in cytoskeleton, motility, and phagocytosis. Results: Compared to the baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 exhibited no apparent changes in the micro- or macroscopic appearance, including histology. Primary TM cells exposed to RKI-1447 showed a significant disruption of the actin cytoskeleton both in the presence and absence of pigment exposure (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI reversed. Conclusions: RKI-1447 is a novel ROCK inhibitor that significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it especially useful for the treatment of secondary glaucomas with an increased phagocytosis load but also for other open angle glaucomas.

Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI). ω3-Polyunsaturated fatty acids (ω3-PUFAs) show protective effects against various renal injuries. It was recently reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI) and evaluated associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt) were divided into four groups: wt sham (n = 10), fat-1 sham (n = 10), wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15), and fat-1 IRI (n = 15). Kidneys and blood were harvested 24 h after IRI. Renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of LC3, Beclin-1, and Atg7; lower amounts of p62; and higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more AMPK activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR). Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.

Populations with essential hypertension have a high risk of nonalcoholic steatohepatitis (NASH). In this study, we investigated the mechanism that underlies the progression of hypertension-associated NASH by comparing differences in the development of high fat and cholesterol (HFC) diet-induced NASH among three strains of rats, i.e., two hypertensive strains comprising spontaneously hypertensive rats and the stroke-prone spontaneously hypertensive 5/Dmcr, and the original Wistar Kyoto rats as the normotensive control. We investigated histopathological changes and molecular signals related to inflammation in the liver after feeding with the HFC diet for 8 weeks. The diet induced severe lobular inflammation and fibrosis in the livers of the hypertensive rats, whereas it only caused mild steatohepatitis in the normotensive rats. Increased activation of proinflammatory signaling (transforming growth factor-β1/mitogen-activated protein kinases pathway) was observed in the hypertensive strains fed with the HFC diet. In addition, the HFC diet suppressed the nuclear factor erythroid 2-related factor 2 pathway in the hypertensive rats and led to lower increases in the hepatic expression of heme oxygenase-1, which has anti-oxidative and anti-inflammatory activities. In conclusion, these signaling pathways might play crucial roles in the development of hypertension-associated NASH.

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility and differentiation. Dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the “rotation model”). This model is also able to explain how oncogenic mutations activate the receptor in the absence of ligand without assuming that the mutations induce receptor dimerization. In this review, we discuss mechanisms underlying ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model.

Fasudil, a potent Rho kinase (ROCK) inhibitor, can ameliorate LPS-induced acute lung injury (ALI) in mice, but the mechanism remains obscure. In this study, a mice model of ALI was established by intra-tracheal instillation of LPS. Histological changes, cytokine levels, lung permeability, and endothelial apoptosis were determined to evaluate the effects of fasudil on lung injury. The cellular and molecular biological mechanisms were explored by culturing human pulmonary microvascular endothelial cells (PMECs). The results showed that fasudil reduced LPS-induced lung inflammation, pulmonary hyperpermeability, and endothelial apoptosis in mice. In cultured human PMECs, fasudil inhibited LPS-induced caspse-3 cleavage and cell apoptosis. It also decreased LPS-induced hyperpermeability of human PMECs monolayer by reversing the down-regulation of intercellular junctions. Moreover, fasudil inhibited LPS-induced overexpression of chemokines and intercellular adhesion molecule (ICAM)-1 in human PMECs, which in turn suppressed neutrophil chemotaxis and neutrophil-endothelial adhesion. Further molecular researches showed fasudil inhibited LPS-induced activation of ROCK, NF-κB, and p38 in human PMECs. Our findings demonstrated that fasudil alleviated LPS-induced ALI by protecting endothelial function via inhibiting endothelial apoptosis, maintaining endothelial barrier integrity, and reducing endothelial inflammation. These effects of fasudil could be attributed to the inhibition of ROCK and its downstream NF-κB and p38 signaling pathways.

Abnormality of fibroblast growth factor receptors (FGFRs) mediated signaling pathways were frequently found in various human malignancies, making FGFRs hot targets for cancer treatment. To address the consistent need of new chemotype of FGFR inhibitors, here, we started with a hit structure identified from our internal c-Met inhibitor project, and conducted a chemical optimization. After exploring three parts of the hit compound, we finally discovered a new series of pyrrolo[2,3-b] pyrazine FGFR inhibitors, which contain a novel scaffold and unique molecular shape. We believed that our findings can benefit others to further develop selective FGFR inhibitors.

Targeted image-guided oncologic surgery (IGOS) relies on the recognition of cell surface-associated proteins, which should be abundantly present on the tumor cells but preferably absent on cells in surrounding healthy tissue. The transmembrane receptor tyrosine kinase EphA2, a member of the A class of the Eph receptor family, has been reported to be highly overexpressed in several tumor types including breast, lung, brain, prostate, and colon cancer, and is considered amongst the most promising cell membrane-associated tumor antigens by the NIH. Another member of the Eph receptor family belonging to the B class, EphB4, has also been found to be up-regulated in multiple cancer types. In this study, EphaA2 and EphB4 are evaluated as target for IGOS of colorectal cancer by immunohistochemistry (IHC) using a tissue microarray (TMA) consisting of 168 pairs of tumor and normal tissue. The IHC sections were scored for staining intensity and percentage of cells stained. The results show a significantly enhanced staining intensity and more widespread distribution in tumor tissue compared with adjacent normal tissue for EphA2 as well as EphB4. Based on its more consistently higher score in colorectal tumor tissue compared to normal tissue, EphB4 appears to be an especially promising candidate for IGOS of colorectal cancer.