Discovery of 3-Amino-1H-pyrazole-Based Kinase Inhibitors to Illuminate the Understudied PCTAIRE Family.

The PCTAIRE subfamily belongs to the CDK (cyclin-dependent kinase) family and represents an understudied class of kinases of the dark kinome. They exhibit a highly conserved binding pocket and are activated by cyclin Y binding. CDK16 is targeted to the plasma membrane after binding to N-myristoylated cyclin Y and is highly expressed in post-mitotic tissues, such as the brain and testis. Dysregulation is associated with several diseases, including breast, prostate, and cervical cancer. Here, we used the N-(1H-pyrazol-3-yl)pyrimidin-4-amine moiety from the promiscuous inhibitor 1 to target CDK16, by varying different residues. Further optimization steps led to 43d, which exhibited high cellular potency for CDK16 (EC50 = 33 nM) and the other members of the PCTAIRE and PFTAIRE family with 20-120 nM and 50-180 nM, respectively. A DSF screen against a representative panel of approximately 100 kinases exhibited a selective inhibition over the other kinases. In a viability assessment, 43d decreased the cell count in a dose-dependent manner. A FUCCI cell cycle assay revealed a G2/M phase cell cycle arrest at all tested concentrations for 43d, caused by inhibition of CDK16.

Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.

Metformin, the first-line drug to treat type 2 diabetes (T2D), inhibits mitochondrial glycerolphosphate dehydrogenase in the liver to suppress gluconeogenesis. However, the direct target and the underlying mechanisms by which metformin increases glucose uptake in peripheral tissues remain uncharacterized. Lipid phosphatase Src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2) is upregulated in diabetic rodent models and suppresses insulin signaling by reducing Akt activation, leading to insulin resistance and diminished glucose uptake. Here, we demonstrate that metformin directly binds to and reduces the catalytic activity of the recombinant SHIP2 phosphatase domain in vitro. Metformin inhibits SHIP2 in cultured cells and in skeletal muscle and kidney of db/db mice. In SHIP2-overexpressing myotubes, metformin ameliorates reduced glucose uptake by slowing down glucose transporter 4 endocytosis. SHIP2 overexpression reduces Akt activity and enhances podocyte apoptosis, and both are restored to normal levels by metformin. SHIP2 activity is elevated in glomeruli of patients with T2D receiving nonmetformin medication, but not in patients receiving metformin, compared with people without diabetes. Furthermore, podocyte loss in kidneys of metformin-treated T2D patients is reduced compared with patients receiving nonmetformin medication. Our data unravel a novel molecular mechanism by which metformin enhances glucose uptake and acts renoprotectively by reducing SHIP2 activity.-Polianskyte-Prause, Z., Tolvanen, T. A., Lindfors, S., Dumont, V., Van, M., Wang, H., Dash, S. N., Berg, M., Naams, J.-B., Hautala, L. C., Nisen, H., Mirtti, T., Groop, P.-H., Wähälä, K., Tienari, J., Lehtonen, S. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.

Elevated TLR5 expression in vivo and loss of NF-κΒ activation via TLR5 in vitro detected in HPV-negative oropharyngeal squamous cell carcinoma.

In oropharyngeal squamous cell carcinoma (OPSCC), the expression pattern of toll-like receptors (TLRs), in comparison between human papillomavirus (HPV)-positive and -negative tumors differs. TLRs control innate immune responses by activating, among others, the nuclear factor-κΒ (NF-κΒ) signaling pathway. Elevated NF-κΒ activity is detectable in several cancers and regulates cancer development and progression. We studied TLR5 expression in 143 unselected consecutive OPSCC tumors, and its relation to HPV-DNA and p16 status, clinicopathological parameters, and patient outcome, and studied TLR5 stimulation and consecutive NF-κB cascade activation in vitro in two human OPSCC cell lines and immortalized human keratinocytes (HaCat). Clinicopathological data came from hospital registries, and TLR5 immunoexpression was evaluated by immunohistochemistry. Flagellin served to stimulate TLR5 in cultured cells, followed by analysis of the activity of the NF-κB signaling cascade with In-Cell Western for IκΒ and p-IκΒ. High TLR5 expression was associated with poor disease-specific survival in HPV-positive OPSCC, which typically shows low TLR5 immunoexpression. High TLR5 immunoexpression was more common in HPV-negative OPSCC, known for its less-favorable prognosis. In vitro, we detected NF-κΒ cascade activation in the HPV-positive OPSCC cell line and in HaCat cells, but not in the HPV-negative OPSCC cell line. Our results suggest that elevated TLR5 immunoexpression may be related to reduced NF-κΒ activity in HPV-negative OPSCC. The possible prognosis-worsening mechanisms among these high-risk OPSCC patients however, require further evaluation.

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.-Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

Lack of CD2AP disrupts Glut4 trafficking and attenuates glucose uptake in podocytes.

The adapter protein CD2-associated protein (CD2AP) functions in various signaling and vesicle trafficking pathways, including endosomal sorting and/or trafficking and degradation pathways. Here, we investigated the role of CD2AP in insulin-dependent glucose transporter 4 (Glut4, also known as SLC2A4) trafficking and glucose uptake. Glucose uptake was attenuated in CD2AP(-/-) podocytes compared with wild-type podocytes in the basal state, and CD2AP(-/-) podocytes failed to increase glucose uptake in response to insulin. Live-cell imaging revealed dynamic trafficking of HA-Glut4-GFP in wild-type podocytes, whereas in CD2AP(-/-) podocytes, HA-Glut4-GFP clustered perinuclearly. In subcellular membrane fractionations, CD2AP co-fractionated with Glut4, IRAP (also known as LNPEP) and sortilin, constituents of Glut4 storage vesicles (GSVs). We further found that CD2AP forms a complex with GGA2, a clathrin adaptor, which sorts Glut4 to GSVs, suggesting a role for CD2AP in this process. We also found that CD2AP forms a complex with clathrin and connects clathrin to actin in the perinuclear region. Furthermore, clathrin recycling back to trans-Golgi membranes from the vesicular fraction containing GSVs was defective in the absence of CD2AP. This leads to reduced insulin-stimulated trafficking of GSVs and attenuated glucose uptake into CD2AP(-/-) podocytes.

LRG1 and SDR16C5 protein expressions differ according to HPV status in oropharyngeal squamous cell carcinoma.

The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is primarily due to human papillomavirus, and understanding the tumor biology caused by the virus is crucial. Our goal was to investigate the proteins present in the serum of patients with OPSCC, which were not previously studied in OPSCC tissue. We examined the difference in expression of these proteins between HPV-positive and -negative tumors and their correlation with clinicopathological parameters and patient survival. The study included 157 formalin-fixed, paraffin-embedded tissue samples and clinicopathological data. Based on the protein levels in the sera of OPSCC patients, we selected 12 proteins and studied their expression in HPV-negative and HPV-positive OPSCC cell lines. LRG1, SDR16C5, PIP4K2C and MVD proteins were selected for immunohistochemical analysis in HPV-positive and -negative OPSCC tissue samples. These protein´s expression levels were compared with clinicopathological parameters and patient survival to investigate their clinical relevance. LRG1 expression was strong in HPV-negative whereas SDR16C5 expression was strong in HPV-positive tumors. Correlation was observed between LRG1, SDR16C5, and PIP4K2C expression and patient survival. High expression of PIP4K2C was found to be an independent prognostic factor for overall survival and expression correlated with HPV-positive tumor status. The data suggest the possible role of LRG1, SDR16C5 and PIP4K2C in OPSCC biology.

Current Advances in CETSA.

Knowing that the drug candidate binds to its intended target is a vital part of drug discovery. Thus, several labeled and label-free methods have been developed to study target engagement. In recent years, the cellular thermal shift assay (CETSA) with its variations has been widely adapted to drug discovery workflows. Western blot-based CETSA is used primarily to validate the target binding of a molecule to its target protein whereas CETSA based on bead chemistry detection methods (CETSA HT) has been used to screen molecular libraries to find novel molecules binding to a pre-determined target. Mass spectrometry-based CETSA also known as thermal proteome profiling (TPP) has emerged as a powerful tool for target deconvolution and finding novel binding partners for old and novel molecules. With this technology, it is possible to probe thermal shifts among over 7,000 proteins from one sample and to identify the wanted target binding but also binding to unwanted off-targets known to cause adverse effects. In addition, this proteome-wide method can provide information on the biological process initiated by the ligand binding. The continued development of mass spectrometry labeling reagents, such as isobaric tandem mass tag technology (TMT) continues to increase the throughput of CETSA MS, allowing its use for structure-activity relationship (SAR) studies with a limited number of molecules. In this review, we discussed the differences between different label-free methods to study target engagement, but our focus was on CETSA and recent advances in the CETSA method.

Ebselen enhances insulin sensitivity and decreases oxidative stress by inhibiting SHIP2 and protects from inflammation in diabetic mice.

Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.

Functional human cell-based vascularised cardiac tissue model for biomedical research and testing.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) are widely used in in vitro biomedical research and testing. However, fully matured, adult cardiomyocyte characteristics have not been achieved. To improve the maturity and physiological relevance of hiPSC-derived cardiomyocytes, we co-cultured them with preconstructed vascular-like networks to form a functional, human cell-based cardiac tissue model. The morphology and gene expression profiles indicated advanced maturation in the cardiac tissue model compared to those of a cardiomyocyte monoculture. The cardiac tissue model's functionality was confirmed by measuring the effects of 32 compounds with multielectrode array and comparing results to human data. Our model predicted the cardiac effects with a predictive accuracy of 91%, sensitivity of 90% and specificity of 100%. The correlation between the effective concentration (EC50) and the reported clinical plasma concentrations was 0.952 (R2 = 0.905). The developed advanced human cell-based cardiac tissue model showed characteristics and functionality of human cardiac tissue enabling accurate transferability of gained in vitro data to human settings. The model is standardized and thus, it would be highly useful in biomedical research and cardiotoxicity testing.

Novel Sulfonanilide Inhibitors of SHIP2 Enhance Glucose Uptake into Cultured Myotubes.

A series of substituted sulfonanilide analogs were prepared and evaluated as novel potent inhibitors of SH2 domain-containing inositol polyphosphate 5'-phosphatase 2 (SHIP2). SHIP2 has been shown to be a new attractive target for the treatment of insulin resistance in type 2 diabetes mellitus (T2D), which can lead to life-threatening diabetic kidney disease (DKD). Amongst the synthesized compounds, the two most promising candidates, 10 and 11, inhibited SHIP2 significantly. Additionally, these compounds induced Akt activation in a dose-dependent manner, increased the presence of glucose transporter 4 at the plasma membrane, and enhanced glucose uptake in cultured myotubes in vitro at lower concentrations than metformin, the most widely used antidiabetic drug. These results show that the novel SHIP2 inhibitors have insulin sensitizing capacity and provide prototypes for further drug development for T2D and DKD.

Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

Lack of CD2-associated protein (CD2AP) in mice increases podocyte apoptosis and leads to glomerulosclerosis and renal failure. We showed previously that SHIP2, a negative regulator of the PI3K/AKT signalling pathway, interacts with CD2AP. Here, we found that the expression level and activity of SHIP2 and production of reactive oxygen species (ROS) are increased in cultured CD2AP knockout (CD2AP-/-) mouse podocytes. Oxidative stress was also increased in CD2AP-/- mouse glomeruli in vivo. We found that puromycin aminonucleoside (PA), known to increase ROS production and apoptosis, increases SHIP2 activity and reduces CD2AP expression in cultured human podocytes. PDK1 and CDK2, central regulators of AKT, were downregulated in CD2AP-/- or PA-treated podocytes. Downregulation of PDK1 and CDK2, ROS generation and apoptosis were prevented by CD2AP overexpression in both models. Notably, inhibition of SHIP2 activity with a small molecule inhibitor AS1949490 ameliorated ROS production in CD2AP-/- podocytes, but, surprisingly, further reduced PDK1 expression and aggravated apoptosis. AKT- and ERK-mediated signalling was diminished and remained reduced after AS1949490 treatment in the absence of CD2AP. The data suggest that inhibition of the catalytic activity of SHIP2 is beneficial in reducing oxidative stress, but leads to deleterious increase in apoptosis in podocytes with reduced expression of CD2AP.