A novel small-molecule MRCK inhibitor blocks cancer cell invasion

Background

Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate.

Results

To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H₂O₂ production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype.

Conclusions

This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.     

Adenosine receptor A2b confers ovarian cancer survival and PARP inhibitor resistance through IL-6-STAT3 signalling

Ovarian cancer is the deadliest gynecologic cancer worldwide, and the therapeutic options are limited. PARP inhibitor (PARPi) represents an effective therapeutic strategy and has been approved for maintenance therapy. However, the intrinsic or acquired resistance to PARPi becomes a big challenge. To investigate the mechanisms for PARPi resistance, we analysed public databases and established Olaparib-resistant ovarian cancer cells for exploration. Our results showed that the inflammatory pathway and adenosine receptor A2b (Adora2b/A_2B) expression were significantly increased in Olaparib-resistant cells. A_2B was highly expressed in recurrent ovarian tumours and negatively correlated with the clinical outcomes in cancer patients. Olaparib treatment enhanced A_2B expression through NF-κB activation. The elevated A_2B contributed to Olaparib resistance by sensing adenosine signal and promoting tumour cell survival, growth and migration via IL-6-STAT3 signalling. Therefore, inhibition of A_2B-IL-6-STAT3 axis could overcome Olaparib resistance and synergize with Olaparib to reduce cancer cell growth and lead to cell death. Our findings reveal a critical role of A_2B signalling in mediating PARPi resistance independent of DNA damage repair, providing insights into developing novel therapies in ovarian cancers.     

Large-scale network models of IL-1 and IL-6 signalling and their hepatocellular specification

The pro-inflammatory cytokines interleukin 1 (IL-1) and 6 (IL-6) are crucially involved in the regulation of a multitude of physiological processes, in particular coordinating the immune response upon bacterial infection and tissue injury. Both interleukins induce complex signalling cascades and trigger the production of mitogenic, pro-proliferative, anti-apoptotic, chemotactic, and pro-angiogenic factors thereby affecting the delicate balance between regeneration vs. invasive growth, tumourigenesis and metastasis. Moreover, several links to insulin resistance have been found within their associated signalling networks. Focusing on this from a systems biology perspective, we introduce comprehensive large-scale network models of IL-1 and IL-6 signalling which are based on a logical modelling approach and reflect the current biological knowledge. Theoretical network analysis enabled us to uncover general topological features and to make testable predictions on the stimulus-response behaviour of the networks. In this context, non-intuitive network-wide species dependencies as well as structures of regulatory feedback and feed-forward mechanisms could be characterised. By integrating high-throughput phosphoproteomic data from primary human hepatocytes we optimised the model structures to obtain models with high prediction accuracy for hepatocytes. Our model-based data analysis, for instance, suggested model modifications regarding (i) Akt contribution to IL-1-stimulated p38 MAPK activation and (ii) insignificant p38 MAPK activation in response to IL-6. In light of the presented results and in conjunction with the detailed model documentations, both models hold great potential for theoretical studies and practical applications.     

A selective small-molecule inhibitor of c-Jun N-terminal kinase 1

Indiscriminately suppressing total c-Jun N-terminal kinase (JNK) activity is not an appropriate strategy because each JNK appears to have a distinct function in cancer, asthma, diabetes, or Parkinson’s disease. Herein, we report that 7-(6-N-phenylaminohexyl)amino-2H-anthra[1,9-cd]pyrazol-6-one (AV-7) inhibited JNK1 activity, but not JNK2 or JNK3. We found that ultraviolet B (UVB) induced c-Jun phosphorylation and sub-G1 accumulation in JNK2^−/− murine embryonic fibroblasts, which contain an abundance of JNK1, but not JNK2. These results demonstrate that AV-7 is an isoform selective small-molecule inhibitor of JNK1 activity, which might be developed as a therapeutic against diabetes.

Structured summary

MINT-7148332: JNK3 (uniprotkb:P53779) phosphorylates (MI:0217) c-JUN (uniprotkb:P05412) by protein kinase assay (MI:0424)MINT-7148323: JNK2 (uniprotkb:P45984) phosphorylates (MI:0217) c-JUN (uniprotkb:P05412) by protein kinase assay (MI:0424)MINT-7148314: JNK1 (uniprotkb:P45983) phosphorylates (MI:0217) c-JUN (uniprotkb:P05412) by protein kinase assay (MI:0424)     

The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1

BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.     

A novel human glassy-cell carcinoma cell line producing IL-6 and IL-8 from uterine cervix

Summary A novel human cell line, TOM-2, was established from a rare uterine cervical cancer, glassy cell carcinoma (GCC). TOM-2 is the second established GCC cell line so far reported. The cells were intermediately or poorly differentiated with dysplastic nuclei and polygonal shape and secreted two tumor markers and cytokines, i.e., CA-125 and SCC, interleukin (IL)-1α, -6, and -8, and TNF-α. Growth of TOM-2 was so strongly dependent on population density that it was not possible to determine the plating efficiency. In mass culture, the following characteristics were observed: doubling time, 83 h; mode of chromosome number, 79; human papillomavirus type 18 DNA, detectable; tumorigenicity, easily transplantable into subcutis of nude mice; chemosensitivity in vitro, considerably sensitive to Cisplatin and 5-FU but not to 9 other antineoplastic agents. This novel cell line will be useful for developing new therapeutic strategies for the rare cancer, GCC.     

A novel small-molecule selective activator of homomeric GIRK4 channels

G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.     

A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.     

Development of a sphingosine kinase 1 specific small-molecule inhibitor

The sphingolipid metabolic pathway represents a potential source of new therapeutic targets for numerous hyperproliferative/inflammatory diseases. Targets such as the sphingosine kinases (SphKs) have been extensively studied and numerous strategies have been employed to develop inhibitors against these enzymes. Herein, we report on the optimization of our novel small-molecule inhibitor SKI-I (N'-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide) and the identification of a SphK1-specific analog, SKI-178, that is active in vitro and in vivo. This SphK1 specific small-molecule, non-lipid like, inhibitor will be of use to elucidate the roles of SphK1 and SphK2 in the development/progression of hyperproliferative and/or inflammatory diseases.     

A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos

To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.     

A novel inhibitor of mammalian collagenase

N-[[[(5-chloro-2-benzothiazolyl)thiolphenyllacetyll-L-cysteine (WY-45,368) is a potent inhibitor of human skin fibroblast collagenase. Kinetic data show that the inhibition is competitive, with a Ki of 3.5 microM. WY-45,368 inhibits neither of two other metalloproteinases, thermolysin and angiotensin converting enzyme, nor does it inhibit clostridial collagenase--thus indicating specificity for mammalian collagenase.