A novel selective multikinase inhibitor of ROCK and MRCK effectively blocks cancer cell migration and invasion.
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ABSTRACT:
SUBMITTER: Kale VP
PROVIDER: S-EPMC4182185 | biostudies-literature | 2014 Nov
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.ResultsScreening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 μM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.ConclusionsBDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.
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Project description:Exchange protein directly activated by cAMP (EPAC) and cAMP-dependent protein kinase (PKA) are two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. Herein, we report the identification and characterization of 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile (ESI-09), a novel noncyclic nucleotide EPAC antagonist that is capable of specifically blocking intracellular EPAC-mediated Rap1 activation and Akt phosphorylation, as well as EPAC-mediated insulin secretion in pancreatic β cells. Using this novel EPAC-specific inhibitor, we have probed the functional roles of overexpression of EPAC1 in pancreatic cancer cells. Our studies show that EPAC1 plays an important role in pancreatic cancer cell migration and invasion, and thus represents a potential target for developing novel therapeutic strategies for pancreatic cancer.
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Project description:Clear cell renal cell carcinoma (RCC) is characterized by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL loss drives tumor angiogenesis and accounts for the clinical activity of VEGF receptor (VEGFR) tyrosine kinase inhibitors (TKIs), the first-line standard of care for advanced RCC. Within the last year, three new second-line treatments have received FDA approval for use after anti-angiogenic therapy: the immune checkpoint inhibitor nivolumab, the TKI cabozantinib, and the combination of the TKI lenvatinib and the mTOR inhibitor everolimus. Cabozantinib inhibits VEGFRs, MET, and AXL, kinases that promote tumorigenesis, angiogenesis, metastasis, and drug resistance. Compared with everolimus, cabozantinib has shown statistically significant improvements in the three key efficacy endpoints of overall survival, progression-free survival, and objective response rate in patients with RCC who were previously treated with a VEGFR TKI. Herein, we summarize the translational research and clinical development that led to approval of cabozantinib as second-line therapy in RCC.
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Project description:The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.
| S-EPMC10572734 | biostudies-literature
Project description:Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.
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Project description:BACKGROUND: Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members. RESULTS: We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. CONCLUSIONS: Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.
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Project description:Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib (PS-341), a dipeptide-boronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293T (293T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells. Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded non-structural protein (NS) mediated degradation of retinoic acid-inducible gene-1 (RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.
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Project description:Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an important role in circulatory dysfunction in Alzheimer's disease (AD). Previous work has shown that the interaction between fibrinogen and ?-amyloid (A?), a hallmark pathological protein in AD, induces plasmin-resistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates cognitive function in mouse models of AD. Since A? oligomers have a much stronger affinity for fibrinogen than A? monomers, we tested whether amyloid aggregation inhibitors could block the A?-fibrinogen interaction and found that some A? aggregation inhibitors showed moderate inhibitory efficacy against this interaction. We then modified a hit compound so that it not only showed a strong inhibitory efficacy toward the A?-fibrinogen interaction but also retained its potency toward the A?42 aggregation inhibition process. Furthermore, our best hit compound, TDI-2760, modulated A?42-induced contact system activation, a pathological condition observed in some AD patients, in addition to inhibiting the A?-fibrinogen interaction and A? aggregation. Thus, TDI-2760 has the potential to lessen vascular abnormalities as well as A? aggregation-driven pathology in AD.
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Project description:In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA-ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.
| S-EPMC2064414 | biostudies-literature
Project description:Cellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell's transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5. IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.
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