SummaryCentrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics.
Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.
Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light chain in the seminiferous epithelium of transgenic mice. Spermatogenesis was severely impaired and mature spermatozoa were completely absent. Late spermatids exhibited pleomorphic shapes and acrosomal distortions. The number of Leydig cells was greatly increased. In situ hybridization analysis revealed that the toxin acts on Sertoli cells. Affected cells exhibited an aberrant distribution of actin filaments and many cells contained large vacuoles. Our results demonstrate that tetanus toxin is active in non‐neuronal cells and suggest an important function for members of the synaptobrevin gene family during the late stages of spermatogenesis.
ABSTRACT:As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.Tumour cell invasion and metastasis are the primary causes of mortality in cancer patients. During progression of tumour cells to a metastatic phenotype, they undergo a series of changes that begin with loss of contact inhibition and increased motility, allowing them to migrate from the primary tumour site, invade distant organs and induce neovascularization resulting in metastasis. 1 Despite numerous developments, cancer cell invasion and metastasis is still a poorly studied process. Most strategies to treat cancer do not rely on inhibiting invasion and metastasis as the primary phenotype due to the requirement for lengthy and complicated clinical trials. However a detailed understanding of the drivers of cancer cell invasion and migration is essential to develop new treatments for cancer patients. The LIM kinases (LIMK1 and LIMK2; collectively LIMK) are TKL kinases that act downstream of Rho GTPases. LIM kinases phosphorylate and inactivate the filamentous-actin severing protein cofilin. Cycles of cofilin inactivation and activation enable dynamic actin rearrangements that are required for cell motility (Figure 1). Once phosphorylated at Ser3 by the LIM kinases cofilin can no longer bind to actin leading to the accumulation of actin polymers. LIM kinases are therefore centrally positioned regulators of actin cytoskeletal dynamics and also play important roles in microtubule organization. 2,3 LIMK1 has been reported to play a key regulatory role in tumour cell invasion and the level of LIMK1 is increased in invasive breast, 4 prostate, 5 and pancreatic 6 cancer cell lines in comparison with less invasive cells. Overexpression of LIMK1 in MCF-7 and in MDA MB-231 human breast cancer cell lines increased their motility, while inhibition of LIMK1 activity in MDA MB-231 cells by expression of a dominant negative LIMK1 resulted in decreased motility and formation of osteolytic bone lesions in an animal model of tumour invasion. 7 As such, the LIM kinases have been proposed to be attractive drug targets to block tumour cell invasion and metastasis. A number of groups have previously reported inhibitors of the LIM kinases 9-13 as treatments for cancer and for their ability to lower intraocular pressure for glaucoma. Herein we describe the discovery and development of a series of LIMK inhibitors that demonstrate inhibition of p-cofilin and inhibit invasion of cancer cells in a 3D inverse invasion assay. We used a commercially available kinase Glo ® kit measuring ATP depletion using full length LIMK and cofilin to screen 60,000 compounds. 14 From this we identified two lead series, a series of pyrimid...
Almost a century ago Theodor Boveri suggested that tumor cells differ from normal cells in their high incidence of centrosome amplification. However, only recently have new therapeutic strategies been explored in an attempt to exploit these differences and the role of kinesins in mitosis. Intense interest in the field has led to development of KSP and CENP-E inhibitors that have been tested clinically as treatments for human cancer. Success has been limited because both motor proteins are essential to normal mitosis and inhibition leads to mitotic arrest and associated neutropenia toxicity in normal cells. In contrast HSET is essential for survival of cancer cells with centrosome amplification and has been shown to be dispensable in normal cells. Hence, HSET inhibition offers a unique opportunity to selectively damage malignant cells with supernumerary centrosomes without affecting normal cells. In keeping with these findings we report discovery of a novel allosteric inhibitor of HSET, CW069, that does not disrupt division in normal human fibroblast cells, or in MCF-7 cells with normal centrosome numbers. In fact, CW069 induces multipolar mitosis exclusively in cancer cells with extra centrosomes, causing apoptosis via catastrophic aneuploidy. The increased multipolar mitoses induced in N1E-115 cells by inhibitor CW069 recapitulates the phenotype described here, and by others, for siRNA depletion of HSET. The inhibitor also reduces cell growth and centrosome clustering in cancer cells with a lower incidence of centrosome amplification, including BT549 and MDA-MB-231 breast cancer cells. This is consistent with recent reports that depletion of HSET in DNA damage repair deficient cells may be lethal even to cancer cells with low-level centrosome amplification. Taken together, these data indicate that CW069 inhibition of HSET is not restricted to use in N1E-115 cells with high centrosome amplification, and could be broadly applicable to a range of human cancers. What is more, CW069 does not decrease the clonogenic capacity of primary adult human bone marrow cells, suggesting that it would not cause neutropenia toxicity in normal cells. It is anticipated that HSET inhibition could have a greater therapeutic margin than KSP or CENP-E inhibition, and, to the best of our knowledge we have described the first allosteric inhibitor of HSET that reduces centrosome clustering but does not induce the mitotic phenotypes associated with inhibition of KSP or CENP-E. This selectivity for HSET is consistent with our computational model, which indicates that the HSET loop 5 displays dynamic conformational selection for CW069 that cannot be achieved by closely related KSP. In summary, CW069 not only represents a substantial advance toward new cancer therapeutics, but also offers researchers a unique tool to unveil the full details of HSET function in mitosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B96. Citation Format: Ciorsdaidh A. Watts, Frances M. Richards, Andreas Bender, Peter J. Bond, Oliver Korb, Oliver Kern, Michelle Riddick, Paul Owen, Rebecca M. Myers, Jordan Raff, Fanni Gergely, Duncan I. Jodrell, Steven V. Ley. Design, synthesis and biological evaluation of a novel allosteric inhibitor of HSET that damages cancer cells with supernumerary centrosomes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B96.
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