Diversity through a branched reaction pathway: generation of multicyclic scaffolds and identification of antimigratory agents.

A library of 91 heterocyclic compounds composed of 16 distinct scaffolds has been synthesized through a sequence of phosphine-catalyzed ring-forming reactions, Tebbe reactions, Diels-Alder reactions, and, in some cases, hydrolysis. This effort in diversity-oriented synthesis produced a collection of compounds that exhibited high levels of structural variation both in terms of stereochemistry and the range of scaffolds represented. A simple but powerful sequence of reactions thus led to a high-diversity library of relatively modest size with which to explore biologically relevant regions of chemical space. From this library, several molecules were identified that inhibit the migration and invasion of breast cancer cells and may serve as leads for the development of antimetastatic agents.

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion.

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.

Synthesis and structure-activity relationships of metal-ligand complexes that potently inhibit cell migration.

We screened the National Cancer Institute Diversity Set compound collection for small molecules that affect mammalian cell migration and identified NSC 295642 as an inhibitor of cell motility with nanomolar potency. We found by LC-MS and X-ray crystallography that NSC 295642, a Cu(II) complex of the Schiff base product of condensation of S-benzyl dithiocarbazate and 2-acetylpyridine, has a bridged dimeric Cu2Cl2(L)2 structure with distorted square pyramidal geometry. Each of the two copper atoms is five-coordinated to one of the two tridentate chelating ligands and both bridging chlorine atoms. To define structure-activity relationships,we investigated the bioactivity of related metal-ligand complexes derived from different metal(II) atoms and different ligands. Complexation of the NSC 295642 ligand with Zn(II) or Ni(II), delivered as metal(II) chloride salts under conditions identical to those used for preparation of the original Cu(II) complex, instead results in distorted octahedral bis-chelate structures, where a single metal atom is six-coordinated to two ligands. The Zn(L)2 complex possesses a potency similar to that of the Cu2Cl2(L)2 complex, while the Ni(L)2 has no antimigratory activity at all. We carried out density functional theory calculations to obtain the electronic ground state geometry of the complexes, both in vacuum and implicit water solvent. The X-ray crystal and energy-minimized structures are very similar and exhibit a transoid orientation of the S-benzyl groups relative to the central metal-coordinated rings for both of the bioactive Cu2Cl2(L)2 and Zn(L)2 complexes, despite their different coordination geometries. In contrast, the biologically inactive Ni(L)2 complex adopts a cisoid conformation. Varying the ligand structure, we found that hydrophobic S-alkylaryl groups are required for activity. Complexes with a simple S-methyl group, S-benzyl groups with polar substitutions or a carboxylated pyridine ring exhibit dramatically reduced activity. We tested the most potent metal-ligand complex in a number of cancer cell lines and found cell-type selectivity in its effect on cell motility. Collectively, these results suggest that a two-ligand structure with bulky nonpolar S-substituents in a transoid conformation is important for the antimigratory activity of these metal-ligand complexes.

Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Locostatin Disrupts Association of Raf Kinase Inhibitor Protein With Binding Proteins by Modifying a Conserved Histidine Residue in the Ligand-Binding Pocket.

Raf kinase inhibitor protein (RKIP) interacts with a number of different proteins and regulates multiple signaling pathways. Here, we show that locostatin, a small molecule that covalently binds RKIP, not only disrupts interactions of RKIP with Raf-1 kinase, but also with G protein-coupled receptor kinase 2. In contrast, we found that locostatin does not disrupt binding of RKIP to two other proteins: inhibitor of κB kinase α and transforming growth factor ß-activated kinase 1. These results thus imply that different proteins interact with different regions of RKIP. Locostatin's mechanism of action involves modification of a nucleophilic residue on RKIP. We observed that after binding RKIP, part of locostatin is slowly hydrolyzed, leaving a smaller RKIP-butyrate adduct. We identified the residue alkylated by locostatin as His86, a highly conserved residue in RKIP's ligand-binding pocket. Computational modeling of the binding of locostatin to RKIP suggested that the recognition interaction between small molecule and protein ensures that locostatin's electrophilic site is poised to react with His86. Furthermore, binding of locostatin would sterically hinder binding of other ligands in the pocket. These data provide a basis for understanding how locostatin disrupts particular interactions of RKIP with RKIP-binding proteins and demonstrate its utility as a probe of specific RKIP interactions and functions.

Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics.

BACKGROUND: Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton. METHODOLOGY/PRINCIPAL FINDINGS: We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I. CONCLUSIONS/SIGNIFICANCE: Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.

Raf kinase inhibitor protein suppresses nuclear factor-κB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression.

Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor-κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP's function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.