CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes.

The inhibitors, CK-666 and CK-869, are widely used to probe the function of Arp2/3 complex mediated actin nucleation in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 also only inhibits Arp3- but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.

FYCO1 regulates migration, invasion, and invadopodia formation in HeLa cells through CDC42/N-WASP/Arp2/3 signaling pathway.

FYCO1, an autophagy adaptor, plays an essential role in the trafficking toward the plus-end of microtubules and the fusion of autophagosomes. Autophagic dysfunction is involved in numerous disease states, including cancers. Previous studies have implicated FYCO1 as one of the critical genes involved in the adenoma to carcinoma transition, but the biological function and mechanism of FYCO1 in carcinogenesis remain unclear. This study aims to elucidate the role and mechanism of up- and downregulation of FYCO1 in mediating tumor effects in HeLa cells. Functionally, FYCO1 promotes cellular migration, invasion, epithelial-mesenchymal transition, invadopodia formation, and matrix degradation, which are detected through wound healing, transwell, immunofluorescence, and Western blot approaches. Interestingly, the data show that although FYCO1 does not affect HeLa cell proliferation, cell cycle distribution, nor vessels' formation, FYCO1 can block the apoptotic function. FYCO1 inhibits cleavage of PARP, caspase3, and caspase9 and increases Bcl-2/Bax ratio. Then, we used CK666, an Arp2/3 specific inhibitor, to confirm that FYCO1 may promote the migration and invasion of HeLa cells through the CDC42/N-WASP/Arp2/3 signaling pathway. Taken together, these results provide a new insight that FYCO1, an autophagy adaptor, may also be a new regulator of tumor metastasis.

Cellular and pathophysiological consequences of Arp2/3 complex inhibition: role of inhibitory proteins and pharmacological compounds.

The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.

Intranuclear Actin Structure Modulates Mesenchymal Stem Cell Differentiation.

Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA-seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin-induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevented by silencing of the formin mDia1, but not its paralog mDia2. Furthermore, while inhibition of actin, branching is a potent adipogenic stimulus, silencing of either mDia1 or mDia2 blocks adipogenic gene expression. We propose that mDia1, which localizes in the cytoplasm of multipotential MSCs and traffics into the nucleus after cytoskeletal disruption, joins intranuclear mDia2 to facilitate primary filament formation before mediating subsequent branching via Arp2/3 complex recruitment. The resulting intranuclear branched actin network specifies osteogenic differentiation, while actin polymerization in the absence of Arp2/3 complex-mediated secondary branching causes adipogenic differentiation. Stem Cells 2017;35:1624-1635.

Inhibition of the Arp2/3 complex represses human lung myofibroblast differentiation and attenuates bleomycin-induced pulmonary fibrosis.

BACKGROUND AND PURPOSE: The Arp2/3 multiprotein complex regulates branched polymerisation of the actin cytoskeleton and may contribute to collagen synthesis and fibrogenesis in the lung. EXPERIMENTAL APPROACH: Expression of Arp2/3 components was assessed in human lung fibroblasts and in the bleomycin-induced pulmonary fibrosis model in mice. The Arp2/3 complex was repressed with the allosteric inhibitor CK666 and with interfering RNAs targeting the ARP2, ARP3 and ARPC2 subunits (siARP2, siARP3 and siARPC2) in CCD-16Lu human lung fibroblasts in vitro. Mice received daily intraperitoneal injections of CK666 from the 7th to the 14th day after tracheal bleomycin instillation. KEY RESULTS: Expression of Arp2/3 complex subunits mRNAs was increased in fibroblasts treated with TGF-ß1 and in the lungs of bleomycin-treated mice compared with controls. In vitro, CK666 and siARPC2 inhibited cell growth and TGF-ß1-induced α-smooth muscle actin (ACTA2) and collagen-1 (COL1) expression. CK666 also decreased ACTA2 and COL1 expression in unstimulated cells. CK666 reduced Akt phosphorylation and repressed phospho-GSK3ß, ß-catenin and MRTF-A levels in unstimulated fibroblasts. In vivo, CK666 reduced levels of both procollagen-1 and insoluble collagen in bleomycin-treated mice. CONCLUSION AND IMPLICATIONS: Expression of the Arp2/3 complex was increased in profibrotic environments in vitro and in vivo. Inhibition of the Arp2/3 complex repressed ACTA2 and COL1 expression and repressed an Akt/phospho-GSK3ß/ß-catenin/MRTF-A pathway in lung fibroblasts. CK666 exerted antifibrotic properties in the lung in vivo. Inhibition of the Arp2/3 complex could represent an interesting new therapy for idiopathic pulmonary fibrosis and other fibrotic interstitial lung diseases.

Synthesis, Screening and Characterization of Novel Potent Arp2/3 Inhibitory Compounds Analogous to CK-666.

Branched actin networks polymerized by the Actin-related protein 2 and 3 (Arp2/3) complex play key roles in force generation and membrane remodeling. These networks are particularly important for cell migration, where they drive membrane protrusions of lamellipodia. Several Arp2/3 inhibitory compounds have been identified. Among them, the most widely used is CK-666 (2-Fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide), whose mode of action is to prevent Arp2/3 from reaching its active conformation. Here 74 compounds structurally related to CK-666 were screened using a variety of assays. The primary screen involved EdU (5-ethynyl-2'-deoxyuridine) incorporation in untransformed MCF10A cells. The resulting nine positive hits were all blocking lamellipodial protrusions and cell migration in B16-F1 melanoma cells in secondary screens, showing that cell cycle progression can be a useful read-out of Arp2/3 activity. Selected compounds were also characterized on sea urchin embryos, where Arp2/3 inhibition yields specific phenotypes such as the lack of triradiate spicules and inhibition of archenteron elongation. Several compounds were filtered out due to their toxicity in cell cultures or on sea urchin development. Two CK-666 analogs, 59 (N-{2-[5-(Benzyloxy)-2-methyl-1H-indol-3-yl] ethyl}-3-bromobenzamide) and 69 (2,4-Dichloro-N-[2-(7-chloro-2-methyl-1H-indol-3-yl) ethyl]-5-[(dimethylamino) sulfonyl] benzamide), were active in all assays and significantly more efficient in vivo than CK-666. These best hits with increased in vivo potency were, however, slightly less efficient in vitro than CK-666 in the classical pyrene-actin assay. Induced-fit docking of selected compounds and their possible metabolites revealed interaction with Arp2/3 that suppresses Arp2/3 activation. The data obtained in our screening validated the applicability of original assays for Arp2/3 activity. Several previously unexplored CK-666 structural analogs were found to suppress Arp2/3 activation, and two of them were identified as Arp2/3 inhibitors with improved in vivo efficiency.