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Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

ABSTRACT: Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.

SUBMITTER: Hetrick B

PROVIDER: S-EPMC3684959 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

Hetrick Byron B Han Min Suk MS Helgeson Luke A LA Nolen Brad J BJ

Chemistry & biology 20130425 5

Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short ...[more]

PMID: 23623350

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Project description: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.

Project description:The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.

Dataset Information

Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

Publications

Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

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