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

by Selleckchem | Jan 07 2024

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.

Comments:

This is a detailed and technical description of a study focused on identifying compounds related to CK-666 that inhibit the activity of the Actin-related protein 2 and 3 (Arp2/3) complex. The primary goal appears to be finding compounds that can prevent Arp2/3 from adopting its active conformation, thus affecting cellular processes like cell migration, membrane protrusion, and cell cycle progression.

The research involved screening 74 compounds structurally related to CK-666 using various assays. The initial screening used EdU incorporation in MCF10A cells, and nine compounds showed promising results. These compounds were further tested on B16-F1 melanoma cells, confirming their ability to block lamellipodial protrusions and cell migration.

Additionally, the study investigated the effects of these compounds on sea urchin embryos, known to exhibit specific phenotypes upon Arp2/3 inhibition, such as the lack of triradiate spicules and inhibition of archenteron elongation. Some compounds were ruled out due to toxicity in cell cultures or on sea urchin development.

Two CK-666 analogs, compounds 59 and 69, were found to be active in all assays and more efficient in vivo than CK-666, despite being slightly less efficient in vitro. They were identified as potent Arp2/3 inhibitors with improved in vivo potency through induced-fit docking, which revealed their interaction with Arp2/3, suppressing its activation.

Overall, the study demonstrates the validity of original assays for assessing Arp2/3 activity, identifies new compounds that can suppress Arp2/3 activation, and highlights two analogs with enhanced in vivo efficiency compared to CK-666.