Fokin AI et al. (2022), Synthesis, Screening and Characterization of No...

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Front Pharmacol 2022 Jan 01;13:896994. doi: 10.3389/fphar.2022.896994.

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

Fokin AI , Chuprov-Netochin RN , Malyshev AS , Romero S , Semenova MN , Konyushkin LD , Leonov SV , Semenov VV , Gautreau AM .

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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.

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	Scheme 1. Synthesis of substituted 2-methyltryptamines 1–74 and CK-666.
	FIGURE 1. Screening of Arp2/3 inhibitors in MCF10A cells based on EdU incorporation. CK-666 and nine out of 74 tested analogs block cell cycle progression in a dose-dependent manner. Mean ± S.D. of three technical repeats. Two biological replicates with three technical repeats were performed and gave similar results. Only one is displayed. Dashed and dotted lines represent Mean ± SD of control cells treated with DMSO.
	FIGURE 2. Effect of selected CK-666 analogs on lamellipodium protrusion and cell migration in the mouse melanoma B16-F1 cell line. (A) Immunofluorescence using antibodies targeting ARPC2 (a subunit of the Arp2/3 complex) or cortactin (marker of branched actin), phalloidin (probe targeting filamentous actin) and DAPI (marker of nuclei). Cells were treated for 1 h with 100 μM of the inactive control CK-689, CK-666 and its analog #59. Scale bar: 20 μm. (B) Quantification of cells displaying lamellipodia. For each compound, more than 120 cells from seven fields of view (n = 7) were analyzed. Mean ± S.D. Kruskal–Wallis test. Two biological replicates with three technical repeats were performed and gave similar results. Only one is displayed. p < 0.05, p < 0.01, p < 0.001, *p < 0.0001, ns non-significant. (C) Viability of cells treated with 100 μM of compound for 4 h was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)). Mean ± S.D. of a single experiment with three technical repeats (n = 3). Kruskal–Wallis test. p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001, ns non-significant. (D) Dose-dependent lamellipodium formation. The four selected compounds, 59, 69, 15 and 71 were compared to the active CK-666 and the inactive CK-689 compounds. For each compound, more than 130 cells from seven fields of view (n = 7) were analyzed per condition. Mean ± S.D. of a single experiment. p-values are given in the Supplementary Material (Supplementary Table S2). (E) Wound healing. CK-689 and CK-666 were at 100 μM, the four compounds 59, 69, 15 and 71 were at 25 μM. Nine wounded fields (n = 9) were imaged per condition and healing was measured over time. Mean ± S.D. is plotted for each time point. Several experiments were performed with different concentrations of analogs (25, 50 and 100 μM) in comparison with 100 μM of CK-666. One representative experiment with 25 μM of compounds is shown. p-values are given in the Supplementary Material (Supplementary Table S3). Plots of 15 and 69 are hardly visible, because they are superimposed to the plot of CK-666.
	FIGURE 3. Arp2/3 inhibition in vitro. Four selected compounds, 59, 69, 15 and 71, were compared to the active CK-666 and its inactive analog, CK-689. Polymerization of 1.5 μM actin (10% pyrenyl-labeled) was induced by 20 nM Arp2/3 and 250 nM N-WASP VCA and monitored by fluorescence increase of pyrenyl-labeled actin. The maximum polymerization rate was converted in nM/s. One representative experiment is shown, another experiment with slightly different conditions gave similar results. Compounds were dissolved in DMSO and total amount of DMSO was kept constant in the different conditions. The inactive inhibitor curve is not fitted.
	FIGURE 4. Effect of CK-666 analogs on sea urchin embryos. Embryos were treated with compounds at zygote stage (8–15 min post fertilization) and incubated at 22°C for the indicated time. (A) Untreated gastrula. Primary mesenchyme cells (PMC) form two ventrolateral clusters with triradiate spicule rudiments (Sp). Secondary mesenchyme cells (SMC) at the tip of archenteron (Ar) participate to archenteron elongation. (B) Defective gastrulation induced by compound 69 at 12 μM. (C) Defective gastrulation induced by CK-666 at 100 μM. Note altered distribution of primary and secondary mesenchyme cells, lack of spicules, and half-size archenteron. (D) Non-specific defects induced by compound 15 at 20 μM. Note the lack of obvious structures and low transparency. (E) Untreated larva at the early pluteus stage. Scale bar 100 μm.
	FIGURE 5. Models of Arp2/3 complex bound to CK-666 (A), compound 59, (B), debenzylated metabolite of 59 (C), and compound 69 (D). Induced-fit docking was used to model binding of CK-666 analogs to the structure obtained by co-crystallizing CK-666 with the Arp2/3 complex (PDB code 3UKR).

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