Characterization of two classes of small molecule inhibitors of Arp2/3 complex

Abstract

Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

Figure 1

Two classes of small molecules inhibit nucleation of actin filaments by Arp2/3 complex. a, Structures of CK-636, CK-548, CK-666 and CK-869. b, Inhibition of HsArp2/3 complex by CK-636 and CK-548. The time course of actin polymerization was monitored by the fluorescence increase of pyrenyl-actin. Conditions: 20 μM compound or DMSO, 2.5 μM 15% pyrenyl-actin alone or with 100 nM Cdc12(FH2) or 6 nM HsArp2/3 complex and 300 nM WASp-VCAThe maximum polymerization rate is expressed in arbitrary units. Error bars, s.d., n=4) c, Inhibition of Bos taurus (Bt) and Schizosaccharomyces pombe (Sp) Arp2/3 complexes by CK-636 and CK-548. The time course of polymerization was measured as in (1b). Conditions: 4 μM 15% pyrenyl-actin, 5 nM SpArp2/3 complex, and 1 μM N-WASp-VCA, or 3 μM 30% pyrenyl-actin, 5 nM BtArp2/3 complex and 1 μM N-WASp-VCA. CK548 was insoluble at 200 μM under the conditions used for this assay. The maximum polymerization rate of actin alone under these conditions was 4.6 nM/s. d, Effect of CK-666 and CK-869 on the polymerization of actin with bovine and yeast Arp2/3 complexes. Conditions as in 1(c) with either 3 μM 30% pyrenyl-actin and 5 nM BtArp2/3 complex or 4 μM 20% pyrenyl-actin plus 20 nM SpArp2/3 complex or 5 nM ScArp2/3 complex. Both compounds reduced the maximum polymerization rate of samples with BtArp2/3 complex to the basal rate without Arp2/3 complex but CK869 did not inhibit either yeast Arp2/3 complex. e, Fluorescence micrographs of the products of actin polymerization assays stained with Alexa 488-phalloidin. Actin (3.6 μM) was polymerized with 6 nM HsArp2/3 complex, 100 nM WASp-105-502, 300 nM Cdc42 and 100 μM CK-636 Scale bar = 20 μm.

Figure 2

Inhibition of actin assembly in live cells by CK-548, CK-636 and CK-666. a–g, Formation of actin filament comet tails by Listeria infecting SKOV3 cells. a–b, Fluorescence micrographs of fixed cells stained with rhodamine phalloidin. a, Cells incubated at 37°C for 90 min with 0.1% DMSO had Listeria comet tails. b, Cells incubated at 37°C for 90 min with 100 μM CK548 in 0.1% DMSO had no actin comet tails. c, Dependence of the fraction of Listeria with comet tails on the concentrations of CK-636 and CK-548. Error bars, s.d., n=3. d–g, Effects of CK-666 on actin fluorescence around Listeria in SKOV3 cells. Infected cells were treated with 40 μM CK-666 for 60 min followed by a 60 min washout. The pairs of fluorescence micrographs show anti-Listeria fluorescence (top) and Alexa 488 phalloidin fluorescence (bottom). d, Control without CK-666 for 60 minutes. e, CK-666 for 60 minutes. f, CK-666 for 60 minutes followed by 60 minutes washout. g, Dependence of the mean actin fluorescence around each Listeria cell on the concentrations of CK-666 (active) and CK-689 (inactive) for 60 minutes. Fluorescence recovered when CK-666 was washed out for 60 minutes. Error bars are standard deviations of the mean fluorescence values from four separate experiments each with around 5000 Listeria per condition. A fluorescence value of 0.6 corresponds to background actin fluorescence. h–k, Effect of compounds on podosome formation by THP-1 derived monocyte cells. Adhered cells were treated with DMSO or compounds for 15 minutes, then fixed and stained with Alexa 568 phalloidin. h, 0.1 % DMSO control. i, 100 μM CK-636 and j, 100 μM CK-548 reduce formation of podosomes. k, Fraction of cells with >5 podosomes versus inhibitor concentration, normalized to DMSO control. Error bars, s.d., n=3. Scale bar = 20 μm.

Figure 3

Crystal structure of BtArp2/3 complex with bound CK-636. Color code: Arp3, orange; Arp2, pink; ARPC1, green; ARPC2, cyan; ARPC3, magenta, ARPC4, blue; ARPC5, yellow. a, Ribbon diagram with a detail of binding pocket. b, Stereo diagram of an Fo-Fc electron density map showing changes caused by CK-636. Positive (blue) and negative (red) difference densities indicate the position of CK-636 and small conformational changes of the protein. The apo-structure (1K8K pdb) is shown in green stick representation and the final structure in yellow. The density is contoured at 3.5 σ and was generated using structure factors calculated after one round of rigid body refinement and the data for the CK-636-soaked crystal.

Figure 4

Crystal structure of BtArp2/3 complex with bound CK548. The color coding is the same as in Figure 3. a, Ribbon diagram with detail of binding pocket. Black arrow marks the loop (residues 76–85) connecting β6 (73–75) and αB (86–98) in subdomain 1 of Arp3, which flips up to accommodate inhibitor binding. b, Ribbon diagrams of Arp3 with bound CK-548 (orange) overlaid onto the apo-BtArp2/3 structure (1K8K.pdb, grey). Black dotted line indicates the binding pocket for ATP or ADP. Orange dotted lines indicate disordered regions of the structure. Small red arrowheads indicate alternative conformations of the sensor loop. CK-548 is shown in stick representation with grey carbon atoms and select residues in the sensor loop from the 1K8K structure are shown as sticks with yellow carbon atoms.