The emergence of tumor resistance to conventional microtubule-targeting drugs restricts their clinical use. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we identified Pyr1, a cell permeable inhibitor of LIM kinase, which is the enzyme that phosphorylates and inactivates the actin-depolymerizing factor cofilin. Pyr1 reversibly stabilized microtubules, blocked actin microfilament dynamics, inhibited cell motility in vitro and showed anticancer properties in vivo, in the absence of major side effects. Pyr1 inhibition of LIM kinase caused a microtubule-stabilizing effect, which was independent of any direct effects on the actin cytoskeleton. In addition, Pyr1 retained its activity in multidrug-resistant cancer cells that were resistant to conventional microtubule-targeting agents. Our findings suggest that LIM kinase functions as a signaling node that controls both actin and microtubule dynamics. LIM kinase may therefore represent a targetable enzyme for cancer treatment. Cancer Res; 72(17); 4429–39. ©2012 AACR.

Microtubules are filaments composed of α-tubulin and β-tubulin heterodimers. They are highly dynamic polymers, and their dynamics is tightly regulated by a balance between activities of microtubules-stabilizing and microtubule-destabilizing proteins (1–3).

Moreover, the interaction of the microtubule growing tips with some proteins such as CLIP 170 results in microtubule stabilization (4, 5). The binding of all of these proteins to tubulin is tightly regulated by phosphorylation/dephosphorylation processes (6, 7) or, directly, by tubulin modifications such as the tubulin tyrosination cycle (8, 9).

A detailed knowledge of the different players and how they interact both spatially and temporally to regulate microtubule functions is, however, still lacking.

Because of microtubules key role in mitosis, microtubule-targeting agents are powerful anticancer drugs. Tubulin is now considered as one of the most highly validated cancer target (10, 11). These drugs have, however, some limitations due to side effects, principally myelosuppression and neurotoxicity. Moreover, many cancers are, or become, resistant to these drugs (12). This is often the result of multidrug resistance caused by overexpression of ATP-binding cassette transporters (13). Several strategies have been proposed for the development of more effective and less toxic anticancer drugs. One of them is to identify drugs targeting proteins that regulate microtubule functions and that would lead to mitotic arrest and/or apoptosis.

The aim of this study was to identify chemical compounds able to interact with regulators of microtubule dynamics. We therefore conducted a screening using a cell-based assay that probes the microtubule polymerization status (14). We identified a compound (Pyr1) that slows down microtubule dynamics. Pyr1 is toxic for cancerous cell lines, including drug-resistant cell lines. Pilot studies on mice with xenografted tumors indicate that Pyr1 prevented tumor growth and was well tolerated. Pyr1 also affected the actin cytoskeleton and inhibited cell motility. We have identified LIM kinases (LIMK) as the main targets of Pyr1. The activity of LIMK1/2 is regulated mainly by the Rho-dependent kinases ROCK1 (15) and ROCK2 (16) and the p21-activated kinases PAK1 (17) and PAK4 (18) and inactivated mainly by the slingshot phosphatases (19). Phosphorylation and inhibition of the actin-depolymerizing protein cofilin by LIMKs is the last step of a cascade that regulates actin polymerization. Here, we show that Pyr1 inhibits cofilin phosphorylation. We established that the microtubule-stabilizing effect of Pyr1 results from its inhibitory effect on LIMKs and is independent of its effect on the actin cytoskeleton. Thus, LIMK inhibition by Pyr1 explains the observed phenotypes on both actin and tubulin.

Screen for chemical modulators of microtubule dynamics

The screen was conducted as described in the work of Vassal and colleagues (14). For a summarized description, see the Supplementary Methods.

Chemical reagents, recombinant and purified proteins, plasmids, antibodies, and cell lines

All reagents used are indicated in the Supplementary Methods.

Immunofluorescence microscopy

To visualize microtubules, cells were seeded either on glass coverslips or in microplates (ViewPlate-96, Perkin-Elmer Inc.). They were permeabilized in warm OPT buffer (80 mmol/L Pipes, 1 mol/L EGTA 1 mol/L MgCl2, 0.5% Triton X-100, and 10% glycerol, pH 6.8) and fixed for 6 minutes in −20°C methanol. To visualize F-actin, cells were washed in warm PBS and fixed with 4% paraformaldehyde in PBS at 37°C, followed by cell permeabilization with 0.1% Triton X-100 in PBS. After incubation with primary antibodies, cells were incubated with Cy3 (Jackson ImmunoResearch Laboratories) or Alexa 488 (Invitrogen) secondary antibodies. To visualize F-actin, Alexa 488-phalloidin (Invitrogen) was included with the secondary antibody.

Images were captured either with a charge-coupled device camera (CoolSNAP ES; Roper Scientific) in a straight microscope (Nikon Eclipse 90i) controlled by Nikon software (Universal Imaging Corp.) using a ×40, ×60, or ×100 Plan-Neofluar oil objectives, with an IN Cell Analyzer 1000 automated microscope (GE-Healthcare) using a ×40 objective, controlled by IN Cell software allowing automated cell imaging or with an Orca R2 N/B camera (Hamamatsu) in a straight microscope (Zeiss AxioImager Z1) controlled by Axiovision software (Carl Zeiss) using a ×63 oil objective. When necessary, cells were observed with a confocal microscope (Leica).

Immunofluorescence analysis of growing microtubules + ends

HeLa cells were incubated for 2 hours with the tested compound, fixed for 6 minutes in −20°C methanol, and processed for immunofluorescence using anti-EB1–specific antibody.

Tubulin polymerization assay

Microtubule polymerization assay was adapted from the work of Bonne and colleagues (20). Briefly, microtubule assembly was carried out in a half-area, 96-well black plate (Greiner, #675090) using a microplate reader FLUOstar OPTIMA (BMG Labtechnologies). Wells were charged with either microtubule proteins or pure tubulin (final concentration of 25 and 30 μmol/L, respectively) in MEM buffer with 10 μmol/L 4′,6-diamidino-2-phenylindole (DAPI) and variable concentrations of compounds. Following 10-minute incubation, assembly was started by injection of GTP and MgCl2 to a final concentration of 1 and 5 mmol/L, respectively, in 100 μL. The excitation and emission wavelengths were set at 360 and 450 nm, respectively, and the fluorescence of microtubule-bound DAPI was monitored as a function of time at 37°C. Fluorescence signal at time 0 was subtracted from each of the subsequent fluorescence readings. Each compound was assayed in triplicate.

Actin-pyrene polymerization assay

Mg-ATP-monomeric actin (2 μmol/L, 10% pyrene labeled) in MME buffer containing 0.2 mmol/L ATP was polymerized at room temperature after addition of one-tenth volume of ×10 buffer (500 mmol/L KCl, 20 mmol/MgCl2, 2 mmol/L EGTA, 100 mmol/L imidazole, pH 7.5). Experiments were carried out in the presence or absence of different compounds, as indicated in the figure legend. The polymerization was followed by changes in pyrene fluorescence using a Xenius SAFAS fluorimeter (Safas SA).

Tumor xenograft experiments

Twelve B6D2F1 female mice were injected subcutaneously in the right flank with 2 × 106 L1210 cells. Mice were then randomly separated into 2 groups of 6 mice each. In one group, Pyr1 (10 mg/kg/d in PEG400) was injected intraperitoneally, on the left flank, daily for 10 days. In the other group, PEG400 alone was injected, daily for 10 days. Each day, the mice were observed for survival. They were weighted twice a week. The experiment was stopped 20 days after the last vehicle-treated mice death. The ethics committee of Lyon University (Lyon, France) validated this experimental protocol.

All the other experimental procedures are described in the Supplementary Methods.

Hits selection process

The screening assay is based on the properties of the tubulin enzymes involved in the tubulin tyrosination cycle, tubulin tyrosine ligase, and tubulin carboxypeptidase. Because of their substrate properties, dynamic microtubules are composed of tyrosinated tubulin (Tyr-tubulin), whereas stabilized microtubules are composed of detyrosinated tubulin (Detyr-tubulin; ref. 21). Thus depolymerization or stabilization of the microtubule network can be distinguished by double immunofluorescence using specific Tyr- and Detyr-tubulin antibodies (14). We selected 16 compounds that enhance the Detyr-tubulin signal by 20%. After checking the compounds structure, only 5 chemically nonreactive compounds that generate stable Detyr-microtubules in cells were selected for further characterization (Supplementary Table S1).

It is well established that sodium azide can stabilize microtubules, thus generating Detyr-tubulin (22). To eliminate compounds acting via similar mechanisms, we analyzed compounds effects on mitochondria and discarded compounds targeting mitochondria as did sodium azide (Supplementary Table S1). We also eliminated compounds with no analogues, leaving one remaining compound, 9-benzoyloxy-5,11-dimethyl-2H,6H-pyrido[4,3-b]carbazol-1-one, (Pyr1; Fig. 1A). Incubation of HeLa cells with 25 μmol/L of Pyr1 resulted in Detyr-microtubules generation (Fig. 1B).

Figure 1.

Effect of Pyr1 on cellular Tyr- and Detyr-microtubule content. A, chemical structure of Pyr1. B, cells treated with Pyr1 have increased Detyr-microtubule content. HeLa cells were treated for 2 hours with 25 μmol/L Pyr1 or with 0.25% DMSO alone (DMSO, control) as indicated. Left, Tyr-tubulin staining; right, Detyr-tubulin staining. Bar, 10 μm.

Figure 1.

Effect of Pyr1 on cellular Tyr- and Detyr-microtubule content. A, chemical structure of Pyr1. B, cells treated with Pyr1 have increased Detyr-microtubule content. HeLa cells were treated for 2 hours with 25 μmol/L Pyr1 or with 0.25% DMSO alone (DMSO, control) as indicated. Left, Tyr-tubulin staining; right, Detyr-tubulin staining. Bar, 10 μm.

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Pyr1 suppresses microtubule dynamic instability without directly targeting tubulin

To check whether the enrichment of Detyr-microtubules results from an effect on the tubulin tyrosination enzymes or from an effect on microtubules dynamics, we first studied the resistance of the microtubule network to nocodazole-induced depolymerization. Nocodazole binds free tubulin and prevents its incorporation into microtubules, inducing microtubule depolymerization. Microtubules with slow dynamics have reduced exchanges with the free tubulin pool and are thus less sensitive to nocodazole-induced depolymerization (23). We found that a pretreatment of cells with Pyr1 protects the microtubule network from nocodazole-induced depolymerization (Fig. 2A). We next examined the distribution of the plus-end tracking protein (+TIP) EB1 (end binding protein 1) that specifically probes growing microtubule plus ends. Cells treated with dimethyl sulfoxide (DMSO), 5 μmol/L paclitaxel, or 10 and 25 μmol/L Pyr1 were stained with an anti-EB1–specific antibody (Fig. 2B). We observed a decrease of the number of EB1 comets per cell in paclitaxel-treated cells (92% reduction of the number of EB1 comets) as well as in cells treated with Pyr1 (84% reduction of the number of EB1 comets for 25 μmol/L Pyr1), together with a size reduction of remaining EB1 comets (45% and 27% reduction of the comets surface for 5 μmol/L paclitaxel and 25 μmol/L Pyr1, respectively), suggesting a strong reduction of microtubule growth dynamics (Supplementary Fig. S1).

Figure 2.

Effect of Pyr1 on the stabilization of cellular microtubules in cells and in vitro and its effect on the cell cycle. A, immunofluorescence analysis of cell microtubules and their resistance to nocodazole-induced depolymerization. HeLa cells were incubated for 2 hours with DMSO, 5 μmol/L paclitaxel, or 25 μmol/L Pyr1. Nocodazole (10 μmol/L) was then added for 30 minutes. Cells were then stained for total tubulin (red). Nuclei were stained with Hoechst. B, immunofluorescence analysis of EB1 comets HeLa cells were incubated for 2 hours with DMSO, 5 μmol/L paclitaxel, or 10 and 25 μmol/L Pyr1 as indicated and then stained for EB1. C, comparison of nocodazole, paclitaxel, and Pyr1 effects on the mitotic spindles. HeLa cells were incubated for 2 hours with DMSO, 10 μmol/L nocodazole, 5 μmol/L paclitaxel, or 25 μmol/L Pyr1 as indicated and then stained for total tubulin. Representative metaphase spindles are shown. D, Pyr1 induces a cell-cycle arrest at S- to G2–M phases. HeLa cells were cultured for 26 hours with 0.25% DMSO or 10 μmol/L of nocodazole, paclitaxel, or Pyr1 as indicated. The cell-cycle parameters were analyzed by flow cytometry. Histograms represent the percentage of the total cell population in each cell-cycle phase: sub-G1 (yellow), G1 (blue), S (green), G2–M (red), >4N (purple). E and F, microtubule proteins (E) and pure in vitro tubulin (F) polymerization assay. Tubulin (0.5 mg/mL for microtubule proteins and 1 mg/mL for pure tubulin) was allowed to polymerize at 37°C in the presence of 1 μmol/L paclitaxel (red), 25 μmol/L Pyr1 (green), or equivalent amount of DMSO (0.25%, orange). Results are presented as mean ± SEM of 3 independent experiments. Bars, 10 μm. A.U., arbitrary units.

Figure 2.

Effect of Pyr1 on the stabilization of cellular microtubules in cells and in vitro and its effect on the cell cycle. A, immunofluorescence analysis of cell microtubules and their resistance to nocodazole-induced depolymerization. HeLa cells were incubated for 2 hours with DMSO, 5 μmol/L paclitaxel, or 25 μmol/L Pyr1. Nocodazole (10 μmol/L) was then added for 30 minutes. Cells were then stained for total tubulin (red). Nuclei were stained with Hoechst. B, immunofluorescence analysis of EB1 comets HeLa cells were incubated for 2 hours with DMSO, 5 μmol/L paclitaxel, or 10 and 25 μmol/L Pyr1 as indicated and then stained for EB1. C, comparison of nocodazole, paclitaxel, and Pyr1 effects on the mitotic spindles. HeLa cells were incubated for 2 hours with DMSO, 10 μmol/L nocodazole, 5 μmol/L paclitaxel, or 25 μmol/L Pyr1 as indicated and then stained for total tubulin. Representative metaphase spindles are shown. D, Pyr1 induces a cell-cycle arrest at S- to G2–M phases. HeLa cells were cultured for 26 hours with 0.25% DMSO or 10 μmol/L of nocodazole, paclitaxel, or Pyr1 as indicated. The cell-cycle parameters were analyzed by flow cytometry. Histograms represent the percentage of the total cell population in each cell-cycle phase: sub-G1 (yellow), G1 (blue), S (green), G2–M (red), >4N (purple). E and F, microtubule proteins (E) and pure in vitro tubulin (F) polymerization assay. Tubulin (0.5 mg/mL for microtubule proteins and 1 mg/mL for pure tubulin) was allowed to polymerize at 37°C in the presence of 1 μmol/L paclitaxel (red), 25 μmol/L Pyr1 (green), or equivalent amount of DMSO (0.25%, orange). Results are presented as mean ± SEM of 3 independent experiments. Bars, 10 μm. A.U., arbitrary units.

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We then analyzed the effect of Pyr1 on microtubule dynamic instability parameters, using time lapse fluorescence microscopy on GFP-EB3–transfected cells (ref. 24; Supplementary Movie S1). As expected, Pyr1 suppressed the microtubule growth rate, strongly reduced the microtubule growth length as indicated by the dose-dependent increase of the distance-based catastrophe frequency, and increased time spent in pause. Such effects have been previously described for microtubule-stabilizing agents (ref. 25; Supplementary Table S2). Taken together, these experiments clearly show that Pyr1 suppresses microtubule dynamics instability.

Close analysis of the morphology of the mitotic spindles revealed that the spindles of cells treated with the compound were abnormal, with disorganized asters. We found that 84.2% of the mitotic spindles were normal in DMSO-treated cells and 12.8% abnormal, whereas 9.7% of the mitotic spindles were normal in Pyr1-treated cells and 82.9% were abnormal. These defects were clearly different from the multiple asters observed in cells treated with paclitaxel. Spindles depolymerize in nocodazole-treated cells (Fig. 2C).

As agents interfering with microtubule dynamics lead to a G2–M cell-cycle arrest, we analyzed Pyr1 effect on the cell cycle, by flow cytometry. Although Pyr1 effect was not as strong as that of nocodazole or paclitaxel, we found that it induced a cell-cycle arrest at the S- to G2–M phases (Fig. 2D).

To test whether Pyr1 directly interacts with tubulin, we assayed it in an in vitro tubulin assembly. Paclitaxel is a well-characterized microtubule-stabilizing agent that can induce in vitro tubulin assembly at low tubulin concentrations that would not induce spontaneous tubulin assembly. We found that Pyr1 could not induce in vitro tubulin assembly, neither when assayed on tubulin in the presence of associated protein (microtubule proteins, Fig. 2E) nor on pure tubulin (Fig. 2F). This experiment strongly suggests that Pyr1 does not interact directly with tubulin.

Pyr1 overcomes drug-resistant cell phenotype

Because Pyr1 was able to stabilize microtubules, with a mechanism of action different to that of taxanes, we tested whether it could be of therapeutic interest. We first examined its toxicity on several cancerous cell lines, including drug-resistant cell lines overexpressing the glycoprotein P or transporters such as ABCG2 and MRP1 that renders cells insensitive to current chemotherapies.

Pyr1 reduced the viability of these cell lines in a dose-dependent manner, with a GI50 (50% of growth inhibition) in the micromolar range (Table 1).

Table 1.

Effect of Pyr1 on the viability of different cell lines

Cell lineTypeConcentration of Pyr1 that inhibits 50% of the cell growth, μmol/L
HeLa Human cervical adenocarcinoma 
786-O Human renal adenocarcinoma 55 
NCI H460 Human lung carcinoma 
MCF7 Human breast adenocarcinoma 62 
MDA-MB-231 Human breast carcinoma 25 
MES-SA Human uterine sarcoma 26 
MES-SA/DX5 Multidrug-resistant cell line derived from the MES-SA 27 
HEK-293 Cell line derived from human embryonic kidney 
HEK-293-ABCG2 HEK-293 stably transfected with the ABCG2 transporter 
BHK-21 Cell line derived from baby hamster kidney 
BHK-21-MRP1 BHK-21 stably transfected with MRP1 (multidrug resistance protein 1) 
Cell lineTypeConcentration of Pyr1 that inhibits 50% of the cell growth, μmol/L
HeLa Human cervical adenocarcinoma 
786-O Human renal adenocarcinoma 55 
NCI H460 Human lung carcinoma 
MCF7 Human breast adenocarcinoma 62 
MDA-MB-231 Human breast carcinoma 25 
MES-SA Human uterine sarcoma 26 
MES-SA/DX5 Multidrug-resistant cell line derived from the MES-SA 27 
HEK-293 Cell line derived from human embryonic kidney 
HEK-293-ABCG2 HEK-293 stably transfected with the ABCG2 transporter 
BHK-21 Cell line derived from baby hamster kidney 
BHK-21-MRP1 BHK-21 stably transfected with MRP1 (multidrug resistance protein 1) 

Interestingly, Pyr1 toxicity is the same for the drug-sensitive human cell lines and for their multidrug-resistant counterparts, indicating that it is not substrate of the glycoprotein P or of ABCG2 and MRP1 transporters.

Pyr1 is well tolerated in mice and shows anticancer activity

The core structure of Pyr1 is similar to that of ellipticine, an alkaloid with proven antineoplastic activity, through DNA intercalation and inhibition of topoisomerase II. The ellipticine derivative N2-methyl-9-hydroxyellipticium has been used in the treatment of breast cancer, but it has been retrieved because of its toxicity. Previous studies have shown that Pyr1 is a weak DNA intercalator and has no effect on topoisomerase II (26).

To determine whether Pyr1 could serve as chemotherapeutic agent, we examined its toxicity in mice. Mice received daily intraperitoneal injections of Pyr1 (from 15 to 60 mg/kg), during 7 days. Transient signs of discomfort related to administration of the vehicle were observed in all animals (Supplementary Table S3). The weights of Pyr1-treated animals and vehicle-treated animals were not significantly different (Supplementary Table S4). Hematologic parameters were normal after Pyr1 treatment. The only small changes observed for the highest doses were not considered as having significant toxicity (Supplementary Table S5). After treatment period, a full postmortem examination was conducted. Yellow foci on the liver for mice that received high dose of Pyr1 were observed, which correlated with acute to subacute peritoneal inflammation seen in the hepatic capsule (data not shown) but without signs of severe hepatic damage. Thus, under these experimental conditions, no severe adverse effects related to Pyr1 administration were observed up to the dose of 30 mg/kg/d, indicating that Pyr1 is well tolerated.

We then conducted a pilot study using an in vivo model of leukemia L1210-bearing mice of the B6D2F1strain. The injected dose of Pyr1 was chosen to be 3 times lower than the dose (30 mg/kg/d) for which the first adverse signs were detected. Vehicle-treated mice were all dead at day 70, whereas all the Pyr1-treated mice survived (Fig. 3) and were still alive 20 days later, when the experiment was stopped. Thus, Pyr1 induced a complete survival gain (P < 0.002) with no apparent toxicity (no loss of weight, data not shown).

Figure 3.

Effects of Pyr1 on tumor growth in mice. Survival curve of L1210 syngeneic leukemia–bearing mice treated with Pyr1 or with the vehicle alone as indicated. Both groups significantly differ (P < 0.002) at day 70 (Mann–Whitney U test).

Figure 3.

Effects of Pyr1 on tumor growth in mice. Survival curve of L1210 syngeneic leukemia–bearing mice treated with Pyr1 or with the vehicle alone as indicated. Both groups significantly differ (P < 0.002) at day 70 (Mann–Whitney U test).

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These experiments indicate that Pyr1 has a good therapeutic efficacy and is well tolerated.

Pyr1 affects the organization of F-actin but does not directly target it

We then tried to identify Pyr1 cellular targets. As the microtubule and actin microfilament networks are highly interconnected (27), we investigated the effect of Pyr1 on the actin cytoskeleton. Cells treated with Pyr1 were stained for filamentous actin (F-actin) using fluorescein-phalloidin. Pyr1 affected the organization of the actin microfilaments, which was not observed when cells were exposed to paclitaxel (Fig. 4A).

Figure 4.

Effects of Pyr1 on the actin cytoskeleton and cell motility. A, immunofluorescence analysis of Pyr1 effects on F-actin. HeLa cells were incubated for 2 hours with DMSO, 5 μmol/L paclitaxel, or 25 μmol/LPyr1 as indicated. Cells were then stained with phalloidin (green) and Hoechst (blue). Bar, 20 μm. B, analysis by time lapse microscopy of the random migration of MCF10A cells treated with DMSO (control) or 10 and 25 μmol/L of Pyr1 as indicated. Tracks of individual cells obtained from 3 representative stacks revealed that Pyr1 completely abolished cell motility. C, actin-pyrene polymerization assays with 2 μmol/L actin (10% pyrene-labeled): Control (blue), with 10 μmol/L Pyr1 (orange), with 2 μmol/L phalloidin (green), and with 1 μmol/L latrunculin B (red). A.U., arbitrary units.

Figure 4.

Effects of Pyr1 on the actin cytoskeleton and cell motility. A, immunofluorescence analysis of Pyr1 effects on F-actin. HeLa cells were incubated for 2 hours with DMSO, 5 μmol/L paclitaxel, or 25 μmol/LPyr1 as indicated. Cells were then stained with phalloidin (green) and Hoechst (blue). Bar, 20 μm. B, analysis by time lapse microscopy of the random migration of MCF10A cells treated with DMSO (control) or 10 and 25 μmol/L of Pyr1 as indicated. Tracks of individual cells obtained from 3 representative stacks revealed that Pyr1 completely abolished cell motility. C, actin-pyrene polymerization assays with 2 μmol/L actin (10% pyrene-labeled): Control (blue), with 10 μmol/L Pyr1 (orange), with 2 μmol/L phalloidin (green), and with 1 μmol/L latrunculin B (red). A.U., arbitrary units.

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Because of the central role of actin dynamics in cell motility, we examined the effect of Pyr1 on MCF10A cells motility, using time lapse video microscopy. We found that Pyr1 rapidly and completely blocked cell motility, even at the lowest concentration assayed (10 μmol/L; Fig. 4B).

Pyr1 was not able to directly affect actin assembly in vitro and could not modify actin assembly kinetics, contrary to phalloidin that can stabilize F-actin in vitro and to latrunculin B that inhibits actin assembly (Fig. 4C). These results indicate that Pyr1 does not directly interact with actin.

We have also tested the possibility that the effect of Pyr1 on F-actin influences microtubule dynamics, which could indirectly result in the stabilization of microtubules. We thus pretreated cells with cytochalasin D, which depolymerizes F-actin (28), and examined Pyr1 effect on microtubule dynamics (Supplementary Fig. S2). We observed that even when the microfilaments are completely depolymerized, Pyr1 still induces the formation of Detyr-microtubules, indicating that the microtubule network is stabilized. This result strongly suggests that Pyr1-induced microtubule stabilization is independent of the inhibitor effect on the actin network.

Taken together, these findings indicate that Pyr1 targets regulators of actin as well as tubulin.

Pyr1 selectively inhibits LIM kinases activity

Previous screening identified a class of Pyr1 structurally related compounds that often targets protein kinases (26, 29). We therefore tested the ability of Pyr1 to inhibit the activity of a panel of 66 protein kinases known to be involved in the regulation of the cytoskeleton. Pyr1 inhibited only the activities of a member of NIMA-related kinase family (NEK11), mixed lineage kinase 1 (MLK1), and LIM kinase 1 (LIMK1; Fig. 5A and Supplementary Table S6). The highest inhibition was observed for LIMK1, which showed only 4% residual in vitro kinase activity. A well-established substrate of LIMK1 is cofilin, a protein that regulates actin dynamics. Using cofilin as substrate, we found that Pyr1 inhibited the activity of recombinant LIMK1 and LIMK2 (Fig. 5B C) in vitro with an IC50 of 50 and 75 nmol/L, respectively (Supplementary Fig. S3a). The inhibitor behaved as an ATP competitor (Supplementary Fig. S3b).

Figure 5.

LIM kinase is a target of Pyr1. A, inhibitory effect of Pyr1 on the kinase activity of a panel of 66 kinases including LIMK1. The data are expressed as the percentage of the activity determined in the absence of inhibitor. B and C, Pyr1 inhibits the phosphorylation of GST-cofilin by GST-LIMK1 (B) or GST-LIMK2 (C). Increasing concentrations of Pyr1 were incubated with kinase assay mix. Phosphorylated proteins were then separated on SDS-PAGE and analyzed by autoradiography (top) and Coomassie blue staining (bottom). GST-LIMK1 autophosphorylation is also abrogated in presence of Pyr1. D, inhibition of LIMK1-mediated GST-p25 phosphorylation by Pyr1. Increasing concentrations of Pyr1 were incubated with kinase assay mix. Phosphorylated proteins were then separated by SDS-PAGE and analyzed by autoradiography (top) and Coomassie blue staining (bottom). E, effect of Pyr1 on cofilin phosphorylation in cells. HeLa cells were treated for 2 hours with increasing concentrations of Pyr1, as indicated. Cells lysates were separated on SDS-PAGE and transferred for immunoblotting with anti-phospho-cofilin (Ser3) or cofilin as indicated.

Figure 5.

LIM kinase is a target of Pyr1. A, inhibitory effect of Pyr1 on the kinase activity of a panel of 66 kinases including LIMK1. The data are expressed as the percentage of the activity determined in the absence of inhibitor. B and C, Pyr1 inhibits the phosphorylation of GST-cofilin by GST-LIMK1 (B) or GST-LIMK2 (C). Increasing concentrations of Pyr1 were incubated with kinase assay mix. Phosphorylated proteins were then separated on SDS-PAGE and analyzed by autoradiography (top) and Coomassie blue staining (bottom). GST-LIMK1 autophosphorylation is also abrogated in presence of Pyr1. D, inhibition of LIMK1-mediated GST-p25 phosphorylation by Pyr1. Increasing concentrations of Pyr1 were incubated with kinase assay mix. Phosphorylated proteins were then separated by SDS-PAGE and analyzed by autoradiography (top) and Coomassie blue staining (bottom). E, effect of Pyr1 on cofilin phosphorylation in cells. HeLa cells were treated for 2 hours with increasing concentrations of Pyr1, as indicated. Cells lysates were separated on SDS-PAGE and transferred for immunoblotting with anti-phospho-cofilin (Ser3) or cofilin as indicated.

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As selectivity is a major issue for ATP-competitive kinase inhibitors, we extended the evaluation of the compound on the activity of 45 additional different kinases, using a thermal stability shift assay (30). We did not find significant interaction of the identified inhibitor with any of the screened kinases except LIMK1, which showed a Tm shift of 8.16°C, consisted with the high potency of this inhibitor (Supplementary Table S7).

Another substrate of LIMK1 is p25 (31). We investigated the ability of Pyr1 to inhibit the in vitro phosphorylation of bacterially expressed and purified human GST-p25 by recombinant LIMK1. Although the phosphorylation of GST-p25 was not efficient in control conditions, we found that Pyr1 inhibits it (Fig. 5D).

To ascertain that Pyr1 can also inhibit LIMK activity in cells, we examined the level of phospho-cofilin (p-cofilin) in cells treated for 2 hours with different concentrations of Pyr1. The level of p-cofilin was greatly reduced with increasing amounts of Pyr1, whereas the protein levels of cofilin were not affected. A complete inhibition of cofilin phosphorylation was observed after incubation of cells with 25 μmol/L of Pyr1 (Fig. 5E). This concentration was chosen to determine the time course of Pyr1 effect in cells. Complete inhibition of cofilin phosphorylation was observed 20 minutes after addition of Pyr1 (Supplementary Fig. S3c).

We also investigated whether the inhibition of LIMK1 activity by Pyr1 was reversible in vitro and in cells. To achieve this, the kinase activity of a LIMK1-Pyr1 mix was assayed before and after size exclusion chromatography, allowing the potential separation of the kinase from unbound inhibitor. After gel filtration, a significant increase in LIMK activity was observed (Supplementary Fig. S3d). Western blot analysis of protein lysates prepared from cells first treated with Pyr1 and then re-incubated in fresh medium without Pyr1 showed that cofilin phosphorylation was again detectable 30 minutes after Pyr1 washout and reached basal levels after 2 hours (Supplementary Fig. S3e). These results clearly show that Pyr1 binding to LIMK is reversible.

Similarly, the suppression of microtubule dynamic instability by Pyr1 observed in HeLa cells was also found reversible (Supplementary Table S8).

We also checked whether Pyr1 inhibits NEK11 and MLK1 activities. In vitro, Pyr1 has weak activity on both NEK11 and MLK1 (Supplementary Fig. S4a and S4b). Pyr1 effect was also checked in cells. NEK11 is known to destabilize Cdc25A (32) and a downstream target of MLK1 is Jun kinase. Looking on Cdc25 expression and on the phosphorylation status of Jun kinase, respectively, we found that Pyr1 has no effect on the activity of neither NEK11 nor MLK1 (Supplementary Fig. S4c and S4d).

LIMK inhibition causes microtubule stabilization

The identification of LIMK1/2 as targets of Pyr1 strongly suggested that the inhibition of cofilin phosphorylation is the mechanism that underlies the actin network reorganization and the loss of cell motility of cells treated with Pyr1. The link between LIMK inhibition and the observed microtubule stabilization is, however, less obvious. To investigate this issue, we compared the potency of several Pyr1 structural analogues to inhibit LIMK1 activity in vitro (Supplementary Table S9), to modify actin microfilaments dynamics, as assessed by the resistance of Pyr1-induced actin structures to latrunculin depolymerization, to induce the generation of Detyr-microtubules (Supplementary Fig. S5a), and to inhibit cofilin phosphorylation in cells (Supplementary Fig. S5b). We found a good correlation between the levels of LIMK1 inhibition by the tested compounds and the stabilization of both the actin structures and the microtubules. These experiments also allowed the determination of Pyr1 structure–activity relationships (Supplementary Table S9). In another experiment, we tested whether (N-[5-[2,6-dichloro-phenyl]-5-difluoromethyl-2H-pyrazol-3-yl]thiazol-2-yl)-isobutyramide, a recently described LIMK inhibitor (33, 34), also called LIMKi (Fig. 6A), was able to stabilize microtubules. We showed that LIMKi was indeed able to increase the Detyr-microtubules cell content (Fig. 6B) and to stabilize them to a nocodazole-induced depolymerization (data not shown). These results indicate that an LIMK inhibitor structurally different from Pyr1 is also able to induce a stabilization of the microtubule network.

Figure 6.

LIMK1 inhibition induces microtubule stabilization. A, structure of (N-[5-[2,6-dichloro-phenyl]-5-difluoromethyl-2H-pyrazol-3-yl]thiazol-2-yl)-isobutyramide, also called LIMKi. B, comparison of the effect of Pyr1 and of LIMKi on Detyr-microtubule formation. HeLa cells were treated for 2 hours with DMSO, 5 μmol/L paclitaxel, 25 μmol/L Pyr1 or LIMKi, as indicated. Cells were stained for Detyr-microtubule. C, analysis of LIMK1 content in the different cell extracts. Lysates of untransfected cells and of cells transfected with the control plasmid pEFrFlagPGKpuropAv18 or with pEFrFlagLIMK1PGKpuropAv18 plasmid overexpressing full-length LIMK1 were separated on SDS-PAGE and transferred for immunoblotting with anti-LIMK1 antibody. D, LIMK1 overexpression can reverse Pyr1-induced microtubule stabilization. HeLa cells were transfected with the control plasmid pEFrFlagPGKpuropAv18 or with pEFrFlagLIMK1PGKpuropAv18 plasmid overexpressing full-length LIMK1. Cells were treated for 2 hours with different amounts of Pyr1 as indicated. Nocodazole (10 μmol/L) was then added for 30 minutes. Cells were then stained for total tubulin. Bars, 10 μm.

Figure 6.

LIMK1 inhibition induces microtubule stabilization. A, structure of (N-[5-[2,6-dichloro-phenyl]-5-difluoromethyl-2H-pyrazol-3-yl]thiazol-2-yl)-isobutyramide, also called LIMKi. B, comparison of the effect of Pyr1 and of LIMKi on Detyr-microtubule formation. HeLa cells were treated for 2 hours with DMSO, 5 μmol/L paclitaxel, 25 μmol/L Pyr1 or LIMKi, as indicated. Cells were stained for Detyr-microtubule. C, analysis of LIMK1 content in the different cell extracts. Lysates of untransfected cells and of cells transfected with the control plasmid pEFrFlagPGKpuropAv18 or with pEFrFlagLIMK1PGKpuropAv18 plasmid overexpressing full-length LIMK1 were separated on SDS-PAGE and transferred for immunoblotting with anti-LIMK1 antibody. D, LIMK1 overexpression can reverse Pyr1-induced microtubule stabilization. HeLa cells were transfected with the control plasmid pEFrFlagPGKpuropAv18 or with pEFrFlagLIMK1PGKpuropAv18 plasmid overexpressing full-length LIMK1. Cells were treated for 2 hours with different amounts of Pyr1 as indicated. Nocodazole (10 μmol/L) was then added for 30 minutes. Cells were then stained for total tubulin. Bars, 10 μm.

Close modal

We then tested whether the overexpression of LIMK1 could counteract the microtubule-stabilizing effect of Pyr1. Cells overexpressing LIMK1 (Fig. 6C) were treated with increasing concentrations of Pyr1 and microtubule stability was then probed with nocodazole, as described above. We found that at all the concentrations assayed, the microtubule network of the LIMK1-expressing cells treated with Pyr1 was less stabilized, as compared with control cells (Fig. 6D).

Finally, we analyzed the effect of LIMK invalidation on microtubule stabilization, as assessed by the generation of Detyr-microtubules. We invalidated LIMK1 and LIMK2 into MCF7 cells and found that LIMK invalidation not only decreases cofilin phosphorylation but also increases Detyr-tubulin content (Supplementary Fig. S6). We found that LIMK expression, especially that of LIMK2, was more difficult to silence into HeLa cells. We succeeded, however, using short hairpin RNA lentivirus, to diminish LIMK1 expression in such cells. In these conditions, we found a large amount of cells with Detyr-microtubules, indicating that microtubules are stabilized (Supplementary Fig. S6).

Taken together, our results show that specific inhibition of LIMK activity by the small-molecule inhibitor Pyr1 is responsible of its effects on the cytoskeletal proteins tubulin and actin.

Our strategy based on a screening of a chemical library led to the discovery of a cell-permeable inhibitor of LIMK1/2, enzymes known to phosphorylate and inactivate cofilin, an actin-depolymerizing factor. As the readout of the assay used for the library screening was the immunofluorescence detection of a tubulin modification indicative of the cellular microtubule stability status, it was somewhat surprising that this approach led to the identification of an inhibitor of a known actin regulator. However, such a whole-cell–based assay probes for inhibitors of entire pathways, allowing the cell biology to dictate the best targets (35). Using cytochalasin D, an actin-depolymerizing agent, we showed that the effect of Pyr1 on microtubules was independent of its induced actin cytoskeleton reorganization. Thus, this screening approach allowed the selection of a chemical compound able to target LIMKs, a shared signaling regulation node of the actin and the microtubule cytoskeletons.

Because of the ability of Pyr1 to suppress microtubules dynamic instability, we tested whether it has antimitotic and cytotoxic properties on cancer cell lines. We found that this was the case. Moreover, Pyr1 had the same toxicity for the drug-sensitive human cell lines and for their multidrug-resistant counterparts. This suggests that Pyr1 may be used as an alternative or in addition to standard chemotherapy, in drug-resistant tumors. Our pilot study conducted on tumors xenografts in mice indicated that Pyr1 was indeed able to prevent tumor growth at doses that are well tolerated by the animals. Moreover, because it is active at doses much lower than the doses where the first signs of toxicity appear, Pyr1 has a convenient therapeutic window.

Although we found a close correlation between LIMK inhibition, actin disorganization, and microtubule stability, the mechanism by which Pyr1 induces microtubule stabilization is not completely understood. It was shown that phosphorylation of p25 by LIMK1 inhibits its ability to polymerize microtubules, resulting in reduced levels of stable microtubules (31). Therefore, a Pyr1-induced dephosphorylation of p25 could be responsible of the observed stabilization of microtubules. We cannot, however, rule out the possibility that Pyr1-induced microtubule stabilization results from an effect of Pyr1 on other targets.

The mitotic spindles of cells treated with Pyr1 show a disorganization of astral microtubules. This phenotype is similar to that obtained by Kaji and colleagues after LIMK1 knockdown (36), further supporting our conclusion that the LIMKs are the main cellular targets of Pyr1.

Pyr1 induced rapid and almost complete cofilin dephosphorylation in vitro and in cells. This indicates that the basal activity of cofilin phosphatases (slingshot and chronophin) must be high in the cells tested and that Pyr1 may also target closely related cofilin kinases, that is, TESK.

Pyr1 acts as an ATP-competitive inhibitor of LIMK. A major concern in the use of kinase inhibitors that are ATP antagonist is their target specificity, as the ATP-binding motif is present in all kinases as well as in other proteins. We therefore tested the ability of Pyr1 to inhibit the in vitro activity of 110 protein kinases, and although it is possible that Pyr1 targets other kinases such as TESK, than the ones already assayed, it shows, however, a high selectivity. The kinases assayed include some vital kinases such as the insulin receptor kinase. The absence of Pyr1 effect on these kinases could contribute to the observed limited animal toxicity of the compound.

It is anticipated that inhibition of cofilin phosphorylation that results in activation of cofilin would lead to actin severing and depolymerization of actin filaments. After 2 hours of treatment with Pyr1, we observed a complete disorganization of the actin microfilament network with decreased level of F-actin. This phenotype was not previously observed when LIMK1 and LIMK2 were knocked down by specific siRNA (33). A possible explanation of this disparity is that there are major differences between the mechanism of action of siRNA and pharmacologic inhibitors. Indeed, in addition to differences in kinetics, siRNA experiments reduce protein levels, whereas a small molecule only inhibits protein enzymatic activity. Moreover, LIMKs contain protein–protein interaction domains. They, thus, may have a variety of yet uncharacterized functions, in addition to their enzymatic activity. These other functions might be involved in the observed reorganization of the actin network.

One major advantage of small molecules, particularly when their effect is reversible, as is the case for Pyr1, is that they allow high temporal and spatial control. Their effects can be observed in real time by live cell imaging. To date only 2 cell-permeable inhibitors of LIMK have been described with limited experimental data about their specificity (33, 34, 37, 38). Moreover, LIMK is the last node in the signaling pathway that regulates the actin and the microtubule cytoskeletons. Thus, Pyr1, with its high specificity, is a new reagent that can be added to the biologist toolbox for dissecting cytoskeleton-dependent mechanisms and to investigate the effect of acute and chronic inhibition of LIMKs, on different cells and organisms.

We have shown that LIMK inhibition totally blocks in vitro cell migration. It has been recently shown, using siRNA or a small-molecule inhibitor, that the activity of LIMK1/2 is required for invasive path generation by tumor and tumor-associated stromal cells (33). In contrast to our results, the authors found that LIMK inhibition did not affect cell motility. This disparity can be explained by the differences in the cell lines studied. The activity of cofilin kinases and cofilin phosphatases may vary among different cells. Yet, small changes in the cofilin phosphorylation state may have a great impact on the cell invasiveness (33). Differences in the affinity for LIMKs or in the selectivity of the 2 inhibitors could also explain their different effects on cell motility.

Nevertheless, the cofilin pathway (39) including its upstream effectors ROCK, PAK, and LIMK (40–43) was proposed as a validated target for the treatment of cancer metastasis. Hence, in addition to its use in basic research and possibly its therapeutic use as an alternative antimitotic compound in the treatment of paclitaxel-resistant tumors, Pyr1 has the potential to be used for the inhibition of cancer metastasis.

No potential conflicts of interest were disclosed.

Conception and design: R. Prudent, E. Vassal-Stermann, C.H. Nguyen, C. Barette, E. Soleilhac, O. Filhol, R. Li, L. Lafanechère

Development of methodology: R. Prudent, E. Vassal-Stermann, C.H. Nguyen, A. Martinez, C. Prunier, C. Barette, E. Soleilhac, A. Beghin, J. Antonipillai, R. Li, O. Bernard, L. Lafanechère

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Prudent, A. Martinez, C. Prunier, C. Barette, E. Soleilhac, O. Filhol, G. Valdameri, S. Honoré, J. Antonipillai, R. Li, A. Di Pietro, C. Dumontet, D. Braguer, O. Bernard, L. Lafanechère

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Prudent, A. Martinez, C. Barette, E. Soleilhac, A. Beghin, S. Aci-Sèche, J. Antonipillai, R. Li, A. Di Pietro, C. Dumontet, D. Braguer, S. Knapp, O. Bernard, L. Lafanechère

Writing, review, and/or revision of the manuscript: R. Prudent, E. Vassal-Stermann, S. Honoré, A. Di Pietro, C. Dumontet, D. Braguer, S. Knapp, O. Bernard, L. Lafanechère

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Prudent, C. Pillet, C. Barette, J.-C. Florent

Study supervision: R. Prudent, L. Lafanechère

Microtubule dynamics data: S. Honoré

Putting together and providing the library of potential inhibitors tested in this work: D. Grierson

The authors thank Drs. Laurent Blanchoin, Raja Boujemaa-Paterski, Claude Cochet, Yasmina Saoudi, Manuel Théry, Jérémie Gaillard, Florence Mahuteau, and Marie-Paule Teulade-Fichou for their advices and help; Marianne Bombled and Doriane Poloni for technical assistance. The authors also thank the ChemAxon (http://www.chemaxon.com).

This work was supported by the CNRS, the CEA, the Institut Curie, the INSERM, and by grants from the Association pour la Recherche sur le Cancer, the GRAVIT consortium, and from the French National Agency for Research (grant ANR- 2010-EMMA-013-01). E. Vassal-Stermann was a fellow of the Région Rhône-Alpes. R. Prudent is a fellow of Fondation de France. S. Knapp is funded by the SGC, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada, GlaxoSmithKline, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Eli Lilly, Pfizer, the Novartis Research Foundation and the Wellcome Trust.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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