Phenamacril is a reversible and noncompetitive inhibitor of Fusarium class I myosin

The cyanoacrylate compound phenamacril (also known as JS399–19) is a recently identified fungicide that exerts its antifungal effect on susceptible Fusarium species by inhibiting the ATPase activity of their myosin class I motor domains. Although much is known about the antifungal spectrum of phenamacril, the exact mechanism behind the phenamacril-mediated inhibition remains to be resolved. Here, we describe the characterization of the effect of phenamacril on purified myosin motor constructs from the model plant pathogen and phenamacril-susceptible species Fusarium graminearum, phenamacril-resistant Fusarium species, and the mycetozoan model organism Dictyostelium discoideum. Our results show that phenamacril potently (IC50 ∼360 nm), reversibly, and noncompetitively inhibits ATP turnover, actin binding during ATP turnover, and motor activity of F. graminearum myosin-1. Phenamacril also inhibits the ATPase activity of Fusarium avenaceum myosin-1 but has little or no inhibitory effect on the motor activity of Fusarium solani myosin-1, human myosin-1c, and D. discoideum myosin isoforms 1B, 1E, and 2. Our findings indicate that phenamacril is a species-specific, noncompetitive inhibitor of class I myosin in susceptible Fusarium sp.

The cyanoacrylate phenamacril ( Fig. 1) (1) (formerly known as JS399 -19) has been presented as a novel and environmentally benign fungicide. A growth-inhibitory effect of phenamacril against certain economically devastating phytopathogenic fungi within the genus Fusarium is well-established (2)(3)(4)(5)(6). By inhibiting the ATPase activity of class I myosin in susceptible Fusarium spp., phenamacril disrupts the activity of an essential actin-associated motor protein (3,4). Myosins are ubiquitous eukaryotic motor proteins, which can be divided into ϳ35 classes (7). Although several classes and isoforms may be present in a given organism, Fusarium only encodes single myosin heavy chains (MHC) from class I (4), class II (8), class V (9), and class XVII (10). All myosin isoforms share a functionally and structurally conserved N-terminal motor domain, a neck region which binds EF-hand proteins such as myosin light chains or calmodulin (11,12) and class-specific C-terminal dimerization and/or cargo-binding domains. The Mg 2ϩ -dependent ATPase activity of the motor domain utilizes the energy stored in ATP to produce unidirectional movement along polar actin filaments. Thereby, myosin isoforms facilitate directional cargo-transport processes, local constriction, and other specialized energy-requiring tasks within the cell (8,(13)(14)(15)(16)(17).
Since the establishment of baseline sensitivity of Fusarium graminearum to phenamacril in 2008 (18), both laboratory (3, 4, 18 -20) and field-resistant strains (5) have been characterized in China, where the compound is widely used to control Fusarium-induced infections of cereals (5,6). Resistance development was observed to correlate with amino acid mutations, which are primarily located within the actin-binding cleft of the myosin motor domain (4,5,19). This region is known to play a pivotal role in mediating allosteric communication between the nucleotide-and actin-binding sites (21)(22)(23). It is a well-characterized and conserved allosteric site targeted by other classspecific myosin inhibitors such as blebbistatin (24) and the halogenated pseudilins (21,23,25). Although much is known about the effect of blebbistatin and the halogenated pseudilins on actomyosin kinetics (21,23,24,26,27), the biochemical parameters associated with the phenamacril-mediated inhibition of Fusarium class I myosin have not been characterized.
Here, we describe the elucidation of the mechanism underlying phenamacril-mediated inhibition of Fusarium spp. class I myosin and provide insights into its effect on actomyosin kinetics. To this end, we undertook the production of four active myosin motor domain constructs from both susceptible and phenamacril-resistant species of Fusarium. We copurified the constructs with F. graminearum calmodulin (FgCaM) 4 bound to the lever arm region (28). The soluble and active protein preparations were used for functional analyses. We used an in  cro ARTICLE vitro motility assay (29) to assess the effect of phenamacril on the capacity of the myosin head construct to translocate fluorescently labeled F-actin filaments before and after inhibitor washout. This allowed us to demonstrate that phenamacril acts as a reversible effector of motor function. Finally, we used an NADH-coupled ATPase assay and stopped-flow measurements to establish a nanomolar IC 50 value for the phenamacril-mediated inhibition of F. graminearum class I myosin (FgMyo1) (30) and to demonstrate that phenamacril is a specific and noncompetitive inhibitor of myosin ATPase activity.

Phenamacril reversibly inhibits the motor function of the FgMyo1-FgCaM complex
Using the baculovirus expression system, we produced and purified myosin constructs from F. graminearum, F. avenaceum, and F. solani in Sf9 insect cells. Myosin heavy chain constructs encompassing two IQ-motifs were coproduced with FgCaM. Partial loss of FgCaM during the final size-exclusion chromatography step (Fig. S1) did not affect the solubility and stability of the purified protein. However, motor activity was greatly reduced. The protein could be stored at Ϫ80°C in buffer 30% trehalose in this form. Motor activity was restored when recombinant FgCaM produced in Escherichia coli was added to FaMyo1 IQ2 , FgMyo1 IQ2 , or FsMyo1 IQ2 after thawing. Typically, substoichiometric additions of FgCaM were sufficient for maximal activation.
To assess if phenamacril-mediated inhibition of F. graminearum class I myosin is reversible, we conducted in vitro motility assays, where F-actin filaments move in an ATP-dependent manner on nitrocellulose-coated glass slides decorated with FgMyo1 IQ2 . More than 600 rhodamine-phalloidin-labeled F-actin filaments were tracked, both before and after the infusion of phenamacril, as well as after inhibitor washout. The resulting trajectory-associated velocities could be fitted to Gaussian distributions (Fig. 2). Specifically, we found that phenamacril inhibits the movement of F-actin filaments. In the absence of the inhibitor, actin filaments moved with an average velocity of 436 Ϯ 165 nm⅐s Ϫ1 . In the presence of 1 M and 10 M phenamacril, we observed average velocities of 234 Ϯ 100 nm⅐s Ϫ1 and 133 Ϯ 64 nm⅐s Ϫ1 , respectively. Washout of the inhibitor restored the average sliding velocity to 389 Ϯ 201 nm⅐s Ϫ1 .

Phenamacril is a noncompetitive inhibitor of FgMyo1
To further characterize the inhibitory potential of phenamacril, we established the half-maximal inhibitory concentration (IC 50 value) by using a steady-state NADH-coupled ATPase assay in the presence of 20 M F-actin and increasing concentrations of phenamacril in the range from 0.1 nM to 100 M. To simplify the assay, we used motor domain construct FgMyo1, which lacks both IQ-motifs. FgMyo1 displays the same ATPase activity as FgCaM-saturated construct FgMyo1 IQ2 . Consistent with the data from the in vitro motility assay, phenamacril inhibited the ATPase activity in a dose-dependent manner. By nonlinear regression, we determined the relative IC 50 value of the phenamacril-mediated inhibition of FgMyo1 to 365 Ϯ 39 nM with 0 -10% residual ATPase activity at ϳ10 M phenamacril (Fig. 3).
To elucidate the mechanism of inhibition, we measured the inhibited and noninhibited steady-state ATPase activity of FgMyo1 as a function of the F-actin and ATP concentration (Fig. 4, A and B). Fitting the data with hyperbolic functions demonstrated that the effect of phenamacril on the actin-activated ATPase activity was a lowering of k cat,actin (maximum ATP turnover in the presence of saturating concentrations of F-actin) and an increase in the value for K app,actin (apparent F-actin affinity in the presence of ATP). Specifically, in the presence of 300 and 600 nM phenamacril, K app,actin increased from 4.8 Ϯ 0.5 M to 11.7 Ϯ 0.9 M and 24.9 Ϯ 3.5 M, respectively. k cat,actin decreased from 0.72 Ϯ 0.04 s Ϫ1 in the noninhibited state to 0.51 Ϯ 0.03 s Ϫ1 and 0.49 Ϯ 0.05 s Ϫ1 in the presence of 300 and 600 nM phenamacril, respectively. ATP titration in the presence of phenamacril further demonstrated that although phenamacril reduces k cat,basal (maximum ATP turnover in the absence of F-actin), it does not significantly affect the apparent affinity of FgMyo1 for ATP (K app,basal ) ( Fig. 4B and Fig. S2). This result confirms that phenamacril is a noncompetitive inhibitor of FgMyo1.
We used stopped-flow experiments to probe the impact of phenamacril on the population of the weak actin-binding myosin states. In the absence of ATP, the association of pyrenelabeled fluorescent F-actin with the myosin motor (rigor state) results in quenching of the fluorescence signal. We measured the rate of the ATP-induced increase in fluorescence that results from actomyosin dissociation (Fig. 4C). Exponential fits to the fluorescence transients yielded k obs values that were plotted against the respective ATP concentration (Fig. 4D). Linear fitting of the data gave the second-order rate constant (K 1 k ϩ2 ), which in the absence and presence of 10 M phenamacril was 0.37 Ϯ 0.02 M Ϫ1 ⅐s Ϫ1 and 0.35 Ϯ 0.01 M Ϫ1 ⅐s Ϫ1 , respectively. This shows that phenamacril, like blebbistatin and the halogenated pseudilins, does not inhibit or interfere with ATP-induced dissociation of the actomyosin complex. In line with the steady-state ATPase experiments, it further shows that phenamacril does not compete with ATP for the nucleotide-binding site.

Specificity of Fusarium myosin-1 inhibition by phenamacril
To characterize the specificity of phenamacril, we initially modeled the pre-power stroke state of FgMyo1 and docked phenamacril into 20 conformers covering a 500-ps molecular

Inhibition of Fusarium class I myosin by phenamacril
dynamics trajectory. In line with the noncompetitive mode of inhibition, the best-ranking docking poses clustered at the apex of the actin-binding cleft (Fig. 5, A-C and Fig. S3), in the immediate vicinity of residues that have been associated with resistance development (Fig. 5D). Residues define the allosteric binding pocket and are predicted to pack tightly around phenamacril. In particular, the aliphatic ethyl-ester chain of phenamacril is predicted to make close contact with residues at the rear of the binding pocket. In contrast, the aromatic moiety of the inhibitor occupies a more solvent-exposed position. Most of the interactions are hydrophobic in nature. Hydrogen-bond interactions are contributed by main-chain residues Ser-217, Lys-537, and Cys-423 and the side chain of Cys-423 (Fig. S3).
When compared to F. graminearum, F. avenaceum and F. solani show reduced susceptibility and resistance (respectively) to Phenamacril on Phenamacril-amended agar plates (Fig. 6A). To further assess whether natural resistance in F. solani can be attributed to myosin sequence divergence ( Fig.  S4) or whether other mechanisms of resistance are involved, we examined the effect phenamacril on actin-activated turnover of ATP by F. solani myosin head construct FsMyo1 IQ2 (Fig. 6B). Consistent with the amended agar assay, our results show that the growth-inhibitory effect on F. avenaceum correlates with the inhibition of the enzymatic activity of FaMyo1 IQ2 in vitro. Conversely, although phenamacril also significantly inhibits FsMyo1 IQ2 (p Ͻ 0.005), it still retained 78% ATPase activity at the extreme of 100 M phenamacril. FsMyo1 IQ2 has two S217T and M375K substitutions in the immediate vicinity of the pro-

Inhibition of Fusarium class I myosin by phenamacril
posed phenamacril-binding pocket of F. graminearum. Introduction of either or both substitutions into the docked homology model resulted in a spatial overlap of phenamacril with the exchanged side chains (Fig. S4), suggesting this to be the resistance-conferring mechanism in F. solani.
To further assess the proposed species-specificity of phenamacril for Fusarium, we decided to include a selection of important and well-characterized myosin constructs in our study. These included human myosin-1c (Myo1c) and D. discoideum myosin-1E, myosin-1B (M1B), and myosin-2 (M765) (an amino acid alignment is shown in Fig. S5). Human Myo1c is involved in insulin uptake (12); myosin-1E is involved in phagocytosis (31); myosin-1B in cell translocation and intracellular particle motility (15); and myosin-2 is essential for cell movement, efficient chemotaxis, capping of cell surface receptors, and cytokinesis (13,14). We measured the effect of 100 M phenamacril on their actin-activated motor domain steadystate ATPase activities but found no statistically significant difference between the controls and the phenamacril-amended samples for the D. discoideum myosins (Fig. 6C). Human Myo1c activity is inhibited to a similar extent as FsMyo1 IQ2 . Overall our results indicate that phenamacril is a potent noncompetitive inhibitor of F. graminearum class I myosin but that further studies are warranted in regard to its environmental impact.

Discussion
We purified active Fusarium myosin motor domain constructs and characterized the mechanism and parameters underlying the phenamacril-induced inhibition of myosin ATPase and motor activity. Phenamacril is currently being applied as a fungicide in China for agricultural management of Fusarium-induced plant diseases (5,6). Although the seemingly high specificity associated with phenamacril indicates a noncompetitive mode of inhibition, the mechanism has remained uncharacterized for more than a decade. From an agricultural perspective, insight into this mechanism is of relevance for evaluating the potential risks associated with the application of a fungicide that targets a ubiquitous and structurally conserved domain. ATP-binding motifs including the P-loop, switch-1, and switch-2 are not only highly conserved within the myosin family but among all P-loop nucleotide triphosphatases, such as microtubule-based motor proteins, protein kinases, and G-proteins (7,32). In contrast, allosteric effector-binding sites are less well-conserved (33)(34)(35). In the case of myosin motor domains, at least four nonoverlapping binding sites for druglike modulators of myosin function have been identified (23, 26, 36 -38).
To resolve the involvement of either a competitive or a noncompetitive mode of inhibition, we produced soluble and active

Inhibition of Fusarium class I myosin by phenamacril
Fusarium myosin-1 head constructs in complex with FgCaM and demonstrated that the functional inhibition of FgMyo IQ2 is a reversible process. Moreover, our results show that FgCaM functions as a myosin light chain and binds to IQ-motifs from F. graminearum, F. avenaceum, and F. solani myosin-1. Moreover, we were able to purify an active truncated version of the motor domain (FgMyo1), which allowed for analysis in the absence of the myosin light chain. This allowed us to demonstrate that phenamacril has an inhibitory potency (IC 50 ϳ360 nM) comparable to other known reversible and noncompetitive myosin inhibitors such as blebbistatin (0.5-5 M) (24), pentachloropseudilin (1-5 M) (21), and pentabromopseudilin (1.2 M) (23). More importantly, we could directly show that phenamacril does not compete with ATP for binding to the active site of FgMyo1 nor does it interfere with the ATP-induced dissociation of the actomyosin complex.
It has previously been shown that small molecule inhibitors can reduce myosin-mediated force generation by means of different mechanisms. For example, the more than 50-fold reduction of actin filament sliding velocity observed for D. discoi-  . Error bars denote the S.D. around the mean. The difference between sample means were either statistically significant (** for p Ͻ 0.005), highly significant (*** for p Յ 0.0005), or not significant (ns).

Inhibition of Fusarium class I myosin by phenamacril
deum myosin-5b in the presence of 10 M of the inhibitor pentabromopseudilin has been shown to be the result of a combined effect of the drug on ATP binding, ATP hydrolysis, and ADP dissociation (23). Similarly, the blebbistatin-induced inhibition of the actin filament sliding velocity for skeletal muscle myosin-2 (40) has been attributed to a reduction of the phosphate release step (24). For both inhibitors, no significant effect on the rate of the ATP-induced dissociation was observed. In line with these studies, phenamacril seems to exert its effect not via reducing the rate of ATP-induced dissociation but rather affects one or more of the other steps in the actomyosin ATPase cycle.
Collectively, our study classifies phenamacril as a reversiblebinding and noncompetitive myosin inhibitor. This is in good agreement with blind-docking studies, which predict phenamacril to bind in close proximity to the allosteric binding pockets associated with the halogenated pseudilins (21,23,25) and blebbistatin (26). This region includes several of the residues that are implicated in phenamacril resistance (3-5, 19, 20). This finding shows a path toward the rational design of new and improved cyanoacrylate fungicides based on the phenamacril scaffold.
Although blebbistatin and some halogenated pseudilins are known to be highly class specific, noncompetitive inhibitors of myosin ATPase and motor activity, they are not necessarily species specific (21,(23)(24)(25)(26). The lack of any profound inhibitory activity against human Myo1c and the Dictyostelium myosins belonging to classes I and II seems to confirm the high degree of specificity reported for phenamacril (3,4,6,18). Nonetheless, our results do not exclude toxicological implications for the application of phenamacril as a fungicide. Caution remains warranted in regard to its wider user (5,6).
The correspondence between the amended agar assay and the in vitro ATPase assays indicates that the effect of phenamacril on Fusarium is tied to structural aspects of their myosins. In F. solani, we speculate that a S217T substitution is implicated in its resistance to phenamacril. Although a conservative substitution, this amino acid position has previously been linked to phenamacril resistance in Fusarium (3)(4)(5)19). The myosins from D. discoideum and human Myo1c do not contain substitutions at the aforementioned position but are otherwise highly divergent, have numerous substitutions in the immediate vicinity of the proposed binding pocket, and have three to five substitutions throughout the residues known to be implicated in phenamacril resistance (cf. Fig. S5). Therefore, it seems likely that the lack of inhibitory activity against these myosin constructs is the combined effect of overall sequence divergence and specific amino acid substitutions.
In conclusion, phenamacril is a potent and reversible noncompetitive inhibitor of class I myosin in the model plant pathogen F. graminearum. Identification of its mode of action and allosteric binding pocket will serve not only as a reference foundation for future studies aimed at characterizing myosins in Fusarium but also facilitate the design of novel inhibitors or fungicides based on the phenamacril scaffold.

RNA purification cDNA library preparation
Fifty ml YPG medium, pH 6.5 Ϯ 0.1 (20 g⅐liter Ϫ1 soya peptone, 10 g⅐liter Ϫ1 yeast extract, and 50 ml⅐liter Ϫ1 50% (w/v) D(ϩ)-glucose) was inoculated with five 5 mm mycelia plugs from an actively growing culture and incubated for 5 days at 25°C and 130 rpm. Mycelia was harvested with Miracloth (Merck), washed twice with double-distilled water and freezedried overnight. RNA was subsequently purified from ϳ25 mg freeze-dried mycelium using the RNeasy Plant kit (Qiagen, Hilden, Germany), including the optional on-column DNase treatment. The cDNA library was synthesized with Super-Script TM III Reverse Transcriptase (Thermo Fisher Scientific) and oligo T primers according to manufacturer's protocol.

In silico modeling
The crystal structure of D. discoideum myosin-1E (PDB ID 1LKX) (45) was used as template for homology modeling of the F. graminearum and F. solani class I myosin motor domains in the pre-power stroke state. The C chain of 1LKX with ADPvanadate was energy-minimized and refined in YASARA Structure and WHAT IF version 17.8.15 (Yasara2 force field, 25°C, TIP3P water model, 1000-ps simulation, 40 snapshots) (46,47). The lowest energy conformer was subsequently used as a template in SWISS-MODEL (48). Following repositioning of the ADP-vanadate, the model was refined using the above parameters.
The last 20 refined structures (covering a 500-ps trajectory) were superpositioned and prepared for global ensemble dock-

Inhibition of Fusarium class I myosin by phenamacril
ing with energy-minimized phenamacril. Following docking in YASARA with AutoDock VINA (100 docking runs per conformer) (49), the best-ranking docking pose was energyminimized and redocked to visualize and identify interaction partners.
The FgCaM coding sequence for heterologous protein expression in E. coli BL21(DE3) was cloned into the malEvector with ligase-independent cloning using LIC-qualified T4 DNA polymerase (Merck) to generate overhangs. The construct was fused in-frame with an N-terminally located maltose-binding protein CDS and an interspaced tobacco etch virus (TEV) protease site CDS. All coding regions were verified by Sanger DNA sequencing (Eurofins Genomics, Germany).

ATPase assays
Steady-state NADH-coupled ATPase assays in the presence of variable concentrations of F-actin were performed at 25°C in standard ATPase-buffer (

Stopped-flow kinetics
Transient kinetic experiments were performed at 20°C with a Hi-tech Scientific SF-61 SX single mixing stopped-flow system (TgK Scientific Limited, Bradford on Avon, UK) in assay buffer (20 mM MOPS, pH 7.0, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT). Pyrene fluorescence was excited at 365 nm and detected after passing through a KV 389 nm cut-off filter. Fluorescence transients were fitted with single exponential equations and the resulting kinetic data were analyzed according to models described previously (17,54). The mechanism of ATP-induced pyrene fluorescence enhancement was modeled according to Equation 1, which describes a two-step mechanism for ATP binding to actomyosin: At lower ATP concentrations (Ͻ50 M), the observed rate constants increased linearly, defining the second-order rate binding constant K 1 k ϩ2 .

In vitro motility assays
The in vitro motility experiments were performed according to Kron and Spudich (29) with modifications described by Taft and coworkers (17). Approximately 7.7 M FgMyo1 IQ2 in assay buffer (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM EGTA) containing 5 M FgCaM, 12 M sheared F-actin, 10 mM DTT, and 2 mM ATP was centrifuged for 20 min at 100,000 ϫ g and 4°C. The supernatant contains the active myosin, which was bound via an anti-His antibody (Penta-His, Qiagen, Hilden, Germany) to the flow-cell surface. 20 nM rhodamine-phalloidin-labeled F-actin was subsequently added to the flow cell (ϳ10 l capacity). All buffer solutions used after this step contained 7.5 M BSA and 5 M FgCaM. To probe the effect of phenamacril on motile activity, the flow cells were consecutively infused with 2 mM Mg 2ϩ -ATP and 0, 1, 10, and 0 M (washout) phenamacril. Movement of the rhodamine-phalloidin-labeled F-actin was monitored at 25°C at 300-ms intervals for 30 s with an Olympus IX70 epifluorescence microscope equipped with a 60ϫ Plan Apo 1.49 NA oil immersion objective lens (ApoN, Olympus) and an OrcaFlash 4.0 CMOS camera (Hamamatsu Photonics, Iwata City, Japan). For each flow-cell, filament sliding velocity was tracked in three independent areas with DiaTrack 3.05 (39) (Semasopht, Switzerland). GraphPad Prism 7.03 was used for final binning and data analysis.

Amended agar assay
Mycelia plugs (5 mm) taken from actively growing colonies were placed in the center of 5.5-cm vented Petri dishes containing YPG agar media, pH 6.5 Ϯ 0.1. Plates were amended with either 1-100 M phenamacril or 0.5% ethanol (control). Growth of three replicates was monitored at 25°C until the control reached the edge of the plate (42).

Statistical analysis
Unless otherwise stated, error bars denote mean Ϯ S.D. In vitro motility data are reported as mean Ϯ 1 ⁄ 2FWHM (full width at half maximum). Student's two-tailed t test was used to compare sample means, with statistical significance assigned as ns (not significant) for p Ͼ 0.05, * for p Յ 0.05, ** for p Յ 0.005, and *** for p Յ 0.0005.