Itraconazole inhibits endothelial cell migration by disrupting inositol pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility.


Introduction
Itraconazole is a widely used antifungal drug with a clinical history of over 30 years. Recently, itraconazole is being repurposed as an antiangiogenic agent and exhibits therapeutic efficacies in cancer [1], hereditary hemorrhagic telangiectasia [2], and ocular neovascularization [3]. Despite the identification of many itraconazole target proteins [4][5][6], the downstream functional mechanisms by which itraconazole inhibits angiogenesis are unknown.
In this study, we demonstrate that IP6K1 generates a local pool of 5-InsP 7 to regulate FAK dimerization and the Arp2/3 complex. Itraconazole treatment dislocates IP6K1 and thus disrupts 5-InsP 7 -mediated focal adhesion dynamics and actin cytoskeleton remodelling.

Western blotting
Cells were lysed in lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 0.5% Igepal CA630, 5 mmol/L MgCl 2 and protease/phosphatase inhibitors (Yeasen). Lysates were pulse sonicated and centrifuged at 14,000 g for 10 min at 4ºC. Protein concentrations were normalized using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS loading buffer (Thermo Fisher Scientific) containing 5% β-mercaptoethanol was added, and the samples were boiled for 5 min. Proteins were separated by 8-15% SDS-PAGE gel and transferred to a PVDF membrane (Thermo Fisher Scientific). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature for 1 h, and was incubated with primary antibody overnight at 4 • C. The membrane was washed three times with TBST and incubated with HRP-conjugated secondary antibody for 1 h at room temperature followed by three washes with TBST. Immobilon Western Chemiluminescent HRP substrate (Thermo Fisher Scientific) was used to detect the signal of the secondary antibody. The membrane was then imaged using ChemiDoc™ imaging system (Bio-Rad).

Immunoprecipitation
Cells were lysed in the lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 0.5% Igepal CA630, 5 mmol/L MgCl 2 and protease/phosphatase inhibitors (Yeasen). Lysates were passed through 30gauge needles 20times and centrifuged at 14,000 g for 10 min at 4ºC. The supernatants were collected and pre-cleaned with protein A/G beads (Santa Cruz Biotechnology) for 90 min at 4ºC. Lysates were centrifuged briefly, and the supernatants were collected while the protein A/G beads were discarded. Primary antibody was added to cell lysates and incubated at 4ºC overnight. Protein A/G beads were then added to the cell lysates and incubated for 2 h at 4ºC. The beads were washed with cold lysis buffer 3 times. 1.5x SDS loading buffer containing 5% β-mercaptoethanol was added, and the samples were boiled for 5 min.

Subcellular fractionation
Cell membrane fractions were isolated by ultracentrifuge. Cells were homogenized at 4ºC in buffer containing 250 mM Sucrose, 20 mM HEPES, PH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT and protease/phosphatase inhibitors. Lysates were passed through a 30-gauge needle 20 times, incubated on ice for 20 min, and centrifuged at 14,000 g for 20 min at 4ºC. The supernatant was collected and centrifuged at 200,000 g for 2 h at 4ºC. The resulting supernatant was the cytosolic fraction. The pellet left was washed with fractionation buffer, re-suspended by pipetting, and re-centrifuged at 200,000 g for 2 h at 4ºC. The resulting pellet was the membrane fraction, which contains plasma membrane, microsomes, and small vesicles.

Immunostaining
Cultured cells were washed with PBS followed by fixation with 4% (w/v) paraformaldehyde. The samples were blocked with 10% (v/v) goat serum (MilliporeSigma) for 10 min at room temperature then incubated with primary antibodies at 4 • C overnight. The samples were washed multiple times with PBS for 3 h at room temperature then incubated with fluorescent-dye conjugated secondary antibodies for 1 h at room temperature. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) for 10 min. F-actin was stained by incubating with fluorescein labeled phalloidin (Thermo Fisher Scientific) for 30 min. Slices were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Pictures were taken under a confocal microscope (Zeiss LSM 800).

Lentivirus generation
HEK 293 T/17 cells were plated one day before experiments and allowed to grow to 70% confluence. Lentiviral vectors harboring the gene of interest together with pMD2. G and psPAX2 were transfected into HEK 293 T/17 cells using Lipofectamine 3000. Cell culture medium was replaced with fresh medium 4 h after transfection. The virus containing medium was collected 48 h later and filtered through a 0.45 µm filter then mixed with 1/2 vol of concentration medium containing 25.5% PEG 6000 (MilliporeSigma), 0.9 M NaCl, 2.5 mM Na 2 HPO 4 , and 0.4 mM KH 2 PO 4 . The samples were stored at 4ºC overnight then centrifuged at 17,000 g for 1 h at 4ºC. The resulting pellet containing lentivirus was resuspended with DMEM medium and stored at − 80ºC.
2.9. In vitro enzymatic assays IP6K1 activity was measured using an ADP-Glo™ Max Assay Kit (Promega). GST-fused IP6K1 was produced in HEK 293 cells and harvested by Glutathione Sepharose. PreScission Protease was used to cleave the GST and release IP6K1 into a buffer containing 50 mM Tris (pH 7.4), 10 mM MgCl 2 , and 2.5 mM DTT. IP6K1 activity assay was conducted in a reaction containing ~20 mg/ml protein, 50 μM InsP 6 , and 100 μM ATP for 2 h in 37ºC. Itraconazole or DMSO was added to the reaction. The reaction was quenched with ADP-Glo Reagent for 40 min, and ADP-Glo Max Detection Reagent was added and incubated for 60 min. Using an opaque white 96-well plate (Costar), the bioluminescent signal in relative light units was obtained on a microplate reader with an integration time of 1 s per well.

In vitro binding assay
To assess the binding of p34 with coronin, flag-tagged p34 and GSTfused coronin were produced in HEK293 cells. Cell lysates were precleaned with protein A/G beads (Santa Cruz), anti-flag-tag antibody was added to cell lysates expressing flag-p34 overnight. Flag-p34 was pulled down by protein A/G beads and washed for three times. Separately, Glutathione Sepharose (GE Life Sciences) was used to pull down GST-fused coronin. GST-coronin was released by reduced glutathione (50 mM) in a buffer containing 200 mM NaCl, 50 mM Tris (PH 9.0) and protease inhibitors. Purified coronin was added to protein A/G agarose bound flag-tagged p34 in the presence of InsP 6 or 5-InsP 7 or 5-PCP or CF2 overnight at 4 • C. Beads were then collected and washed three times. The samples were loaded with 1.5x SDS loading buffer containing 5% β-mercaptoethanol and boiled for 5 min.
To assess the binding of flag-FAK with GST-FAK, flag-FAK and GST-FAK were produced in HEK293 cells. Cell lysates were pre-cleaned with protein A/G beads (Santa Cruz), anti-flag antibody was added to cell lysates expressing flag-FAK overnight. Flag-FAK was pulled down by protein A/G beads and washed three times. Separately, Glutathione Sepharose (GE Life Sciences) was used to pull down GST-FAK. GST-FAK was released by reduced glutathione (50 mM) in a buffer containing 200 mM NaCl, 50 mM Tris (PH 9.0) and protease inhibitors. Purified GST-FAK was added to protein A/G agarose bound flag-FAK in the presence of InsP 6 or 5-InsP 7 or 5-PCP or CF2 overnight at 4 • C. Beads were then collected and washed three times. The samples were loaded with 1.5x SDS loading buffer containing 5% β-mercaptoethanol and boiled for 5 min.
To assess binding of p34, coronin, FAK and FAK FERM domain to 5-PCP-InsP 5 , the control and 5-PCP-InsP 5 resin were equilibrated with cell lysis buffer. Purified flag-p34, GST-coronin, GST-FAK and GST-FAK FERM domain were added to the 5-PCP-InsP 5 resin and incubated overnight at 4 • C. The resins were collected and washed three times. The samples were mixed with 1.5x SDS loading buffer containing 5% β-mercaptoethanol and boiled for 5 min.
GST-coronin, GST-FAK and GST-FAK FERM domain were produced in HEK 293 cells and pulled down by Glutathione Sepharose. The GST-fused proteins were then released by reduced glutathione (50 mM) in a buffer containing 200 mM NaCl, 50 mM Tris (PH 9.0) and protease inhibitors.

Live cell imaging
LifeAct-mScarlet or GFP expressing HUVECs or MEF cells were plated onto a glass bottom cell culture dish. Cell images were taken by utilizing a confocal microscope (ZEISS 800) that took one picture per minute for 30 min.

Quantification and statistical analysis
Experiments were repeated for five times. Image J was used to quantify western blots, band intensities were normalized to total or control protein. Volocity software (V6.3, PerkinElmer Inc.) was used to quantify the immunofluorescence data. Statistical analysis was done with Graphpad Prism 7. Data represent means ± standard error of mean (SEM), n = number of independent repeats. Difference between two groups was analyzed by unpaired two-tailed Student's t-test. The Pearson correlation coefficient was used to quantify the degree of colocalization between two proteins. Significance is defined as *p < 0.05; **p < 0.01; ***p < 0.001.

Itraconazole enhances binding of IP6K1 to Arp2
Endothelial cell migration, an essential component for angiogenesis is severely inhibited by itraconazole (Fig S1A, B). Arp2/3 complexmediated lamellipodia formation is required for cell migration [26]. Immunoprecipitations and western blots reveal that itraconazole does not affect the binding between Arp2 and Arp3, two major components of Arp2/3 complex (Fig S2A, B).
We examined whether itraconazole alters proteins interactions with Arp2. Immunoprecipitation of flag-Arp2 co-pulls down several proteins revealed by silver staining (Fig. 1A). A protein band with molecular weight ~55KDa, which was identified as IP6K1 by mass spectrometry, is enriched in the itraconazole-treated samples, suggesting that its binding to Arp2 is enhanced (Fig. 1A). The interaction of IP6K1 with Arp2 is validated (Fig. 1B), and the itraconazole-induced enhanced binding of IP6K1 with Arp2 is confirmed by western blot (Fig. 1C, D).
To test whether IP6K1/5-InsP 7 play a role in mediating the binding of p34 with coronin, we performed immunoprecipitations of endogenous p34 and coronin in WT and IP6K1 KO MEF (mouse embryonic fibroblast) cells (Fig. 1G-J). The results show that the binding between p34 and coronin is increased by itraconazole treatment in WT but not IP6K1 KO preparations (Fig. 1G-J), indicating that IP6K1 is involved in itraconazole-induced augmentation of p34/coronin interaction.
We examined the role of 5-InsP 7 , the major product of IP6K1 by testing whether 5-InsP 7 physically binds p34 and/or coronin. We utilized an affinity resin containing immobilized 5-PCP-InsP 5 (5-PCP) [28], a nonhydrolyzable bisphosphonate analog of 5-InsP 7 as a bait to pull Fig. 1. Itraconazole strengthens interactions of Arp2/3 complex with IP6K1 and coronin. (A) Flag-tagged Arp2 (flag-Arp2) was over-expressed, and the cells were treated with itraconazole (3 μM) for 24 h. Immunoprecipitation of flag-Arp2 and silver staining reveal that a protein at ~55 kDa, which was identified as IP6K1, is increased in the itraconazole preparations (arrow). (B) Myc-tagged IP6K1 (myc-IP6K1) was over-expressed, and the cells were treated with itraconazole (3 μM) for 24 h. Immunoprecipitation of myc-IP6K1 and silver staining show that a protein at ~40 kDa, which was identified as Arp2, is increased in the itraconazole preparations (arrow). (C) Pulling down myc-IP6K1 co-precipitates more Arp2 in the itraconazole treated cells. (D) Immunoprecipitation of flag-Arp2 co-pulls down more IP6K1 in the itraconazole preparations. (E) Cells expressing flag-tagged p34 (flag-p34) were treated with itraconazole (3 μM) for 24 h. Immunoprecipitation of flag-p34 co-pulled down more coronin in itraconazole treated cells. (F) Immunoprecipitation of endogenous coronin co-pulls down more p34 in the itraconazole treated HUVECs. (G) Immunoprecipitation of p34 co-pulls down more coronin in itraconazole treated WT MEF cells. (H) Pulling down coronin co-pulls down more p34 in itraconazole treated WT MEF cells. (I) Immunoprecipitation of p34 co-pulls down similar amounts of coronin in DMSO and itraconazole treated IP6K1 KO MEF cells. (J) Pulling down coronin co-pulls down similar amounts of p34 in DMSO and itraconazole treated IP6K1 KO MEF cells. Statistical data are presented as mean ± SEM, Student's t-test, n = 5 independent repeats, * **p < 0.001, ns=not significant. down p34 and coronin ( Fig. 2A, Fig S4A). 5-PCP resin pulls down endogenous p34 and coronin in the whole cell lysates of HUVECs ( Fig. 2A), and also pulls down purified p34 and coronin in an in vitro protein binding assay (Fig S4A), suggesting that 5-InsP 7 directly binds p34 and coronin.
Consistent with Arp2/3 complex inhibition, itraconazole treatment elicits prominent morphological changes in HUVECs. The DMSO-treated control cells are cobblestone-like, whereas the itraconazole-treated cells display smaller and more irregular shapes (Fig. 2E). Fluorescein phalloidin staining reveals drastically different actin filament architectures in DMSO-and itraconazole-treated HUVECs (Fig. 2F). While F-actin is widely distributed in the DMSO-treated control cells, it largely accumulates at the cell cortex in the itraconazole-treated cells (Fig. 2F), which is similar to Arp2/3 complex inhibition (Fig S5) [30]. Increasing 5-InsP 7 levels by overexpressing IP6K1 in HUVECs, which presumably strengthens the interactions of coronin with Arp2/3 complex, elicits similar effects on F-actin distribution as itraconazole does (Fig. 2G). We utilized WT and IP6K1 KO MEF cells to validate that IP6K1 is involved in itraconazole-elicited alteration of F-actin (Fig S6). Deletion of IP6K1 impairs F-actin formation [31]. Itraconazole disrupts F-actin in WT cells but does not further disrupt F-actin in IP6K1 KO cells (Fig S6).

Itraconazole dissociates IP6K1/α-actinin from focal adhesions
We previously reported that IP6K1 binds α-actinin, which plays a critical role in regulating focal adhesion turnover [31], and ask whether itraconazole affects the interaction of IP6K1 with α-actinin. Immunoprecipitations reveal that the interaction of IP6K1 with α-actinin is strengthened by itraconazole treatment (Fig. 3A, B).
We ask whether the increased binding of IP6K1 to α-actinin induced by itraconazole intensifies IP6K1 at focal adhesions. Unexpectedly, confocal microscopy shows that the co-localization of IP6K1 with FAK is decreased after itraconazole treatment (Fig. 3C). These results suggest that IP6K1 is removed by itraconazole from focal adhesions. This prompted us to assess the interaction between α-actinin and FAK because IP6K1 indirectly associates with FAK through α-actinin [32]. Immunoprecipitation studies reveal that the binding between α-actinin and FAK is disrupted by itraconazole treatment (Fig. 3D, E).
Itraconazole treatment does not seem to affect the expression levels of α-actinin and FAK (Fig S7A). However, itraconazole causes enrichment of α-actinin in the cell membrane, but decreases FAK protein levels in the cell membrane (Fig. 3F). This result further suggests that the interaction of α-actinin with FAK is disrupted by itraconazole. IP6K1 does not seem to play a role in the itraconazole-induced redistribution of α-actinin. Neither overexpression nor knocking down of IP6K1 increases the protein levels of α-actinin in the plasma membrane fractions ( Fig   S7B, C). We utilized confocal microscopy to confirm that itraconazole elicits redistribution of α-actinin (Fig. 3G). In control cells, α-actinin appears diffuse and evenly distributed. In itraconazole-treated cells, α-actinin distributes more heavily along the cell border (Fig. 3G). Consistent with the redistribution of α-actinin, double staining of α-actinin and F-actin reveals that the stress fibers formation is drastically reduced in itraconazole-treated cells (Fig. 3H). Coherently, a large portion of focal adhesions are assembled without connection with stress fibers in itraconazole treated cells (Fig. 3I).
We utilized WT and IP6K1 KO MEF cells to confirm that depleting IP6K1-generated 5-InsP 7 in focal adhesions is responsible for itraconazole-induced inhibition of FAK phosphorylation (Fig. 4C). Western blots show that FAK phosphorylation levels are lower in IP6K1 KO cells than that of WT cells. Itraconazole decreases FAK phosphorylation in WT cells to a level similar to that in the IP6K1 KO cells. In IP6K1 KO cells, itraconazole barely reduces FAK phosphorylation levels (Fig. 4C).
FAK dimerization is critical for its phosphorylation [34]. To test whether itraconazole affects FAK dimerization, we overexpressed GST-FAK and examined the interaction between GST-FAK and endogenous FAK. Pulling down GST-FAK co-precipitates endogenous FAK, confirming that they form dimers (Fig. 4D). Itraconazole treatment drastically reduces the amount of co-precipitated endogenous FAK, suggesting that itraconazole disrupts FAK dimerization (Fig. 4D).
5-InsP 7 strengthens FAK dimer formation, because GST-FAK coprecipitates substantially less endogenous FAK in the IP6K1 KO cells compared to WTs (Fig. 4E). Pharmacologic inhibition of IP6K1 enzymatic activity by TNP treatment also reduces the binding of GST-FAK with endogenous FAK (Fig. 4F).

Itraconazole reduces focal adhesion turnover
Confocal microscopy shows that the density of phosphorylated FAK is markedly decreased in itraconazole-treated HUVECs (Fig. 5A). Similarly, the density of phosphorylated paxillin, a downstream target of FAK, is diminished in itraconazole-treated HUVECs (Fig. 5B). The sizes of focal adhesions, as evidenced by vinculin staining, are relatively larger in the itraconazole-treated cells, indicating fewer turnovers of focal adhesions (Fig. 5C).
Cell spreading requires dynamic reorganization of actin cytoskeleton and coordination of FAK phosphorylation, and is delayed in itraconazole-treated cells (Fig. 5D). At 60 min after plating, phalloidin staining reveals networks of actin filaments in control cells. In contrast, actin filaments exclusively assemble at the cell cortex of itraconazoletreated HUVECs (Fig. 5E), and vinculin staining reveals fewer focal adhesions assemble in itraconazole-treated spreading HUVECs (Fig. 5F).
We double stained phosphorylated FAK and vinculin to validate the role of IP6K1 in itraconazole-induced reduction of focal adhesion turnover (Fig S9). Deletion of IP6K1 reduces FAK phosphorylation, and itraconazole decreases FAK phosphorylation in WT MEF cells but does not further decrease it in IP6K1 KO MEF cells (Fig S9A). Similarly, deletion of IP6K1 reduces paxillin phosphorylation, and itraconazole lowers phosphorylation levels of paxillin in WT MEF cells but does not further reduce it in IP6K1 KO MEF cells (Fig S9B).

Itraconazole disrupts actin remodeling and arrests cell movement
We utilized Lifeact-mScarlet to label actin filaments in living cells and monitored actin dynamics under the confocal microscope (Fig. 5G,  Video 1, 2). Itraconazole treatment severely impedes the active remodeling of actin filaments. Over a 30-minute period, the control cells displayed assembly and disassembly of actin filaments (Fig. 5G, Video 1). In striking contrast, few changes of actin filaments were observed in the itraconazole-treated HUVECs (Fig. 5G, Video 2). We utilized fluorescence microscopy to monitor the cell movement of GFP-expressing HUVECs. The control cells display lamellipodia protrusion and retraction (Fig. 5H, Video 3), but itraconazole-treated cells adhere tightly to the cell culture plate and barely move (Fig. 5H, Video 4). We also confirmed that IP6K1 is involved in the itraconazole-elicited disruption of cytoskeletal remodeling and cell motility (Fig. 6, Video 5-12). Deletion of IP6K1 disrupts active actin remodeling (Fig. 6A, Video 5-8). Itraconazole reduces active actin remodeling in WT MEF cells but does not further reduce it in IP6K1 KO MEF cells (Fig. 6A, Video 5-8). Similarly, deletion of IP6K1 impairs cell motility (Fig. 6B, Video 9-12). Itraconazole delays cell movement in WT MEFs but does not further delay it in IP6K1 KO MEF cells (Fig. 6B, Video 9-12).
Supplementary material related to this article can be found online at doi:10.1016/j.biopha.2023.114449.

Discussion and conclusions
Despite significant efforts to repurpose the anti-fungal drug itraconazole as an anti-angiogenic agent, the mechanisms of action have been elusive. Endothelial cell proliferation and migration are essential components of angiogenesis. In this study, we demonstrate a functional mechanism by which itraconazole inhibits endothelial cell migration. Itraconazole treatment disrupts active remodeling of focal adhesions and the actin cytoskeleton, which requires IP6K1 and its product 5-InsP 7 . IP6K1 generates 5-InsP 7 at focal adhesions to mediate FAK dimerization and phosphorylation. Some IP6K1 protein also binds Arp2 and generates 5-InsP 7 to recruit coronin to Arp2/3 complex. Itraconazole treatment shifts IP6K1 from focal adhesion to Arp2/3 complex, simultaneously reducing 5-InsP 7 -mediated FAK phosphorylation and augmenting 5-InsP 7 -mediated recruitment of coronin to Arp2/3 complex (Fig. 7). As a result, itraconazole disrupts 5-InsP 7 -regulated focal adhesion dynamics and actin cytoskeleton remodeling to impede cell motility.
Energetic remodeling of actin filaments and focal adhesions is essential for cell motility. The Arp2/3 complex generates a dendritic actin network at the leading edge of motile cells to form lamellipodia, which are widely believed to be critical for directional migration [26]. Binding of coronin to Arp2/3 promotes disassembly of branched actin networks [37], and it is required for Arp2/3-mediated actin dynamics in vivo [38]. The coordination of coronin and Arp2/3 complex plays important roles in the leading-edge actin dynamics and overall cell motility [37]. IP6K1 binds Arp2, and generates 5-InsP 7 to perform physiological functions. The inhibition of Arp2/3 by coronin is concentration-dependent [38]. Itraconazole-induced redistribution of IP6K1 to Arp2/3 complex strengthens 5-InsP 7 -mediated recruitment of coronin and shift towards Arp2/3 inhibition. This itraconazole-induced Arp2/3 inhibition may negatively feedback the expression levels of coronin, which displays 30% lower in the itraconazole treated cells.
In living cells, FAK activation is associated with conformational changes [48]. 5-InsP 7 directly binds FAK via its FERM domain and promotes FAK dimerization, which is critical for FAK phosphorylation and its kinase-dependent functions at focal adhesions [49]. This mechanism partially explains the critical roles of 5-InsP 7 in focal adhesion turnover [19,31,33,50]. The extremely short half-life of 5-InsP 7 requires it to be produced near its target proteins. Removing IP6K1 by itraconazole from focal adhesions deprives them of 5-InsP 7 , impeding FAK dimerization. The non-hydrolyzable analogs of 5-InsP 7 are not able to mediate FAK dimerization, indicating that pyrophosphorylation plays a role. Protein pyrophosphorylation is complex, although it has been discovered for a decade, its physiological functions and mechanisms have been elusive [51][52][53][54][55][56]. How pyrophosphorylation affects FAK requires further studies.
Inositol pyrophosphates mediate diverse cellular processes, such as energy production, protein secretion and DNA damage and repair, in particular subcellular areas. Individual IP6K knockout animals display specific phenotypes, and not compensated by other isoforms [57,58]. The consequences of itraconazole-elicited displacement of 5-InsP 7 reiterates the criticality of the compartmentalized production of 5-InsP 7 . Relocation of IP6Ks have been reported in several studies. Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of IP6K1 [59]. Translocation of IP6K2 from nucleus to cytosol causes cell death [60]. The direct target of itraconazole in mediating IP6K1 redistribution is currently unknown, and the mechanism by which itraconazole induces redistribution of IP6K1 requires further studies. It is worth noting that itraconazole does not affect the protein levels nor the kinase activity of IP6K1. data are presented as mean±SEM, Student's t-test, n = 5 independent repeats, ns=not significant, * p < 0.05, * *p < 0.01, * **p < 0.001. Statistical data are presented as means ± SEM, Student's t-test, 10 images from 5 independent experiments were analyzed, * *P < 0.01, * **P < 0.001.
Our study demonstrates that IP6K1-generated 5-InsP 7 is a critical regulator of focal adhesion dynamics and actin cytoskeleton remodeling, and it reveals a functional mechanism by which itraconazole inhibits cell motility.

Funding
This work was supported by the National Natural Science Foundation of China (82070259, 81870232 and 82220108021) and sponsored by Natural Science Foundation of Shanghai (22ZR1440700). W.C. was   Physiologically, IP6K1 generates a local pool of 5-InsP 7 in focal adhesions to promote FAK dimerization and phosphorylation. Some IP6K1 protein also binds Arp2 and generates 5-InsP 7 to mediate the binding of coronin to Arp2/ 3 complex. (B) Itraconazole treatment redistributes IP6K1 from focal adhesions to Arp2/3 complex, simultaneously reducing 5-InsP 7 -mediated FAK activation and enhancing 5-InsP 7 -mediated Arp2/3 inhibition.