Interaction between VPS13A and the XK scramblase is important for VPS13A function in humans.

VPS13 family proteins form conduits between the membranes of different organelles through which lipids are transferred. In humans, there are four VPS13 paralogs, and mutations in the genes encoding each of them are associated with different inherited disorders. VPS13 proteins contain multiple conserved domains. The Vps13 adaptor-binding (VAB) domain binds to adaptor proteins that recruit VPS13 to specific membrane contact sites. This work demonstrates the importance of a different domain in VPS13A function. The pleckstrin homology (PH) domain at the C-terminal region of VPS13A is required to form a complex with the XK scramblase and for the co-localization of VPS13A with XK within the cell. Alphafold modeling was used to predict an interaction surface between VPS13A and XK. Mutations in this region disrupt both complex formation and co-localization of the two proteins. Mutant VPS13A alleles found in patients with VPS13A disease truncate the PH domain. The phenotypic similarities between VPS13A disease and McLeod syndrome caused by mutations in VPS13A and XK, respectively, argue that loss of the VPS13A-XK complex is the basis of both diseases.

Genetic Dissection of Vps13 Regulation in Yeast Using Disease Mutations from Human Orthologs.

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.

XK is a partner for VPS13A: a molecular link between Chorea-Acanthocytosis and McLeod Syndrome.

Vps13 is a highly conserved lipid transfer protein found at multiple interorganelle membrane contact sites where it mediates distinct processes. In yeast, recruitment of Vps13 to different contact sites occurs via various partner proteins. In humans, four VPS13 family members, A-D, are associated with different diseases. In particular, vps13A mutants result in the neurodegenerative disorder Chorea-Acanthocytosis (ChAc). ChAc phenotypes resemble those of McLeod Syndrome, caused by mutations in the XK gene, suggesting that XK could be a partner protein for VPS13A. XK does, in fact, exhibit hallmarks of a VPS13A partner: it forms a complex with VPS13A in human cells and, when overexpressed, relocalizes VPS13A from lipid droplets to subdomains of the endoplasmic reticulum. Introduction of two different ChAc disease-linked missense mutations into VPS13A prevents this XK-induced relocalization. These results suggest that dysregulation of a VPS13A-XK complex is the common basis for ChAc and McLeod Syndrome.

Polymeric nanoparticle-docetaxel for the treatment of advanced solid tumors: phase I clinical trial and preclinical data from an orthotopic pancreatic cancer model.

We assessed the efficacy of the polymeric nanoparticle containing docetaxel (PNP-DTX) in preclinical mouse models and determined the maximum tolerated dose (MTD) through clinical study. Subcutaneous and orthotopic mouse models were dedicated. Tumor growth delay in orthotopic model and quantification of in vivo imaging in orthotopic model were evaluated. Phase I clinical study was a single-center, prospective, open-label trial in advanced solid tumors. PNP-DTX was injected intravenously and the starting dose was 20 mg/m2 escalated to 35 mg/m2, 45 mg/m2, 60 mg/m2 and 75 mg/m2. Pharmacokinetics, tumor response, toxicities were evaluated. Preclinical result revealed the more potent cytotoxic effect of PNP-DTX than docetaxel (DTX). However, there was no difference between PNP-DTX and DTX in subcutaneous model. Tubulin polymerization assay showed that PNP-DTX preserved original mode of action of DTX. For phase I clinical trial, 18 patients were analyzed. The dose of 75 mg/m2 was tentatively determined as the MTD and the most common toxicity was grade 4 neutropenia not lasting over 7days. The Cmax of 60 mg/m2 PNP-DTX and AUClast of 45 mg/m2 PNP-DTX were measured to be comparable to those of 75 mg/m2 DTX. Partial remission (PR) was achieved in 4 (22%) patients. The potency of PNP-DTX was revealed especially in orthotopic mouse model. The MTD of PNP-DTX could not be confirmed, but 75 mg/m2 was tentatively determined. The PNP-DTX of 45 mg/m2 had the same pharmacokinetic profile with that of 75 mg/m2 DTX.

Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites.

The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation. This study demonstrates that VPS13 is also important for mitochondrial integrity. In addition to preventing transfer of DNA from the mitochondrion to the nucleus, VPS13 suppresses mitophagy and functions in parallel with the endoplasmic reticulum-mitochondrion encounter structure (ERMES). In different growth conditions, Vps13 localizes to endosome-mitochondrion contacts and to the nuclear-vacuole junctions, indicating that Vps13 may function at membrane contact sites. The ability of VPS13 to compensate for the absence of ERMES correlates with its intracellular distribution. We propose that Vps13 is present at multiple membrane contact sites and that separation-of-function mutants are due to loss of Vps13 at specific junctions. Introduction of VPS13A mutations identified in ChAc patients at cognate sites in yeast VPS13 are specifically defective in compensating for the lack of ERMES, suggesting that mitochondrial dysfunction might be the basis for ChAc.

Multifunctional hollow gold nanoparticles designed for triple combination therapy and CT imaging.

Hollow gold nanoparticles (HGNP) are a novel class of hybrid metal nanoparticles whose unique optical and morphological properties have spawned new applications including more effective cancer therapy. The shell thickness of HGNPs can tune the surface plasmon resonance to the near infrared light, resulting in photothermal ablation of tumors with optimal light penetration in tissue. The hollow cavity within a HGNP is able to accommodate a high payload of chemotherapeutic agents. They have also been used for enhancing radiosensitization in tumors during radiotherapy due to the high X-ray absorption capability of gold particles. However, no report has yet been published that utilize HGNPs for the triple combination therapy and CT imaging. In this study, we synthesized HGNPs which exhibit better response to radiation for therapy and imaging and demonstrated the effects of combined chemotherapy, thermal and radiotherapy. This combination strategy presented delayed tumor growth by 4.3-fold and reduced tumor's weight by 6.8-fold compared to control tumors. In addition, we demonstrated the feasibility of HGNP as a CT imaging agent. It is expected that translating these capabilities to human cancer patients could dramatically increase the antitumor effect and potentially overcome resistance to chemotherapeutic agents and radiation.

A conserved function in phosphatidylinositol metabolism for mammalian Vps13 family proteins.

The Vps13 protein family is highly conserved in eukaryotic cells. In humans, mutations in the gene encoding the family member VPS13A lead to the neurodegenerative disorder chorea-acanthocytosis. In the yeast Saccharomyces cerevisiae, there is just a single version of VPS13, thereby simplifying the task of unraveling its molecular function(s). While VPS13 was originally identified in yeast by its role in vacuolar sorting, recent studies have revealed a completely different function for VPS13 in sporulation, where VPS13 regulates phosphatidylinositol-4-phosphate (PtdIns(4)P) levels in the prospore membrane. This discovery raises the possibility that the disease phenotype associated with vps13A mutants in humans is due to misregulation of PtdIns(4)P in membranes. To determine whether VPS13A affects PtdIns(4)P in membranes from mammalian neuronal cells, phosphatidylinositol phosphate pools were compared in PC12 tissue culture cells in the absence or presence of VPS13A. Consistent with the yeast results, the localization of PtdIns(4)P is specifically altered in VPS13A knockdown cells while other phosphatidylinositol phosphates appear unaffected. In addition, VPS13A is necessary to prevent the premature degeneration of neurites that develop in response to Nerve Growth Factor. The regulation of PtdIns(4)P is therefore a conserved function of the Vps13 family and may play a role in the maintenance of neuronal processes in mammals.

Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

A cisplatin­incorporated liposome that targets the epidermal growth factor receptor enhances radiotherapeutic efficacy without nephrotoxicity.

Radiotherapy (RT) is one of the major modalities for non­small cell lung cancer (NSCLC), but its efficacy is often compromised by cellular resistance caused by various mechanisms including the overexpression of epidermal growth factor receptor (EGFR). Although cis­diamminedichloroplatinum(Ⅱ) (cisplatin, CDDP) has been well characterized as an effective radiosensitizer, its clinical application is limited by its severe nephrotoxic effects. In our current study, we developed a CDDP­incorporated liposome (LP) conjugated with EGFR antibodies (EGFR:LP­CDDP) and evaluated its potential to radiosensitize EGFR­overexpressing cells without exerting nephrotoxic effects. EGFR:LP­CDDP showed higher cytotoxicity than non­targeting liposomal CDDP (LP­CDDP) in the cells expressing EGFR in vitro. In an A549 cell­derived xenograft tumor mouse model, increased delays in tumor growth were observed in the mice treated with a combination of EGFR:LP­CDDP and radiation. Notably, the EGFR:LP­CDDP­treated animals showed no differences in body weight loss, survival rates of nephrotoxicity compared with untreated control mice. In contrast, the use of CDDP caused lower body weights and poorer survival outcomes accompanied by a significant level of nephrotoxicity [e.g., decreased kidney weight, increased blood urea nitrogen (BUN) and creatinine, and pathological change]. These findings suggest the feasibility of using EGFR:LP­CDDP to radiosensitize cells in a targeted manner without inducing nephrotoxic effects. This compound may therefore have clinical potential as part of a tailored chemoradiotherapy strategy.

Ibulocydine sensitizes human cancers to radiotherapy by induction of mitochondria-mediated apoptosis.

BACKGROUND AND PURPOSE: Ibulocydine (IB), a novel prodrug of CDK inhibitor, has been reported to have anti-cancer effect in human hepatoma cells. In order to address its feasibility as a radiosensitizer to improve radiotherapeutic efficacy for human cancers, this study was designed. MATERIAL AND METHODS: Human cancer cells of lung and colon were treated with IB and/or radiotherapy (RT). The cellular effects were assessed by CCK-8, clonogenic, flow cytometric, and western blotting assays. In vivo radiotherapeutic efficacy was evaluated using the xenograft mouse model. RESULTS: Combined treatment of IB and RT significantly reduced viability and survival fraction of the cells. Apoptotic cell death accompanied with activation of caspases, decrease in Bcl-2/Bax expression, loss of mitochondrial membrane potential (MMP) leading to release of cytochrome c into cytosol was observed. Recovery of Bcl-2 expression level by introducing Bcl-2 expressing plasmid DNA compromised the loss of MMP and apoptosis induced by IB and RT. In vivo therapeutic efficacy of combined treatment was verified in the xenograft mouse model, in which tumor growth was markedly delayed by RT with IB. CONCLUSIONS: IB demonstrated the property of sensitizing human cancer cells to RT by induction of mitochondria-mediated apoptosis, suggesting that IB deserves to be applied for chemoradiotherapy.

SPO71 encodes a developmental stage-specific partner for Vps13 in Saccharomyces cerevisiae.

The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71, a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71Δ cells. SPO71 is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis.

WITHDRAWN: Interferometric biosensing of DNA-damaging chemicals.

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VPS13 regulates membrane morphogenesis during sporulation in Saccharomyces cerevisiae.

The hereditary disorders chorea acanthocytosis and Cohen syndrome are caused by mutations in different members of a family of genes that are orthologs of yeast VPS13. In vegetatively growing yeast, VPS13 is involved in the delivery of proteins to the vacuole. During sporulation, VPS13 is important for formation of the prospore membrane that encapsulates the daughter nuclei to give rise to spores. We report that VPS13 is required for multiple aspects of prospore membrane morphogenesis. VPS13 (1) promotes expansion of the prospore membrane through regulation of phosphatidylinositol phosphates, which in turn activate the phospholipase D, Spo14; (2) is required for a late step in cytokinesis that gives rise to spores; and (3) regulates a membrane-bending activity that generates intralumenal vesicles. These results demonstrate that Vps13 plays a broader role in membrane biology than previously known, which could have important implications for the functions of VPS13 orthologs in humans.

In vivo tumor targeting of gold nanoparticles: effect of particle type and dosing strategy.

Gold nanoparticles (GNPs) have gained significant interest as nanovectors for combined imaging and photothermal therapy of tumors. Delivered systemically, GNPs preferentially accumulate at the tumor site via the enhanced permeability and retention effect, and when irradiated with near infrared light, produce sufficient heat to treat tumor tissue. The efficacy of this process strongly depends on the targeting ability of the GNPs, which is a function of the particle's geometric properties (eg, size) and dosing strategy (eg, number and amount of injections). The purpose of this study was to investigate the effect of GNP type and dosing strategy on in vivo tumor targeting. Specifically, we investigated the in vivo tumor-targeting efficiency of pegylated gold nanoshells (GNSs) and gold nanorods (GNRs) for single and multiple dosing. We used Swiss nu/nu mice with a subcutaneous tumor xenograft model that received intravenous administration for a single and multiple doses of GNS and GNR. We performed neutron activation analysis to quantify the gold present in the tumor and liver. We performed histology to determine if there was acute toxicity as a result of multiple dosing. Neutron activation analysis results showed that the smaller GNRs accumulated in higher concentrations in the tumor compared to the larger GNSs. We observed a significant increase in GNS and GNR accumulation in the liver for higher doses. However, multiple doses increased targeting efficiency with minimal effect beyond three doses of GNPs. These results suggest a significant effect of particle type and multiple doses on increasing particle accumulation and on tumor targeting ability.

Use of macrophages to deliver therapeutic and imaging contrast agents to tumors.

Drug targeting to tumors with limited toxicity and enhanced efficacy of drug is one of the important goals for cancer treatment pharmaceutics. Monocytes/macrophages are able to migrate to tumor sites across the blood barriers by acting as Trojan horses carrying drug cargoes. Taking this advantage, we have intended to develop an efficient administration system using a biologically active carrier of mouse peritoneal macrophage bearing liposomal doxorubicin (macrophage-LP-Dox). We expect that this system could improve the cancer therapeutic efficacy through deeper penetration into tumor even hypoxic region behind tumor blood vessel. We first confirmed that macrophages containing iron oxides could migrate and infiltrate into tumors effectively by MR imaging. Next, we showed that doxorubicin (Dox) encapsulated with liposomes (LP-Dox) was successfully loaded into macrophages, in which the biological activity of macrophage and cytotoxicity of Dox against tumor cells were well preserved. Delivery of Dox into tumor tissue by systemic administration of macrophage-LP-Dox was verified in both subcutaneous and metastasis xenograft tumor models. Importantly, the effective inhibition of in vivo tumor growth was proved with this system. Our results provide the feasibility of macrophages-LP-drug as an active biocarrier for the enhancement of therapeutic effects in cancer treatment and open new perspectives for the active delivery of drugs.

Intra-organ Biodistribution of Gold Nanoparticles Using Intrinsic Two-photon Induced Photoluminescence.

BACKGROUND AND OBJECTIVES: Gold nanoparticles (GNPs) such as gold nanoshells (GNSs) and gold nanorods (GNRs) have been explored in a number of in vitro and in vivo studies as imaging contrast and cancer therapy agents due to their highly desirable spectral and molecular properties. While the organ-level biodistribution of these particles has been reported previously, little is known about the cellular level or intra-organ biodistribution. The objective of this study was to demonstrate the use of intrinsic two-photon induced photoluminescence (TPIP) to study the cellular level biodistribution of GNPs. STUDY DESIGN/MATERIALS AND METHODS: Tumor xenografts were created in twenty-seven male nude mice (Swiss nu/nu) using HCT 116 cells (CCL-247, ATCC, human colorectal cancer cell line). GNSs and GNRs were systemically injected 24 hr. prior to tumor harvesting. A skin flap with the tumor was excised and sectioned as 8 µm thick tissues for imaging GNPs under a custom-built multiphoton microscope. For multiplexed imaging, nuclei, cytoplasm, and blood vessels were demonstrated by hematoxylin and eosin (H&E) staining, YOYO-1 iodide staining and CD31-immunofluorescence staining. RESULTS: Distribution features of GNPs at the tumor site were determined from TPIP images. GNSs and GNRs had a heterogeneous distribution with higher accumulation at the tumor cortex than tumor core. GNPs were also observed in unique patterns surrounding the perivascular region. While most GNSs were confined at the distance of approximately 400 µm inside the tumor edge, GNRs were shown up to 1.5 mm penetration inside the edge. CONCLUSIONS: We have demonstrated the use of TPIP imaging in a multiplexed fashion to image both GNPs and nuclei, cytoplasm, or vasculature simultaneously. We also confirmed that TPIP imaging enabled visualization of GNP distribution patterns within the tumor and other critical organs. These results suggest that direct luminescence-based imaging of metal nanoparticles holds a valuable and promising position in understanding the accumulation kinetics of GNPs. In addition, these techniques will be increasingly important as the use of these particles progress to human clinical trials where standard histopathology techniques are used to analyze their effects.

Essential role for Schizosaccharomyces pombe pik1 in septation.

BACKGROUND: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4(G107S). PRINCIPAL FINDINGS: Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25 degrees C. At 36 degrees C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1(D709A) was kinase-dead, but bound Cdc4. Pik1(R838A) did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1(D709A,R838A) was innocuous, pik1(R838A) was almost innocuous, and pik1(D709A) produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4(G107S). Thus, D709 is essential for kinase activity and septation. CONCLUSIONS: Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.

Near-infrared narrow-band imaging of gold/silica nanoshells in tumors.

Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica)/shell (gold) ratio. In addition to spectral tunability, GNS are inert and bioconjugatable, making them potential labels for in vivo imaging and therapy of tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow-band imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the optimum wavelengths for ex vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and retention (EPR) effect. Ex vivo NBI of tumor xenografts demonstrated heterogeneous distribution of GNS with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as contrast agents to visualize tumors using NBI.

The anaphase promoting complex targeting subunit Ama1 links meiotic exit to cytokinesis during sporulation in Saccharomyces cerevisiae.

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APC(Ama1) is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1Delta mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1Delta mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1Delta mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APC(Ama1) functions to coordinate the exit from meiosis II with cytokinesis.

5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a phospholipase D pharmacological inhibitor that alters cell spreading and inhibits chemotaxis.

The signaling enzyme phospholipase D (PLD) and the lipid second messenger it generates, phosphatidic acid (PA), are implicated in many cell biological processes, including Ras activation, cell spreading, stress fiber formation, chemotaxis, and membrane vesicle trafficking. PLD production of PA is inhibited by the primary alcohol 1-butanol, which has thus been widely employed to identify PLD/PA-driven processes. However, 1-butanol does not always effectively reduce PA accumulation, and its use may result in PLD-independent deleterious effects. Consequently, identification of potent specific small-molecule PLD inhibitors would be an important advance for the field. We examine one such here, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), which was identified recently in an in vitro chemical screen for PLD2 inhibitors, and show that it rapidly blocks in vivo PA production with subnanomolar potency. We were surprised to find that several biological processes blocked by 1-butanol are not affected by FIPI, suggesting the need for re-evaluation of proposed roles for PLD. However, FIPI does inhibit PLD regulation of F-actin cytoskeleton reorganization, cell spreading, and chemotaxis, indicating potential utility for it as a therapeutic for autoimmunity and cancer metastasis.