Design and Synthesis of Arf1-Targeting γ-Dipeptides as Potential Agents against Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related deaths and calls for new druggable targets. We have previously highlighted the critical role of ADP-ribosylation factor-1 (Arf1) activation in HNSCC. In the present study, we address the question whether targeting Arf1 could be proposed as a valuable strategy against HNSCC. METHODS: We rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides to block Arf1 activation. We evaluated the effects of these γ-dipeptides in HNSCC cells: The cell viability was determined in 2D and 3D cell cultures after 72 h treatment and Arf1 protein levels and activity were assessed by GGA3 pull-down and Western blotting assays. RESULTS: Targeting Arf1 offers a valuable strategy to counter HNSCC. Our new Arf1-targeting compounds revealed a strong in vitro cytotoxicity against HNSCC cells, through inhibiting Arf1 activation and its downstream pathways. CONCLUSIONS: Arf1-targeting γ-dipeptides developed in this study may represent a promising targeted therapeutic to improve managing the HNSCC disease.

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics.

BACKGROUND: Although major improvements have been made in surgical management, chemotherapeutic, and radiotherapeutic of prostate cancer, many prostate cancers remain refractory to treatment with standard agents. Therefore, the identification of new molecular targets in cancer progression and development of novel therapeutic strategies to target them are very necessary for achieving better survival for patients with prostate cancer. Activation of small GTPases such as Ras and Arf1 is a critical component of the signaling pathways for most of the receptors shown to be upregulated in advanced prostate cancer. METHODS: The drug effects on cell proliferation were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay. The drug effects on cell migration and invasion were determined by Radius™ 24-well and Matrigel-coated Boyden chambers. The drug effects on apoptosis were assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD and Western blot with antibodies against cleaved PARP and Caspase 3. A NOD/SCID mouse model generated by subcutaneous injection was used to assess the in vivo drug efficacy in tumor growth. ERK activation and tumor cell proliferation in xenografts were examined by immunohistochemistry. RESULTS: We show that Exo2, a small-molecule inhibitor that reduces Arf1 activation, effectively suppresses prostate cancer cell proliferation by blocking ERK1/2 activation. Exo2 also has other effects, inhibiting migration and invasion of PCa cells and inducing apoptosis. The Ras inhibitor salirasib augments Exo2-induced cytotoxicity in prostate cancer cells partially by enhancing the suppression of ERK1/2 phosphorylation. In a xenograft mouse model of prostate cancer, Exo2 reduces prostate tumor burden and inhibits ERK1/2 activation at a dose of 20 mg/kg. Synergistic treatment of salirasib and Exo2 exhibits a superior inhibitory effect on prostate tumor growth compared with either drug alone, which may be attributed to the more efficient inhibition of ERK1/2 phosphorylation. CONCLUSION: This study suggests that simultaneous blockade of Arf1 and Ras activation in prostate cancer cells is a potential targeted therapeutic strategy for preventing prostate cancer development.

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila.

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. Here we investigated stem-cell death in the digestive system of adult Drosophila melanogaster. We found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively killed normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spared differentiated cells. The dying stem cells were engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduced CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers.

Suppression of breast cancer metastasis through the inactivation of ADP-ribosylation factor 1.

Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.

M-COPA, a novel Golgi system disruptor, suppresses apoptosis induced by Shiga toxin.

Shiga toxin (Stx) is a main virulence factor of Stx-producing Escherichia coli (STEC) that contributes to diarrhea and hemorrhagic colitis and occasionally to fatal systemic complications. Therefore, the development of an antidote to neutralize Stx toxicity is urgently needed. After internalization into cells, Stx is transferred to the Golgi apparatus via a retrograde vesicular transport system. We report here that 2-methylcoprophilinamide (M-COPA), a compound that induces disassembly of the Golgi apparatus by inactivating ADP-ribosylation factor 1 (Arf1), suppresses Stx-induced apoptosis. M-COPA inhibited transport of Stx from the plasma membrane to the Golgi apparatus and suppressed degradation of anti-apoptotic proteins and the activation of caspases. These findings suggest that inhibition of Stx retrograde transport by M-COPA could be a novel approach to suppress Stx toxicity.

Brefeldin A exerts differential effects on anaplastic lymphoma kinase positive anaplastic large cell lymphoma and classical Hodgkin lymphoma cell lines.

To obtain further insights into the biological differences of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) and classical Hodgkin lymphoma (HL), we screened microbial culture filtrates to search for compounds that would exert a significantly greater effect on the viability of ALK+ ALCL cell lines compared to HL cell lines and identified Brefeldin A (BFA) as a suitable candidate. BFA inhibited phosphorylation of ALK and its downstream molecule, signal transducer and activator of transcription 3 (STAT3), one of the central pathways for the survival of ALK+ ALCL cells. In HL cell lines BFA did not affect CD30 expression or constitutive nuclear factor (NF)-κB activity, both of which are critical for HL cell survival. BFA induced disruption of the Golgi apparatus in ALK+ ALCL cell lines, which was accompanied by a decrease in active ADP-ribosylation factor 1 (ARF1), whereas BFA had no significant effect on these parameters in HL cell lines. These results add extra insights into the biological distinction between ALK+ ALCL and HL cells and highlight the Golgi apparatus as a target for the treatment of ALK+ ALCL.

ARF1 regulates adhesion of MDA-MB-231 invasive breast cancer cells through formation of focal adhesions.

Adhesion complex formation and disassembly is crucial for maintaining efficient cell movement. During migration, several proteins act in concert to promote remodeling of the actin cytoskeleton and we have previously shown that in highly invasive breast cancer cells, this process is regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. These are overexpressed and highly activated in these cells. Here, we report that one mechanism by which ARF1 regulates migration is by controlling assembly of focal adhesions. In cells depleted of ARF1, paxillin is no longer colocalized with actin at focal adhesion sites. In addition, we demonstrate that this occurs through the ability of ARF1 to regulate the recruitment of key proteins such as paxillin, talin and FAK to ß1-integrin. Furthermore, we show that the interactions between paxillin and talin together and with FAK are significantly impaired in ARF1 knocked down cells. Our findings also indicate that ARF1 is essential for EGF-mediated phosphorylation of FAK and Src. Finally, we report that ARF1 can be found in complex with key focal adhesion proteins such as ß1-integrin, paxillin, talin and FAK. Together our findings uncover a new mechanism by which ARF1 regulates cell migration and provide this GTPase as a target for the development of new therapeutics in triple negative breast cancer.

Fragment-based identification of a locus in the Sec7 domain of Arno for the design of protein-protein interaction inhibitors.

By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. By use of SPR, NMR, and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography. This highlighted the role of stable and transient pockets at the Sec7 domain surface in the discovery and binding of interfering compounds. These results provide structural information on how small organic compounds can interfere with the Arf1-Arno Sec7 domain interaction and may guide the rational drug design of competitive inhibitors of Arno enzymatic activity.

Signaling through Arf6 guanine-nucleotide exchange factor cytohesin-1 regulates migration in Schwann cells.

During development of the peripheral nervous system (PNS), Schwann cells migrate along neuronal axons before initiating myelination of the axons. While intercellular signals controlling migration, between Schwann cells and peripheral neurons, are established, how their intracellular transduction of the signals into Schwann cells still remains to be clarified. Here, we show that cytohesin-1, a guanine-nucleotide exchange factor (GEF), and the effector Arf6 are required for migration of primary Schwann cells. Knockdown of cytohesin-1 or Arf6 in Schwann cells, as well as treatment with the chemical cytohesin inhibitor SecinH3 or knockout of cytohesin-1, inhibits peripheral neuronal conditioned medium-mediated migration. Similar effects are also observed following stimulation with each of growth factors contained in a conditioned medium, suggesting that cytohesin-1 plays a role in transducing soluble ligand signals from neurons. Reintroduction of small interfering (si)RNA-resistant cytohesin-1 into Schwann cells reverses blunted migration in the siRNA-transfected Schwann cells, illustrating the importance of cytohesin-1 in migration. On the other hand, introduction of cytohesin-1 that harbors the Tyr-382 mutation, which is an amino acid that is important for its activation, failed to reverse the reduction in primary Schwann cell migration. These results suggest that signaling through cytohesin-1 is required for Schwann cell migration, revealing a novel mechanism for Schwann cell migration.

Secretion of soluble vascular endothelial growth factor receptor 1 (sVEGFR1/sFlt1) requires Arf1, Arf6, and Rab11 GTPases.

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.

ADP-ribosylation factor 1 protein regulates trypsinogen activation via organellar trafficking of procathepsin B protein and autophagic maturation in acute pancreatitis.

Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy.

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

ADP-ribosylation factor as a novel target for corneal neovascularization regression.

PURPOSE: To evaluate the roles of ADP-ribosylation factor (ARF) in alkali-induced corneal neovascularization (CNV). METHODS: CNV was induced by alkali injury and compared in ARF1 inhibitor- or vehicle-treated mice 3 weeks after injury. Angiogenic and apoptosis factor expression in corneas after injury was quantified with reverse-transcription PCR. Human retinal endothelial cell apoptosis induced by ARF1 inhibitor was detected with flow cytometry. RESULTS: The mRNA expression of ARF1 was augmented in the corneas after alkali injury. Compared with vehicle-treated mice, ARF1 inhibitor-treated mice exhibited impaired CNV 3 weeks after injury, as evidenced by corneal whole mount CD31-staining. Concomitantly, the enhancement of intraocular vascular endothelial growth factor expression was reduced in ARF1 inhibitor-treated mice compared to control mice after injury. Moreover, local administration of the ARF1 inhibitor after alkali injury enhanced intraocular caspase-3 expression. ARF1 inhibitor treatment can significantly induce human retinal endothelial cell apoptosis. CONCLUSIONS: The ARF1 inhibitor can induce the regression of alkali-induced CNV through increased endothelial cell apoptosis and downregulated intracorneal VEGF expression. ARF1 is an effective intervention target for CNV.

Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation.

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/ß Î³ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.

ADP ribosylation factor 1 activity is required to recruit AP-1 to the major histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I trafficking in HIV-1-infected primary T cells.

HIV-1-infected cells are partially resistant to anti-HIV cytotoxic T lymphocytes (CTLs) due to the effects of the HIV Nef protein on antigen presentation by major histocompatibility complex class I (MHC-I), and evidence has been accumulating that this function of Nef is important in vivo. HIV Nef disrupts the normal expression of MHC-I by stabilizing a protein-protein interaction between the clathrin adaptor protein AP-1 and the MHC-I cytoplasmic tail. There is also evidence that Nef activates a phosphatidylinositol 3 kinase (PI3K)-dependent GTPase, ADP ribosylation factor 6 (ARF-6), to stimulate MHC-I internalization. However, the relative importance of these two pathways is unclear. Here we report that a GTPase required for AP-1 activity (ARF-1) was needed for Nef to disrupt MHC-I surface levels, whereas no significant requirement for ARF-6 was observed in Nef-expressing T cell lines and in HIV-infected primary T cells. An ARF-1 inhibitor blocked the ability of Nef to recruit AP-1 to the MHC-I cytoplasmic tail, and a dominant active ARF-1 mutant stabilized the Nef-MHC-I-AP-1 complex. These data support a model in which Nef and ARF-1 stabilize an interaction between MHC-I and AP-1 to disrupt the presentation of HIV-1 epitopes to CTLs.

Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication.

We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.

LG186: An inhibitor of GBF1 function that causes Golgi disassembly in human and canine cells.

Brefeldin A-mediated inhibition of ADP ribosylation factor (Arf) GTPases and their guanine nucleotide exchange factors, Arf-GEFs, has been a cornerstone of membrane trafficking research for many years. Brefeldin A (BFA) is relatively non-selective inhibiting at least three targets in human cells, Golgi brefeldin A resistance factor 1 (GBF1), brefeldin A inhibited guanine nucleotide exchange factor 1 (BIG1) and brefeldin A inhibited guanine nucleotide exchange factor 2 (BIG2). Here, we show that the previously described compound Exo2 acts through inhibition of Arf-GEF function, but causes other phenotypic changes that are not GBF1 related. We describe the engineering of Exo2 to produce LG186, a more selective, reversible inhibitor of Arf-GEF function. Using multiple-cell-based assays and GBF1 mutants, our data are most consistent with LG186 acting by selective inhibition of GBF1. Unlike other Arf-GEF and reported GBF1 inhibitors including BFA, Exo2 and Golgicide A, LG186 induces disassembly of the Golgi stack in both human and canine cells.

Small-molecule inhibitors of protein-protein interactions.

Protein-protein interactions regulate almost all aspects of cellular signaling and aberrant protein-protein interactions have the potential to cause or contribute to human disease. The modulation of these interactions by drug-like molecules would offer previously unavailable opportunities to explore the relevance of pre-selected protein-protein interactions for cellular signaling, as well as benefits to patients. After an initial period of skepticism with regards to feasibility, there is now an encouraging set of data indicating that the effective and selective modulation of protein-protein interactions by drug-like molecules is attainable. This review highlights selected areas of current research that are aimed at identifying potent inhibitors of disease-relevant protein-protein interactions.