Integrating chemical and genetic silencing strategies to identify host kinase-phosphatase inhibitor networks that control bacterial infection.

Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics. These antibiotics target almost all essential bacterial processes, leaving only a few new targets for manipulation. The host proteome has many more potential targets for manipulation in order to control bacterial infection, as exemplified by the observation that inhibiting the host kinase Akt supports the elimination of different intracellular bacteria including Salmonella and M. tuberculosis. If host kinases are involved in the control of bacterial infections, phosphatases could be as well. Here we present an integrated small interference RNA and small molecule screen to identify host phosphatase-inhibitor combinations that control bacterial infection. We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27. Pathway analysis places many kinases and phosphatases controlling bacterial infection in an integrated pathway centered around Akt. This network controls host cell metabolism, survival, and growth and bacterial survival and reflect a natural host cell response to bacterial infection. Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.

Necessity of lysophosphatidic acid receptor 1 for development of arthritis.

OBJECTIVE: Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein-coupled receptors (LPA receptors 1-6 [LPA1-6 ]) and has been implicated as an important mediator of angiogenesis, inflammation, and cancer growth. This study was undertaken to analyze the effects of LPA1 on the development of arthritis. METHODS: Expression of LPA receptors on synovial tissue was analyzed by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The effects of abrogation of LPA1 on collagen-induced arthritis (CIA) were evaluated using LPA1 -deficient mice or LPA1 antagonist. Migrating fluorescence-labeled CD11b+ splenocytes, which were transferred into the synovium of mice with CIA, were counted. CD4+ naive T cells were incubated under Th1-, Th2-, or Th17-polarizing conditions, and T helper cell differentiation was assessed. Osteoclast formation from bone marrow cells was examined. RESULTS: LPA1 was highly expressed in the synovium of patients with rheumatoid arthritis (RA) compared with that of patients with osteoarthritis. LPA1 -deficient mice did not develop arthritis following immunization with type II collagen (CII). LPA1 antagonist also ameliorated murine CIA. Abrogation of LPA1 was associated with reductions in cell infiltration, bone destruction in the joints, and interleukin-17 production from CII-stimulated splenocytes. Infiltration of transferred CD11b+ macrophages from LPA1 -deficient mice into the synovium was suppressed compared with infiltration of macrophages from wild-type mice. LPA1 antagonist inhibited the infiltration of macrophages from wild-type mice. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed under conditions of LPA1 deficiency or LPA1 inhibition in vitro. CONCLUSION: Collectively, these results indicate that LPA/LPA1 signaling contributes to the development of arthritis via cellular infiltration, Th17 differentiation, and osteoclastogenesis. Thus, LPA1 may be a promising target molecule for RA therapy.

Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish.

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffer's vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of ß-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.

Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice.

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.

Structure-based design of novel boronic acid-based inhibitors of autotaxin.

Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug target. We recently described a boronic acid-based inhibitor of ATX, named HA155 (1). Here, we report the design of new inhibitors based on the crystal structure of ATX in complex with inhibitor 1. Furthermore, we describe the syntheses and activities of these new inhibitors, whose potencies can be explained by structural data. To understand the difference in activity between two different isomers with nanomolar potencies, we performed molecular docking experiments. Intriguingly, molecular docking suggested a remarkable binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for inhibitor design.

Controlled supramolecular oligomerization of C3-symmetrical molecules in water: the impact of hydrophobic shielding.

The supramolecular oligomerization of three water-soluble C(3)-symmetrical discotic molecules is reported. The compounds all possess benzene-1,3,5-tricarboxamide cores and peripheral Gd(III)-DTPA (diethylene triamine pentaacetic acid) moieties, but differ in their linker units and thus in their propensity to undergo secondary interactions in H(2)O. The self-assembly behavior of these molecules was studied in solution using circular dichroism, UV/Vis spectroscopy, nuclear magnetic resonance, and cryogenic transmission electron microscopy. The aggregation concentration of these molecules depends on the number of secondary interactions and on the solvophobic character of the polymerizing moieties. Hydrophobic shielding of the hydrogen-bonding motif in the core of the discotic is of paramount importance for yielding stable, helical aggregates that are designed to be restricted in size through anti-cooperative, electrostatic, repulsive interactions.

Structural basis of substrate discrimination and integrin binding by autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Development of an activity-based probe for autotaxin.

Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D that hydrolyses lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX has been implicated in tumour progression and inflammation, and might serve as a biomarker. Here we describe the development of a fluorescent activity-based probe that covalently binds to the active site of ATX. The probe consists of a lysophospholipid-based backbone linked to a trapping moiety that becomes reactive after phosphate ester hydrolysis, and a Cy5 fluorescent dye to allow visualisation of active ATX. The probe reacts specifically with the three known isoforms of ATX, it competes with small-molecule inhibitors for binding to ATX and allows ATX activity in plasma to be determined. Our activity-based reporter will be useful for monitoring ATX activity in biological fluids and for inhibitor screening.

Discovery and optimization of boronic acid based inhibitors of autotaxin.

Autotaxin (ATX) is an extracellular enzyme that hydrolyzes lysophosphatidylcholine (LPC) to produce the lipid mediator lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in diverse physiological and pathological processes, including vascular development, inflammation, fibrotic disease, and tumor progression. Therefore, targeting ATX with small molecule inhibitors is an attractive therapeutic strategy. We recently reported that 2,4-thiazolidinediones inhibit ATX activity in the micromolar range. Interestingly, inhibitory potency was dramatically increased by introduction of a boronic acid moiety, designed to target the active site threonine in ATX. Here we report on the discovery and further optimization of boronic acid based ATX inhibitors. The most potent of these compounds inhibits ATX-mediated LPC hydrolysis in the nanomolar range (IC(50) = 6 nM). The finding that ATX can be targeted by boronic acids may aid the development of ATX inhibitors for therapeutic use.

Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation.

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC(50) approximately 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.