Honglu Zhang

Dual Activity Lysophosphatidic Acid Receptor Pan-Antagonist/Autotaxin Inhibitor Reduces Breast Cancer Cell Migration In vitro and Causes Tumor Regression In vivo

Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric alpha-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein-coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg.

Phosphatidylinositol 4,5-bisphosphate regulates inspiratory burst activity in the neonatal mouse preBötzinger complex.

Neurons of the preBötzinger complex (preBötC) form local excitatory networks and synchronously discharge bursts of action potentials during the inspiratory phase of respiratory network activity. Synaptic input periodically evokes a Ca2+‐activated non‐specific cation current (ICAN) postsynaptically to generate 10–30 mV transient depolarizations, dubbed inspiratory drive potentials, which underlie inspiratory bursts. The molecular identity of ICAN and its regulation by intracellular signalling mechanisms during inspiratory drive potential generation remains unknown. Here we show that mRNAs coding for two members of the transient receptor potential (TRP) family of ion channels, namely TRPM4 and TRPM5, are expressed within the preBötC region of neonatal mice. Hypothesizing that the phosphoinositides maintaining TRPM4 and TRPM5 channel sensitivity to Ca2+ may similarly influence ICAN and thus regulate inspiratory drive potentials, we manipulated intracellular phosphatidylinositol 4,5‐bisphosphate (PIP2) and measured its effect on preBötC neurons in the context of ongoing respiratory‐related rhythms in slice preparations. Consistent with the involvement of TRPM4 and TRPM5, excess PIP2 augmented the inspiratory drive potential and diminution of PIP2 reduced it; sensitivity to flufenamic acid (FFA) suggested that these effects of PIP2 were ICAN mediated. Inositol 1,4,5‐trisphosphate (IP3), the product of PIP2 hydrolysis, ordinarily causes IP3 receptor‐mediated ICAN activation. Simultaneously increasing PIP2 while blocking IP3 receptors intracellularly counteracted the reduction in the inspiratory drive potential that normally resulted from IP3 receptor blockade. We propose that PIP2 protects ICAN from rundown by interacting directly with underlying ion channels and preventing desensitization, which may enhance the robustness of respiratory rhythm.

Inositol polyphosphates, diphosphoinositol polyphosphates and phosphatidylinositol polyphosphate lipids: Structure, synthesis, and development of probes for studying biological activity

Covering: 2006 up to June 2010, but also including representative examples of prior work Polyphosphorylated myo-inositol compounds, including the inositol polyphosphates (InsPs), diphosphoinositol polyphosphates (PP-InsPs), and phosphatidylinositol polyphosphates (PIPns), represent key biomolecules that regulate a litany of critical biological processes. These compounds exist with myriad combinations of phosphorylation patterns, resulting in a complex network of interconverting signaling molecules that control different events. Due to the significance and intricate nature of this molecular family, the elucidation of biological roles has elicited substantial interest in both the biochemical and chemical communities. Within this broad effort, strategies employing chemical synthesis for the production of both natural products and chemically modified structures have proven advantageous for determining activities. Herein, we will discuss recent advancements in these efforts, including (i) a brief overview of structure and biological activity, (ii) current methods for the chemical synthesis of phosphorylated myo-inositols, (iii) strategies for the design of biologically active probe structures, and (iv) case studies in which synthetic probes have been applied to characterize biological properties.

Phosphatase-Resistant Analogues of Lysophosphatidic Acid: Agonists Promote Healing, Antagonists and Autotaxin Inhibitors Treat Cancer

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and also biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues also are feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, a.k.a., autotaxin, ATX), a central regulator of invasion and metastasis. For cancer therapy, the optimal therapeutic profile would be a metabolically-stabilized, pan-LPA receptor antagonist that also inhibited lysoPLD. For protection of gastrointestinal mucosa and lymphocytes, LPA agonists would be desirable to minimize or reverse radiation or chemical-induced injury. Analogues of lysophosphatidic acid (LPA) that are chemically modified to be less susceptible to phospholipases and phosphatases show activity as long-lived receptor-specific agonists and antagonists for LPA receptors, as well as inhibitors for the lysoPLD activity of ATX.

A Scalable Synthesis of the IP7 Isomer, 5-PP-Ins(1,2,3,4,6)P5

The phosphorylated inositol diphosphates, including the diphosphoinositol pentakisphosphate regioisomers, play critical roles in signal transduction and cellular regulation. In particular, the IP(7) isomer 5-PP-Ins(1,2,3,4,6)P(5) is implicated in a nonenzymatic phosphate transfer converting a protein serine phosphate residue to a serine diphosphate. A scalable, practical new synthesis of 5-PP-Ins(1,2,3,4,6)P(5) is described that also allows access to a variety of IP(7) and IP(8) regioisomers. The identity of the synthetic 5-PP-Ins(1,2,3,4,6)P(5) was validated using IP6K1 to catalyze the conversion of IP(7) + ADP to ATP + IP(6).

Fluorometric Detection of Inositol Phosphates and the Activity of Their Enzymes with Synthetic Pores: Discrimination of IP7 and IP6 and Phytate Sensing in Complex Matrices

We report the fluorometric and noninvasive detection of inositol phosphates, which act as privileged blockers of synthetic pores. Phytate (IP6) and IP7 recognition in the pore occurs substoichiometrically in the low nanomolar range, with more than 2 orders of magnitude higher sensitivity than the best available alternative. Zn2+-mediated fluorometric discrimination between IP6 and IP7 demonstrates that significant pore discrimination challenges can be solved with judiciously selected additives. The detectability of inositol phosphate enzyme activity was exemplified with phytase. Phytate sensing was accomplished in complex matrices such as extracts from almonds, soybeans, or lentils, using phytase as a specific signal generator. These results are important because they not only add essential evidence in support of the usefulness of synthetic pores as multianalyte sensors in complex matrices but also reveal the existence of privileged analytes that can provide access to submicromolar sensitivity without the...

5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration.

Metabolically stabilized analogues of PtdIns(3,4,5)P3 have shown long‐lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5‐phosphatase‐resistant analogues of PtdIns(3,4,5)P3, the 5‐methylene phosphonate (MP) and 5‐phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P3 analogues in four contexts. First, the relative binding affinities of the 3‐MP, 3‐PT, 5‐MP, 5‐PT, and 3,4,5‐PT3 analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P3. Third, exogenously delivered analogues severely impair complement factor C5a‐mediated polarization and migration of murine neutrophils. Finally, the new analogues show long‐lived agonist activity in mimicking insulin action in sodium transport in A6 cells.

Tissue-Engineered “Metastases”: Treatment of Hepatic Colon Tumors with a Dual Action Autotaxin Inhibitor-Lysophosphatidic Acid Receptor Antagonist

Lysophosphatidic acid (LPA) acts via G protein coupled receptors (GPCRs) to regulate critical cellularfunctions and pathophysiological levels of LPA or its receptors are linked to cancer initiation, progression andmetastasis. LPA is biosynthesized by the lysophospholipase D activity of autotaxin (ATX/lysoPLD), a known factor fortumorigenesis. By attenuating both LPA signaling and LPA production, we expected to observe synergistic anti-cancertherapeutic effects. In vitro, treatment of human colon cancer cells (HCT 116) with BrP-LPA, a potent dual action ATXinhibitor and pan-LPA GPCR antagonist, significantly reduced cell proliferation, migration and invasion. Next, a tissueengineeredxenograft model to mimic hepatic metastasis of colon cancer was used to evaluate BrP-LPA efficacy in vivo.HCT 116 cells were suspended in Extracel™, a synthetic extracellular matrix (sECM), and injected directly into the liversof nude mice (n = 8). After 1 week, BrP-LPA in saline buffer was administered for two weeks by intraperitoneal injection(10 mg/kg) twice per week. Controls were injected with saline buffer only. The BrP-LPA treated group showed reducedliver tumor weight (p < 0.05) and reduced tumor volume (p < 0.05) relative to controls. This study is the firstdemonstration of the effects of a dual action ATX inhibitor/LPA antagonist on colon cancer cells, and the first example ofa tissue-engineered hepatic colon cancer “metastases” as a platform for anti-cancer drug evaluation. The results suggestthat attenuation of signaling through the LPA pathway offers a promising therapeutic target for reducing colon cancergrowth and metastasis.