Echelon Biosciences

Abstract Phosphoinositides (PIPs) are an integral part of multiple cell signaling pathways and are metabolized by kinases, phosphatases, and phospholipases. PIPs produced by the activities of phosphoinositide 3-kinase (PI3K) serve as second messengers in cell survival, growth, motility, immune response, inflammation, and apoptosis. Conversely, abnormal production of these signaling lipids leads to proliferative, metabolic, and inflammatory disorders. It is critical to know the absolute and relative concentration of individual PIPs in cell samples, however this analysis is difficult due to their low abundance and the inherent physical properties of lipids. Experiments to measure PIPs have involved radiolabeling cells, extraction of radioactive products, and separation using thin-layer chromatography. The amount of PIPn extracted from cell lysates was determined by means of a standard competitive ELISA format, eliminating the need for radioactivity. Using specific antibodies and binding proteins we developed assays able to differentiate closely related PIPs. These nonradioactive assays employ synthetic di-C16 lipids as standards and are able to measure as little as 0.1 pmol of lipid. Cell-extracted PIPs were added as competitors in these quantitative ELISAs. We used the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient) as a model system to evaluate multiple activators of PI3K pathways by looking at levels of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. Using growth factors, hyperosmolarity, and oxidative stress we found that different stimulants targeted different classes of PI3K. All three classes of PI3K were more active in PTEN deficient cell line (468 cells) than PTEN+/+ (231 cells). We found that all stimulants used increased PI3K products. Pre-incubation with wortmannin, a known PI3K inhibitor, decreased PIPn levels. We propose that hyperactivation of class I and class II PI3K in the absence of PTEN regulation (468 cells) leads to excessive 3’ phosphorylated phosphoinositide levels that are known to activate Akt, We expect the ability to quantify individual phosphoinositides from cells will help assign specific biological functions to these important lipid second messengers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2011-3835

BackgroundAlthough the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective.ResultsHerein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor.ConclusionsThus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.

Short QT Syndrome (SQTS) is a novel clinical entity characterized by markedly rapid cardiac repolarization and lethal arrhythmias. A mutation in the Kir2.1 inward rectifier K⁺ channel (D172N) causes one form of SQTS (SQT3). Pharmacologic block of Kir2.1 channels may hold promise as potential therapy for SQT3. We recently reported that the anti-malarial drug chloroquine blocks Kir2.1 channels by plugging the cytoplasmic pore domain. In this study, we tested whether chloroquine blocks D172N Kir2.1 channels in a heterologous expression system and if chloroquine normalizes repolarization properties using a mathematical model of a human ventricular myocyte. Chloroquine caused a dose- and voltage-dependent reduction in wild-type (WT), D172N and WT-D172N heteromeric Kir2.1 current. The potency and kinetics of chloroquine block of D172N and WT-D172N Kir2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N Kir2.1 condition predicted that 3 μM chloroquine normalized inward rectifier K⁺ current magnitude, action potential duration and effective refractory period. Our results suggest that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3.

Stable phosphoinositide (PIP(n))-containing liposomes were prepared using polydiacetylene photochemistry. Tethered pentacosadiynyl inositol polyphosphate (InsP(n)) analogues of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,4,5)P(4) were synthesized, incorporated into vesicles made up of diyne-phosphatidylcholine and -phosphatidylethanolamine, and polymerized by UV irradiation. The polymerized liposome nanoparticles showed markedly increased stability over conventional PIP(n)-containing vesicles as a result of the covalent conjugated ene-yne network in the acyl chains. The polymerized liposomes were specifically recognized by PIP(n) binding PH domains in liposome overlay assays and amplified luminescent proximity homogeneous assays. Moreover, the biotin moiety allowed attachment of the nanoparticles to a streptavidin-coated sensor chips in surface plasmon resonance (SPR) sensor. The PIP(n) headgroups displayed on SPR sensors showed higher affinities for PH domains and PIP(n) monoclonal antibodies than did monomeric PIP(n)-analogues with biotinylated acyl chains.

Originally discovered as oncogene-associated lipid kinases more than 15 years ago, the family of phosphoinositide 3-kinase (PI 3-K) enzymes has recently emerged as an important therapeutic target in human pathophysiology. After more than a decade with only a few inhibitors with low activity, poor isoform or kinase selectivity or unacceptable pharmacological and toxicological profiles, a variety of PI 3-K inhibitors with pan-isoform activity and isoform-selective inhibition have recently been identified. A growing excitement surrounds the success of signal transduction modifiers in cancer therapy, as well as in the therapy of other diseases. In combination with additional genetic evidence implicating the PI 3-K pathway in human disease, the search for more selective and more drug-like PI 3-K inhibitors has been reinvigorated. As a consequence, many biotech and pharmaceutical companies have established PI 3-K pathway drug development programmes. With the help of important insights provided by genetic knockout animals, potential applications of PI 3-K inhibitors for the treatment of cancer, cardiovascular and endocrine disorders, autoimmune and allergic diseases and inflammation have been recognised. Several potential drugs have now been validated in animal models of human disease but numerous pitfalls still await the translation from animal model to human clinical trials. Herein, the progress in the development of PI 3-K inhibitors and their potential applications in medicine are summarised and the opportunities and concerns inherent in targeting this essential signalling pathway are discussed.

Simultaneous measurements of intracellular pH (pHi) and tension development were made in resting cat papillary muscles and resting ventricular myocytes (cat, guinea pig). pH microelectrodes and the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) were used to measure pHi in muscles and myocytes, respectively. pHi-induced changes in isometric muscle tension and myocyte length were elicited by variations in PCO2, HCO3-, and [NH4Cl]. Increased pHi elevated resting tension and decreased resting cell length, whereas decreased pHi decreased tension and increased cell length. The tension-pHi and cell length-pHi relationships were nonlinear and displayed hysteresis. A reduction in extracellular [Ca2+] from 2.7 to 0.5 mM caused little or no change in the tension and cell length responses to elevated pHi. Ca2+ uptake and/or release by the sarcoplasmic reticulum (SR) does not appear to be involved in the tension response to intracellular alkalosis because the response was unaffected by a postpacing rest period and was not inhibited by ryanodine (5 microM), which depletes SR Ca2+ stores. The cross-bridge inhibitor 2,3-butanedione monoxime (15 mM), but not internal dialysis with 14 mM ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, inhibited myocyte contractures elicited by elevated pHi. The latter finding suggests that factors other than pHi-induced increase in myofilament Ca2+ sensitivity may contribute to the resting contractile response to elevated pHi.