Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP.

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.

Colorimetric polymer assay for the diagnosis of plasma lipids atherogenic quality in hypercholesterolemic patients.

OBJECTIVE: Hypercholesterolemia (increased blood cholesterol level) is considered a major risk factor for developing atherosclerotic diseases. As such, alerting individuals on hypercholesterolemic conditions is a crucial component in averting onset of atherosclerosis and its outcome-cardiovascular diseases. While common diagnostic tools such as cholesterol and lipoproteins determination are widely employed for hypercholesterolemia screening, their effectiveness has been questioned since they do not shed light on critical physiological factors like lipid oxidation and inflammation levels, which constitute prominent determinants for development of atherosclerotic diseases. The objective of this study is to develop a simple assay for identifying hypercholesterolemia, and assessing the impact of therapeutic treatments. METHODS: We developed a diagnostic assay based upon color transformations of polydiacetylene, a unique conjugated polymer, upon interactions with blood plasma obtained from healthy individuals, hypercholesterolemic patients, hypercholesterolemic patients treated with statin, and hypercholesterolemic patients treated with statin together with pomegranate extracts. The color transformations of the polymer were monitored through desktop color scanning combined with colorimetric image analysis. RESULTS: We show that the colorimetric assay was able to distinguish among plasma. Bio-analytical characterization reveals that the distinct colorimetric responses likely arise from interactions with plasma lipoproteins. Importantly, the colorimetric changes are not simply correlated with the relative abundance of cholesterol (or other lipids) in the plasma of hypercholesterolemic or healthy patients, but also reflect the presence of oxidized and inflamed species. CONCLUSIONS: This paper introduces a simple color assay for detection of hypercholesterolemia and monitoring the effect of therapies directed at mitigating this physiological condition. The colorimetric system might constitute a novel platform for assessing patient vulnerability towards the development of atherosclerosis.

A novel approach for noninvasive drug delivery and sensing through the amniotic sac.

Current invasive prenatal tests (amniocentesis and chorionic villus sampling) are known for their risk to the fetus. In the last decade, the use and awareness of these prenatal tests have increased, resulting in growing demand for a safe, non-invasive, and accurate prenatal test. Chemical penetration enhancers (CPEs) have long been used to increase transport phenomena across skin and other membranes (e.g., tympanic membrane). The amniotic sac membrane is called the chorioamnion (CA) membrane and serves as the physical barrier between the fetus and the mother. In this research, the effect of CPEs on human CA mass transport was evaluated both in vitro and ex vivo. The results show that the tested CPEs exhibit an enhancing effect on CA mass transport. Based on the permeability results, two mechanisms of action were suggested: "extractors" and "fluidizers". Fourier transform infrared (FTIR) and rapid colorimetric screening measurements supported the mechanisms, based on which, more potent compounds were designed and tested for their enhancing effect. The enhancing mass transport effect of CPEs on CA membrane may be used both for sampling of cell-free DNA and for noninvasively administering drugs and other biological agents to the amniotic sac.

Aggregation of oligoarginines at phospholipid membranes: molecular dynamics simulations, time-dependent fluorescence shift, and biomimetic colorimetric assays.

A time-dependent fluorescence shift method, biomimetic colorimetric assays, and molecular dynamics simulations have been performed in search of explanations why arginine rich peptides with intermediate lengths of about 10 amino acids translocate well through cellular membranes, while analogous lysine rich peptides do not. First, we demonstrate that an important factor for efficient peptide adsorption, as the first prerequisite for translocation across the membrane, is the presence of negatively charged phospholipids in the bilayer. Second, we observe a strong tendency of adsorbed arginine (but not lysine) containing peptides to aggregate at the bilayer surface. We suggest that this aggregation of oligoarginines leads to partial disruption of the bilayer integrity due to the accumulated large positive charge at its surface, which increases membrane-surface interactions due to the increased effective charge of the aggregates. As a result, membrane penetration and translocation of medium length oligoarginines becomes facilitated in comparison to single arginine and very long polyarginines, as well as to lysine containing peptides.