Multiparameter Fluorescence-Activated Cell Sorting of Human Lymphatic Endothelial Cells.

Multiparameter fluorescence-activated cell sorting (FACS) procedure separates target cells from a total population of cells by using specific signatures that the target cell expresses on their cell surface. For human lymphatic endothelial cells (LECs) this relates to cell surface expression of the CD34LowCD31HighVEGFR-3HighPodoplaninHigh profile that permits their separation from blood vascular endothelial cells and other cells likely to be present in the digested tissue sample. In addition, FACS allows the evaluation of LEC size, volume, granularity, and purity at the time of sorting.

α/ß-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and ß-amino acid residues ("α/ß-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/ß-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/ß-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/ß-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/ß-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

Inhibitors of Leishmania GDP-mannose pyrophosphorylase identified by high-throughput screening of small-molecule chemical library.

The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing approximately 80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 microM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.