Isidoros Vlattas

Mapping the subsite preferences of protein tyrosine phosphatase PTP-1B using combinatorial chemistry approaches.

Protein tyrosine phosphatases (PTPases) are important regulators of signal transduction systems, but the specificity of their action is largely unexplored. We have approached this problem by attempting to map the subsite preferences of these enzymes using combinatorial chemistry approaches. Protein-tyrosine peptidomimetics containing nonhydrolyzable phosphotyrosine analogues bind to PTPases with high affinity and act as competitive inhibitors of phosphatase activity. Human PTP-1B, a PTPase implicated to play an important role in the regulation of growth factor signal transduction pathways, was used to screen a synthetic combinatorial library containing malonyltyrosine as a phosphotyrosine mimic. Using two cross-validating combinatorial chemistry screening approaches, one using an iterative method and the other employing library affinity selection-mass spectrometric detection, peptides with high affinity for PTP-1B were identified and subsite preferences were detailed by quantitatively comparing residues of different character. Consistent with previous observations, acidic residues were preferred in subsites X-3 and X-2. In contrast, aromatic substitutions were clearly preferred at the X-1 subsite. This information supports the concept that this class of enzymes may have high substrate specificity as dictated by the sequence proximal to the phosphorylation site. The results are discussed with regards to the use of combinatorial techniques in order to elucidate the interplay between enzyme subsites.

The use of thioesters in solid phase organic synthesis

Abstract Mercaptan-resin linkers are used for the synthesis of C-terminal thioesters which are then cleaved with alcohols, amines and organometallic reagents to generate esters, amides, ketones, aldehydes and alcohols as the final products of solid phase synthesis.

Inhibition of mevalonate 5-pyrophosphate decarboxylase by a proline-containing transition state analog

Abstract Mevalonate 5-pyrophosphate decarboxylase is involved in the conversion of MevPP to IPP in the biosynthesis of steroids and terpenoids from mevalonate. Based on a hypothetical model of the transition state of this enzymatic transformation we have synthesized novel hydroxyacetyl proline analogs that are potent inhibitors of the enzyme.

Understanding the cellular uptake of phosphopeptides

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells—a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.

Letter to the Editor: Backbone resonance assignment of the 298 amino acid catalytic domain of protein tyrosine phosphatase 1B (PTP1B)

Sebastian Meiera, Yu-Chin Lib, James Koehnb, Isidoros Vlattasc, James Wareingc, Wolfgang Jahnked, Lawrence P. Wennoglec & Stephan Grzesieka,∗ aDivision of Structural Biology and Division of Biophysical Chemistry, Biozentrum, University of Basel, 4056 Basel, Switzerland; bCore Technology Department and cNovartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, 556 Morris Avenue, Summit, NJ 07901-1398, U.S.A.; dCore Technologies, Novartis Pharma AG, CH-4002 Basel, Switzerland

Thia-prostanoid analogs with combined thromboxane receptor antagonist/thromboxane synthase inhibitor activities. Synthesis and pharmacological evaluation

This report describes the incorporation of thia-prostanoid, amino-prostanoid and thromboxane synthase inhibitor features into a new series of thia-prostanoids that combines potent thromboxane receptor antagonist/thromboxane synthase inhibitor activities. Synthesis and biological evaluation is discussed.

OXA-PROSTANOID ANALOGS. IDENTIFICATION OF AN ORALLY EFFECTIVE, DUAL THROMBOXANE RECEPTOR ANTAGONIST/THROMBOXANE SYNTHASE INHIBITOR

Abstract The synthesis and pharmacological evaluation of oxa-prostanoid analogs with dual thromboxane receptor antagonist (TxRA)/thromboxane synthase inhibitor (TxSI) activities are described. One analog 6 with the desirable long lasting oral TxRA/TxSI activity has been identified.