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.

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.

Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection.

Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Their use has greatly facilitated the study of protein-protein interactions. We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates. The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of individual components without the need for chemical tags. This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine. The described method has broad applicability to combinatorial library screening.

Characterization of the S3 subsite specificity of cathepsin B.

Five synthetic substrates containing different amino acid residues at the P3 position (acetyl-X-Arg-Arg-AMC, where X is Gly, Glu, Arg, Val, and Tyr and where AMC represents 7-amindo-4-methylcoumarin) were used to investigate the S3 subsite specificity of cathepsin B. At pH 6.0, the specificity constant, kcat/Km, for tripeptide substrate hydrolysis was observed to increase in the order Glu < Gly < Arg < Val < Tyr. Molecular modeling studies of substrates containing a P3 Glu, Arg, or Tyr covalently bound as the tetrahedral intermediate to the enzyme suggest that the specificity for a P3 Tyr is because of a favorable aromatic-aromatic interaction with Tyr75 on the enzyme as well as a possible H bond between the P3 Tyr hydroxyl and the side chain carboxyl of Asp69.

Identification of a receptor-binding region in the core segment of the human anaphylatoxin C5a.

In order to identify regions of C5a that contribute to receptor binding and functional activity of the anaphylatoxin, a series of peptides was synthesized in which core segments have been attached to C-terminal segments via native peptidic or disulfide bonds. It has been found that residues Arg40 and Arg46 in the loop-3 region of the core induce a 1000-fold increase in the affinity of the disordered C-terminal segment of C5a. The results obtained from this work lead to the conclusion that the loop-3 region is most likely the core binding site of C5a.

Solvent effects on the conformation of cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-). An NMR spectroscopy and molecular modeling study.

The conformations of cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-) in dimethyl sulfoxide-d6 (DMSO-d6) and water were determined using two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Comparisons were made between conformations of the cyclic pentapeptide in both solvents. The NMR study revealed that, while the backbone remained relatively unchanged in both solvents, the side-chains adopted distinctly different orientations in DMSO-d6 vs. H2O. A modeling study, minus NOE constraints, produced a set of low-energy conformers possessing agreement in backbone conformation with the NMR-derived structures; however, lowest-energy conformers did not have this agreement. These results show that different solvents can significantly affect the preferred side-chain conformation of small cyclic peptides in solution. This finding will impact the selection of solvent when determining structures for use as templates in rational drug design.

A series of hexahydro[1,4]oxazino[3,4-a]isoquinolines as potential neuroleptics.

The synthesis and stereochemistry of trans-N,N-diethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro[1,4]oxazino[3,4-a]isoquinoline-3-carboxamide hydrochloride (16) and a series of analogues are described. 16 and its (+) isomer had neuroleptic properties in the Sidman avoidance test in gerbils. A few closely related amides of the trans series were active but cis amides were inactive as neuroleptics.

Total synthesis of natural (levo) and enantiomeric (dextro) forms of prostaglandin El.

PIP: 1 newly-discovered method of synthesizing PGE1 (prostaglandin E1) is explained and diagrammed chemically. Recrystallization of the synthetic product produced material identical in all respects with natural PGE1. The synthetic product showed the same infrared spectrum and chromatographic values as racemic and natural forms of PGE1. The biological activity of the synthetic product was found to be .1% of that of the naturally-occurring PGE1 in the stimulation of smooth muscle contraction. This is the 1st report of a total synthesis of PGE1. Research continues on improvements in this synthesizing method and on discovering other synthetic approaches to PGs.^ieng