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Biomedical subjects

B A Lynch

Publications and source records attributed to B A Lynch.

9 recordsLinked to original sources
PubMed1999

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.

Animals↗
PubMed1999

Discovery of potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70.

A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.

Enzyme Inhibitors↗
PubMed1999

Structure-activity relationships of a novel class of Src SH2 inhibitors.

The structure-activity relationships (SAR) of a novel class of Src SH2 inhibitors are described. Variation at the pY+1 and pY+3 side chain positions using 2,4- and 2,5-substituted thiazoles and 1,2,4-oxadiazoles as scaffolds resulted in inhibitors that bound as well as the standard tetrapeptide Ac-pYEEI-NH2.

Structure-Activity Relationship↗
PubMed1997

A fluorescence polarization based Src-SH2 binding assay.

The tyrosine kinase pp60c.src has been implicated as being a potential therapeutic target in several human diseases including cancer and osteoporosis. An important region within this kinase is the SH2 domain (Src homology 2) which binds to phosphorylated tyrosine residues contained within specific peptide sequences. Homologous domains are found in a variety of cytoplasmic proteins and have been shown to be essential for controlling many important signaling pathways. Developing specific inhibitors of SH2 interactions would therefore be extremely useful for modulating a variety of signaling pathways and potentially be useful for the treatment of human disease. Current methodology for the development of organic molecules as drug leads requires the ability to test thousands of individual compounds or natural product extracts in biochemical assays. Such tests must be reproducible, simple, and versatile. This paper describes an assay based on fluorescence polarization for measuring the binding of compounds to the Src-SH2 domain. The assay is insensitive to changes in fluorescence intensity working even in solutions with moderate optical density and functions in the presence of up to 20% dimethyl sulfoxide. These features make it especially useful for high-throughput screening of both natural and synthetic compound libraries.

Amino Acid Sequence↗
PubMed1992

The fifth Datta Lecture. Structural similarities between the aspartate receptor of bacterial chemotaxis and the trp repressor of E. coli. Implications for transmembrane signaling.

A high resolution structure of the N-terminal ligand-binding domain of the aspartate receptor which mediates aspartate chemotaxis in Salmonella typhimurium has recently been reported. A least-squares superposition of the alpha-amino nitrogen, alpha-carbon, beta-carbon, and alpha-carboxylate carbon of the aspartate bound to the aspartate receptor onto the equivalent atoms in the tryptophan bound to the trp repressor provides evidence for similarity between key parts of the active sites that bind to the alpha-amino and alpha-carboxylates of the respective ligands. Because the N-terminal domain of the aspartate receptor and the trp repressor also share other structural similarities, we hypothesize that the similarity between the aspartate receptor and the trp repressor derives from a similarity in ligand-induced conformational changes at the active sites of these proteins. This hypothesis also implies that an important signaling event in the aspartate receptor occurs through tertiary conformational changes within a single subunit.

Aspartic Acid↗
PubMed1991

Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli.

The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS. These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function.

Amino Acid Sequence↗
PubMed1985

Effects of low protein diets or feed restriction on rat lung glutathione and oxygen toxicity.

Male rats weighing 200-250 g were fed a 25% casein diet in restricted amounts or ad libitum or one of two low protein diets (3 and 0% casein) ad libitum. Decreased tolerance to hyperoxic stress was observed only in the rats fed low protein diets. These animals had a median death time of 49-50 h compared to 58-69 h for feed-restricted or normal control groups. Death was due to accelerated development of lung edema. Changes in total lung levels of glutathione reductase, glucose-6-phosphate dehydrogenase or catalase did not correlate with oxygen sensitivity. Lung glutathione levels were related to the amount of sulfur-containing amino acids in the diet and were depressed in the feed-restricted as well as the protein-restricted groups. However, feed restriction alone did not enhance oxygen toxicity. We conclude that a decrease in lung glutathione may be partially responsible for the increased oxygen sensitivity in the protein-deficient rats, but that other factors are necessary for explanation of the relative oxygen tolerance of the feed-restricted animals with reduced levels of glutathione in the lung.

Amino Acids↗
PubMed1985

Transient depletion of lung glutathione by diethylmaleate enhances oxygen toxicity.

Diethylmaleate (DEM) decreases glutathione (GSH) levels in various organs by enzymatic conjugation with reduced GSH catalyzed by GSH transferase. We have examined levels of GSH, glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PD) in lungs of 200-250-g rats after intraperitoneal injection of 0.5 or 1 g DEM/kg body wt. The GSH levels are severely depressed at 2 and 4 h but have essentially recovered by 12 and 24 h after either dose of DEM. The GR and G6PD activities in the 1 g/kg group are depressed at 4 h to a lesser extent than the GSH levels and also return to normal by 12 and 24 h. These enzymes are not affected in the 0.5 g/kg group. To determine whether these transient decreases in GSH and related enzymes affected O2 tolerance, we exposed rats injected with DEM to greater than 98% O2 and found that halftime (t1/2) for survival was decreased in rats receiving both 0.5 and 1 g DEM/kg body wt when compared with untreated or saline-injected controls (t1/2 control, 74 h; 0.5 g DEM, 59 h; 1 g DEM, 53 h). No deaths occurred in air controls at 1 mg/kg DEM for up to 5 days. DEM, in itself, caused no morphological alteration of the lung. Thus a decrease in lung GSH and related enzymes, occurring by 4 h and reversed by 12 h, has a significant effect on the subsequent progression of lung pathology and indicates that early biochemical events occurring in lungs exposed to hyperoxia may be very important in determining the degree of longer-term damage to rat lungs.

Animals↗