The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60C-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and pl85HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosinephosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the maEjor sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp6(C-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.Signaling mediated by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), requires receptor autophosphorylation on tyrosine (1). These phosphotyrosine residues serve as unique binding sites for proteins that contain SRC homology 2 (SH2) domains. This protein motif recognizes phosphotyrosine in a sequence-specific manner, and the in vivo specificity of proteins for their cognate phosphotyrosine residue in the receptor is maintained in in vitro binding assays using the isolated SH2 domains of these proteins. Such domains are found in a number of proteins involved in intracellular signaling including p85, the noncatalytic subunit of phosphatidylinositol 3-kinase; GAP, the GTPase-activating protein of p2lras; and phospholipase Cy (2). Constitutive activation of these signaling pathways is apparent in many malignancies. Human breast cancers often overexpress two closely related receptor tyrosine kinases, EGFR or p185HER2/neu, and amplification of these genes is correlated with poor clinical prognosis (3-5). It has recently been reported that many primary human breast tumors also show elevated activity of pp6c-src (6), suggesting that this protein may play a role in carcinoma of the breast. These results indicate that activation of downstream events mediated by both receptor and nonreceptor tyrosine kinases may be critical in some types of human neoplasia. Evidence for similar interactions between receptor and nonreceptor tyro-The publication costs of this article were defrayed in part by page charge payment. This article must the...
The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a >2-log 10 reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC 90 , 0.015 g/ml) and coagulase-negative staphylococci (MIC 90 , 0.12 g/ml), regardless of their drug resistance, hospital-or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are equipotent to CCK as anorectic agents in rats following intraperitoneal administration.
Drug discovery increasingly relies on the ability to rapidly identify "quality" molecules that possess the desired attributes of bioavailability, chemical tractability, selectivity, and potency. Traditional methods used to determine the pharmacokinetics (oral bioavailability, clearance, volume of distribution, and half-life) of molecules have presented a bottleneck in some drug discovery programs. We have increased throughput in in vivo pharmacokinetic screening of structurally related compounds by dosing mixtures of compounds intravenously to a single animal and using atmospheric pressure ionization (API) tandem liquid chromatography/ mass spectrometry (LC/MS/MS) for analysis. We have referred to this as N-in-one dosing where N is the number of compounds coadministered. The method was used to simultaneously determine the clearance (CL), steady-state volume of distribution (V ss ), and elimination half-life (t 1/2 ) of five R 1a receptor antagonists (compounds possessing selective R 1a antagonist properties may have potential therapeutic importance in the treatment of benign prostatic hyperplasia). 1-3 The mixture approach provides an opportunity to study the pharmacokinetics of several compounds under identical conditions while minimizing sample processing time and the number of animals required. To the best of our abilities, we found limited literature that capitialized on the advantages gained through simultaneously dosing compounds to determine pharmacokinetics and bioavailability after intravenous and intraduodenal administration, 4,5 and one of these utilized LC/MS. 6 Five R 1a receptor antagonists ( Figure 1) that had been previously studied by individual dosing (known range of CL, V ss , and t 1/2 ) were dosed intravenously as a mixture to a single dog. In the traditional individual dosing studies, plasma samples were analyzed for R 1a antagonist by reverse-phase HPLC with fluorescence detection. Plasma sample analysis of the R 1a antagonists in the mixture study relied heavily on the application of atmospheric pressure ionization (API) LC/MS methodology using a triple quadrupole instrument. LC/ MS with its inherent detection specificity, selectivity, and sensitivity enabled rapid analytical method development prior to dosing the animal as well as high throughput sample analysis.The compounds studied exhibited good mass spectrometric response in the positive ion mode using the API technique. To increase analyte specificity from the biological matrix, a reverse-phase LC/MS/MS method was developed. For this series of compounds, a characteristic neutral loss of CF 3 CH 2 OH (100 amu) was observed in the product ion mass spectra. This transition was optimized for sensitivity in the selected reaction monitoring (SRM) mode. Among the compounds studied, a pair of isobars (compounds 2 and 3 of molecular weight 638) produced the same abundant fragment ion at m/z 539. Consequently, the HPLC mobile phase conditions were optimized to resolve compounds 2 and 3, extending the analysis time to 6 min. An internal standar...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.