Salicylketoximes as inhibitors of Glucose Transporters

Some derivatives of the 4-arylsalicylketoximes series displayed inhibitory effects on glucose transport and on cell proliferation in several biological assays,[1] resulting to be effective GLUT1 inhibitors also in GLUT1-containing giant vesicles. GLUT1 is one of the 14 glucose transporter isoforms, widely overexpressed in many cancer types. Thus, for the discovered properties, the oximes of interest represent interesting candidates for anticancer therapy. Variously substituted 4-arylsalicylketoximes (3, Fig.1) were synthetized via Suzuki cross-coupling and a subsequent condensation of the resulting biaryl-ketone intermediates with hydroxylamine hydrochloride. [1] Rat GLUT1 membrane proteins were produced by Pichia Pastoris cultures, and purified following GLUT1 purification protocols, [2] which were largely revised to avoid the protein cleavage. Compounds 3a, 3b, 3e, and 3f efficiently inhibited glucose uptake in GLUT1-containing giant vesicle assays. [3] To study the nature of the binding process between GLUT1 and the synthetic compounds, many crystallization attempts were set up with 3a and 3e using Lipidic Cubic Phase method, which produced many small crystals. Since many isoforms of GLUTs are overexpressed in cancer cells, inhibition of other GLUT isoforms, such as GLUT3, will be tested in the near future. In conclusion, 4-arylsalicylketoximes showed good inhibition of GLUT1 isoform. First results from GLUT3-giant vesicles assays revealed that, within this series of compounds, 3a is the most selective GLUT1-inhibitor. Further assays with GLUTs-containing giant vesicle and crystallization attempts are currently underway. [1] Granchi C, Qian Y, Lee H.Y, Paterni I, Pasero C, Iegre J, Carlson K. E, Tuccinardi T, Chen X, Katzenellenbogen J. A, Hergenrother P. J, Minutolo F, ChemMedChem. 2015; 1892–1900. [2] Venskutonyté R, Elbing K, Lindkvist-Petersson K, Methods Mol Biol. 2018; 1713, 1–13. [3] Hansen J.H, Elbing K, Thompson J.R, Malmstadt N, Lindkvist-Petersson K, Chem. Commun. 2015; 51, 2316–2319

Glucose transporters: production, crystallization and inhibition

Glucose transporters (GLUTs) comprise a family of 14 membrane proteins that regulate glucose uptake into the cell. Different types of GLUTs are expressed in various tissues and play a crucial role in glucose metabolism. Cancer cells are highly dependant on glucose and therefore GLUTs are possible drug targets for cancer therapy. In order to block the glucose uptake facilitated by GLUTs, various inhibitors are studied and both natural and synthetic compounds having an inhibitory effect on glucose uptake have been discovered. High resolution X-ray structure of the GLUT-inhibitor complex would provide a detailed understanding of protein-inhibitor interactions and contribute to facilitating the development of new derivatives. The focus of this study is on a glucose transporter 1 (GLUT1). The GLUT1 has been produced and crystallization trials set up, which resulted in microcrystals. A series of salicylketoxime based compounds have been shown to inhibit GLUT1 and two lead compounds displaying the highest inhibition have been identified in a giant vesicle assay. The main goal of the study is to determine the structure of the GLUT1 with selected inhibitors. Moreover, studies on one more glucose transporter GLUT3 are carried out to investigate the selectivity of the salcylketoxime compounds

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Atomic-resolution X-ray crystallography, functional analyses, and molecular dynamics simulations suggest a novel mechanism for the regulation of water flux through the yeast Aqy1 water channel

Expression and purification of rat glucose transporter 1 in Pichia pastoris

Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods

Glucose transport activity measured in giant vesicles

Incorporation of membrane proteins and internal reporter systems directly into giant vesicles, during their formation from a hydrogel surface, has emerged as a promising new concept in membrane protein characterization. Here, we provide the detailed protocol for a glucose transporter activity assay based on giant vesicles containing a fluorescent enzyme-linked reporter system internally. This assay is applicable for the functional analysis of a variety of hexose-transporting proteins. We furthermore believe that it can aid in the development of drugs targeting hexose transporters

Structure of Staphylococcal Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen Recognition

<div><p>T cells are crucial players in cell-mediated immunity. The specificity of their receptor, the T cell receptor (TCR), is central for the immune system to distinguish foreign from host antigens. Superantigens are bacterial toxins capable of inducing a toxic immune response by cross-linking the TCR and the major histocompatibility complex (MHC) class II and circumventing the antigen specificity. Here, we present the structure of staphylococcal enterotoxin E (SEE) in complex with a human T cell receptor, as well as the unligated T cell receptor structure. There are clear structural changes in the TCR loops upon superantigen binding. In particular, the HV4 loop moves to circumvent steric clashes upon complex formation. In addition, a predicted ternary model of SEE in complex with both TCR and MHC class II displays intermolecular contacts between the TCR α-chain and the MHC, suggesting that the TCR α-chain is of importance for complex formation.</p></div

Correction: Thermal stability and structural changes in bacterial toxins responsible for food poisoning.

[This corrects the article DOI: 10.1371/journal.pone.0172445.]