Extract2Chip—Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance

Funding Information: This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Publisher Copyright: © 2023 by the authors.Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein–protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein–protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.publishersversionpublishe

Structural insights into the early steps of receptor–transducer signal transfer in archaeal phototaxis

Electron paramagnetic resonance-based inter-residue distance measurements between site-directed spin-labelled sites of sensory rhodopsin II (NpSRII) and its transducer NpHtrII from Natronobacterium pharaonis revealed a 2:2 complex with 2-fold symmetry. The core of the complex is formed by the four transmembrane helices of a transducer dimer. Upon light excitation, the previously reported flap-like movement of helix F of NpSRII induces a conformational change in the transmembrane domain of the transducer. The inter-residue distance changes determined provide strong evidence for a rotary motion of the second transmembrane helix of the transducer. This helix rotation becomes uncoupled from changes in the receptor during the last step of the photocycle

A gene-environment study of cytoglobin in the human and rat hippocampus.

BACKGROUND: Cytoglobin (Cygb) was discovered a decade ago as the fourth vertebrate heme-globin. The function of Cygb is still unknown, but accumulating evidence from in vitro studies point to a putative role in scavenging of reactive oxygen species and nitric oxide metabolism and in vivo studies have shown Cygb to be up regulated by hypoxic stress. This study addresses three main questions related to Cygb expression in the hippocampus: 1) Is the rat hippocampus a valid neuroanatomical model for the human hippocampus; 2) What is the degree of co-expression of Cygb and neuronal nitric oxide synthase (nNOS) in the rat hippocampus; 3) The effect of chronic restraint stress (CRS) on Cygb and nNOS expression. METHODS: Immunohistochemistry was used to compare Cygb expression in the human and rat hippocampi as well as Cygb and nNOS co-expression in the rat hippocampus. Transcription and translation of Cygb and nNOS were investigated using quantitative real-time polymerase chain reaction (real-time qPCR) and Western blotting on hippocampi from Flinders (FSL/FRL) rats exposed to CRS. PRINCIPAL FINDINGS: Cygb expression pattern in the human and rat hippocampus was found to be similar. A high degree of Cygb and nNOS co-expression was observed in the rat hippocampus. The protein levels of nNOS and Cygb were significantly up-regulated in FSL animals in the dorsal hippocampus. In the ventral hippocampus Cygb protein levels were significantly up-regulated in the FSL compared to the FRL, following CRS. SIGNIFICANCE: The rodent hippocampus can be used to probe questions related to Cygb protein localization in human hippocampus. The high degree of Cygb and nNOS co-expression gives support for Cygb involvement in nitric oxide metabolism. CRS induced Cygb and nNOS expression indicating that Cygb expression is stress responsive. Cygb and nNOS may be important in physiological response to stress

Ablation of the ASCT2 (SLC1A5) gene encoding a neutral amino acid transporter reveals transporter plasticity and redundancy in cancer cells

The neutral amino acid transporter solute carrier family 1 member 5 (SLC1A5 or ASCT2) is overexpressed in many cancers. To identify its roles in tumors, we employed 143B osteosarcoma cells and HCC1806 triple-negative breast cancer cells with or without ASCT2 deletion. ASCT2ko 143B cells grew well in standard culture media, but ASCT2 was required for optimal growth at < 0.5 mM glutamine, with tumor spheroid growth and monolayer migration of 143B ASCT2ko cells being strongly impaired at lower glutamine concentrations. However, the ASCT2 deletion did not affect matrix-dependent invasion. ASCT2ko 143B xenografts in nude mice exhibited a slower onset of growth and a higher number of small tumors than ASCT2wt 143B xenografts, but did not differ in average tumor size 25 days after xenotransplantation. ASCT2 deficiency was compensated by increased levels of sodium neutral amino acid transporter 1 (SNAT1 or SLC38A1) and SNAT2 (SLC38A2) in ASCT2ko 143B cells, mediated by a GCN2 EIF2alpha kinase (GCN2)-dependent pathway, but this compensation was not observed in ASCT2ko HCC1806 cells. Combined SNAT1 silencing and GCN2 inhibition significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. Similarly, pharmacological inhibition of L-type amino acid transporter 1 (LAT1) and GCN2 significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. We conclude that cancer cells with reduced transporter plasticity are more vulnerable to disruption of amino acid homeostasis than cells with a full capacity to upregulate redundant transporters by an integrated stress response.This work was supported in part by a Merck KGaA speed grant (to S. B.), Australian Research Council Discovery Project Grant DP180101702 (to S. B.), and Cancer Council New South Wales Grants RG17-04 and RG18-06 (to J. H.)

Validation of the immunohistochemistry method.

<p><b>A</b> and <b>B</b> shows Cygb-IR (red) and nNOS-IR (green), respectively, in a coronal section of the rat pituitary gland. <b>C</b> is the merged image of <b>A</b> and <b>B</b>. Note complete separation of the red and green signal showing no false cross reactivity from the secondary antibodies or artifact due to problematic filter settings of the microscope. Scale bar 50 µm.</p

Cygb immunoreactivity and mRNA expression in the rat hippocampus.

<p><b>A</b> Overview of Cygb-IR in the rat hippocampus. High levels of Cygb-IR can be seen in most layers of the hippocampus. The areas within the white square is magnified in <b>B</b> showing Cygb-IR in cell bodies and there processes, marked by white arrows, of CA1 neurons. In <b>C</b> an <i>in situ hybridization</i> of Cygb mRNA expression is shown in the rat hippocampus. Abbreviations: Cornu ammonis1–3 (CA1–3), dentate gyrus (DG), fascio larumcinereum (FC), striatum granulosum of DG (GrDG), molecular layer of DG (MoDG), oriens layer of the hippocampus (Or), polymorph layer of DG (PoDG), pyramidal cell layer (Py), stratum radiatum (Rad). Scale bar 100 µm.</p

Analysis of Cygb and nNOS co-localization.

<p><b>A–C</b> shows high-resolution stacks of the areas from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063288#pone-0063288-g003" target="_blank">figure 3</a>. <b>A</b> Cygb-IR (red) and nNOS-IR (green) in the CA1. Yellow arrowheads denote neurons with co-localized Cygb-IR and nNOS-IR. <b>B</b> shows the stratum radiatum (Rad). Note most nNOS-IR fibers do not co-express Cygb-IR. <b>C</b> shows the dente gyrus (DG). A1–C1 shows calculated co-localization (white). Scale bar 50 µm.</p