Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems

Schmidl, Sina; Ursu, Oleg; Iancu, Cristina V.; Oreb, Mislav; Oprea, Tudor I.; Choe, Juhui

doi:10.1038/s41598-021-93063-5

Cited by 16 publications

(50 citation statements)

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“…Virtual screening involves computational estimation of the optimal binding of a library of potential drugs against targets of interest. This procedure can greatly reduce the time and cost of the drug development compared to in vitro screening alone and has been frequently used to identify compounds with therapeutic potential [21] , [22] , [23] , [24] , [25] . Natural compounds are a valuable source of molecules for drug screening.…”

Section: Introductionmentioning

confidence: 99%

Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein

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et al. 2022

Bioorganic Chemistry

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Section: Introductionmentioning

confidence: 99%

Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein

Power

Turville³

et al. 2022

Bioorganic Chemistry

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“…However, functional studies on the GLUT2∆loopS_Q455R variant in yeast revealed similar substrate specificities and affinities as those of the WT transporter (Schmidl et al, 2021c). Presumably, the large extracellular loop mediates a correct folding and protein trafficking to the membrane in human cells but impedes the same processes in yeast due to differences in the membrane composition and/or signaling reactions.…”

Section: 11mentioning

confidence: 97%

“…screening (Schmidl et al, 2021c), revealing the demand for an additional, subsequent investigation platform. Requirements for such a system are high.…”

Section: Systems For Assaying Glut Activity and Identifying Modulatorsmentioning

confidence: 99%

“…However, the biggest drawback of human cell lines as investigation platform of GLUTs is the endogenous expression of sugar transporters in these cells (Schmidl et al, 2018). This background activity complicates the assessment of the GLUT in question, especially when the latter only shows a low affinity for its substrate, as is the case for GLUT2 (Schmidl et al, 2021c). Xenopus laevis oocytes exhibit a very low endogenous GLUT expression and provide the benefit of a large cell size (enabling electrophysiological investigations) (Long et al, 2018).…”

Section: Animal Cell-based Systemsmentioning

confidence: 99%

“…It is convenient for the application of several assays, including simple growth tests or radiolabeled glucose uptake assays . Furthermore, the hxt 0 system can be applied as a screening platform to examine compound libraries for their effects on the transporter in question as it has been performed for GLUT2, in this thesis (Schmidl et al, 2021c). Whereas for GLUT effectors, medical applications for metabolic diseases or their use in GLUT-related studies are conceivable more areas like the containment of infectious diseases could be tackled by using the hxt 0 screening platform on other transporters.…”

Section: The Hxt 0 Yeast System As a Microbial Investigation Platform For Transportersmentioning

confidence: 99%

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Yeast-based assay systems for screening and characterization of ligands targeting human glucose transporters GLUT2 and GLUT3

Schmidl¹

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Human GLUTs represent a family of specialized transporters that facilitate the diffusion of hexoses through membranes along a concentration gradient. The 14 isoforms share high sequence identity but differ in substrate specificity and affinity, and tissue distribution. According to their structure similarity, GLUTs are divided into three classes, with class 1 comprising the most intensively studied isoforms GLUTs1 4. An abnormal function of different GLUT members has been related to the pathogenesis of various diseases, including cancer and diabetes. Hence, GLUTs are the subject of intensive research, and efforts concentrate on identifying GLUT-selective ligands for putative medical purposes and their application in studies aiming to further unravel the metabolic roles of these transporters. The hexose transporter deficient (hxt0) yeast strain EBY.VW4000 is devoid of all its endogenous hexose transporters and unable to grow on glucose or related hexoses. This strain has proven to be a valuable platform to investigate heterologous transporters due to its easy handling, increased robustness, and versatile applications. However, the functional expression of GLUTs in yeast requires certain modifications. Single point mutations of GLUT1 and GLUT5 led to their functional expression in EBY.VW4000, whereas the native GLUT1 was actively expressed in EBY.S7, a hxt0 strain carrying the fgy1 mutation that putatively reduces the phosphatidylinositol-4-phosphate (PI4P) content in the plasma membrane. GLUT4 was only actively expressed in the hxt0 strain SDY.022, which also contains the fgy1 mutation and in which ERG4 is additionally deleted. Erg4 is one of the late enzymes in the ergosterol pathway, and therefore SDY.022 probably has an altered sterol composition in its membrane. The goal of this thesis was to actively express GLUT2 and GLUT3 in a hxt0 yeast strain, providing a convenient system for their ligand screening. A PCR-derived amino acid exchange in the sequence of GLUT3 enabled its functional expression in EBY.VW4000 and the unmodified GLUT3 protein was active in EBY.S7. Functional expression of GLUT2 was achieved by rational design. The extracellular loop between the transmembrane regions 1 and 2 is significantly larger in GLUT2 than in other class 1 GLUTs. By truncating this loop by 34 amino acids and exchanging an alanine for a serine, a GLUT3-like loop was implemented. The resulting construct GLUT2∆loopS was functional in EBY.S7. With an additional point mutation in the transmembrane region 11, GLUT2∆loopS_Q455R was also actively expressed in EBY.VW4000. Inhibition studies with the known GLUT inhibitors phloretin and quercetin showed a reduced transporter activity for GLUT2 and GLUT3 in uptake assays and growth tests when inhibitors were present, demonstrating that both systems are amenable for ligand screening experiments. The newly established GLUT2 yeast system was then used to screen a library of compounds pre-selected by in silico screening. Thereby, eleven identified GLUT2 inhibitors exhibited strong potencies with IC50 values ranging from 0.61 to 19.3 µM. By employing the other yeast systems, these compounds were tested for their effects on GLUT1, and GLUTs3-5, revealing that nine of the identified ligands were GLUT2-selective. In contrast, one was a pan-class 1 inhibitor (inhibiting GLUTs1-4), and one affected GLUT2 and GLUT5, the two fructose transporting isoforms. These compounds will serve as useful tools for investigations on the role of GLUT2 in metabolic diseases and might even evolve into pharmaceutical agents targeting GLUT2-associated diseases. Due to the beneficial effect of the putatively changed sterol composition in SDY.022 (by ERG4 deletion) on the functional expression of GLUT4, it was hypothesized that the presence of the human sterol cholesterol, or cholesterol-like sterols, might have a beneficial effect on GLUT expression, too. Thus, it was attempted to generate hxt0 strains that synthesize these sterols by genetic modifications targeting the ergosterol pathway. In the scope of these experiments, several strains with different sterol compositions were generated. Drop tests on glucose medium with the different strains expressing GLUT1 or GLUT4 revealed that the deletion of ERG6 is clearly advantageous for a functional expression of GLUT1 (but not GLUT4). This indicates that the methyl group at the ergosterol side chain (introduced by Erg6 and reduced by Erg4) negatively influences GLUT1 activity. However, this effect on GLUT1 activity was less pronounced than the putative altered PI4P content in EBY.S7. Additionally, in this thesis, a new tool to measure glucose transport rates of transporters expressed in the hxt0 yeast system was developed to facilitate their kinetic characterization. For this, the pH-sensitive GFP variant pHluorin was employed as a biosensor for the cytosolic pH (pHcyt) by measuring the ratio (R390/470) of emission intensities at 512 nm from two different excitation wavelengths (390 and 470 nm). Sugar-starved cells exhibit a slightly acidic pHcyt because ATP production is depleted, reducing the activity of ATP-dependent proton pumps. ...

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Structural aspects of the glucose and monocarboxylate transporters involved in the Warburg effect

et al. 2022

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Cancer cells shift their glucose catabolism from aerobic respiration to lactic fermentation even in the presence of oxygen, and this is known as the “Warburg effect”. To accommodate the high glucose demands and to avoid lactate accumulation, the expression levels of human glucose transporters (GLUTs) and human monocarboxylate transporters (MCTs) are elevated to maintain metabolic homeostasis. Therefore, inhibition of GLUTs and/or MCTs provides potential therapeutic strategies for cancer treatment. Here, we summarize recent advances in the structural characterization of GLUTs and MCTs, providing a comprehensive understanding of their transport and inhibition mechanisms to facilitate further development of anticancer therapies.

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Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems

Cited by 16 publications

References 59 publications

Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein

Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein

Yeast-based assay systems for screening and characterization of ligands targeting human glucose transporters GLUT2 and GLUT3

Structural aspects of the glucose and monocarboxylate transporters involved in the Warburg effect

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