A preparative chiral separation of hydroxychloroquine using supercritical fluid chromatography

Wilson, Laura J.; Mi, Charles; Kraml, Christina M.

doi:10.1016/j.chroma.2020.461661

Cited by 11 publications

(6 citation statements)

References 13 publications

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“…Separated enantiomers of racemic SW016789 were obtained from Lotus Separations using supercritical fluid chromatography [2]. Purified enantiomers were resuspended in DMSO and subjected to acute and chronic InsGLuc-MIN6 experiments as described.…”

Section: Esm Methodsmentioning

confidence: 99%

Small molecule-mediated insulin hypersecretion induces transient unfolded protein response and loss of function in pancreatic islet beta cells

Rodrigues-dos-Santos

Roy

Binns

et al. 2022

Preprint

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Aims/hypothesisInsulin production and its regulated exocytosis by pancreatic islet beta cells is the major physiological mechanism controlling blood glucose concentrations. Beta cells are susceptible to failure due to genetic and environmental influences, leading to type 1 and 2 diabetes. In this work we aimed to advance understanding of the regulation of beta-cell function by identifying small molecule modulators of secretion.MethodsFor high-throughput screening and routine medium-throughput beta cell function assays we used mouse MIN6 beta cells stably expressing a Gaussia luciferase-linked insulin secretion reporter (InsGLuc-MIN6). We validated hit small molecules using cadaveric human islets in static culture insulin secretion assays. We measured effects on calcium influx and cAMP in MIN6 cells using Fura-2 and a bioluminescence resonance energy transfer reporter assay, respectively. Metabolism was analyzed by targeted metabolomics and Seahorse oxygen consumption assays. We measured relative gene expression and protein amounts by quantitative RT-PCR and SDS-PAGE immunoblotting, respectively.ResultsThrough our high-throughput screen we discovered 2-(3-benzyl-2-iminobenzimidazol-1-yl)-1-thiophen-2-ylethanol (SW016789) which acutely potentiated nutrient-induced calcium influx and insulin secretion. More than 2 hours of exposure to SW016789 transiently induced the unfolded protein response of the endoplasmic reticulum and shut down of insulin secretory function. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or death as determined by multiplexed cytotoxicity assays and a lack of induction of cleaved PARP. This may be due in part to a more rapid induction of Hspa5, and a lesser induction of signalling through the eIF2α kinase PERK and lack of expression of oxidative stress genes like Txnip. We determined that SW016789 acted upstream of the voltage-dependent calcium channel (VDCC) and potentiated nutrient-stimulated, but not KCl-stimulated, calcium influx. The potentiating effects of SW016789 were not due to altered metabolic pathways, mitochondrial function, or actions on G protein-coupled receptors. In chemical co-treatment experiments we discovered synergy between SW016789 and small molecule activators of protein kinase C and VDCCs, suggesting potential involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as co-treatment with dihydropyridine-class VDCC inhibitors protected MIN6 beta cells and human islets from loss of function.Conclusions/interpretationThese data suggest the major mechanism of action of SW016789 in beta cells is to potentiate opening of VDCCs. This activity may partially depend on protein kinase C. Beta cells under pharmacological hypersecretory conditions have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. Further study of the mechanisms underlying these processes will increase understanding of beta cell function in normal and pathophysiological states.Research in contextWhat is already known about this subject? Pharmacologic and environmental chemicals can stimulate aberrant beta cell activity.Hypersecretion of insulin from pancreatic beta cells can cause ER stress and lead to beta cell dysfunction.Defects in beta cell ER stress handling contribute to diabetes pathogenesis.What is the key question? Can the chemical tool SW016789 help uncover novel insulin secretion regulatory pathways and ER proteostasis factors?What are the new findings? SW016789 caused hypersecretion of insulin via enhanced nutrient-stimulated Ca2+ influx followed by transient ER stress and shutdown of beta cell function without apoptosis.Blockade of voltage-dependent Ca2+ channels protected human islets and beta cells from hypersecretion-induced dysfunction.How might this impact on clinical practice in the foreseeable future? These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

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Section: Esm Methodsmentioning

confidence: 99%

Small molecule-mediated insulin hypersecretion induces transient unfolded protein response and loss of function in pancreatic islet beta cells

Rodrigues-dos-Santos

Roy

Binns

et al. 2022

Preprint

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“…Among these methods, chromatographic separation techniques are common and include methods such as high-performance liquid chromatography (HPLC), [3][4][5][6][7] gas chromatography (GC), [8][9][10][11][12][13] capillary electrochromatography (CEC), [14][15][16][17][18][19] and supercritical fluid chromatography (SFC). [20][21][22][23][24][25] They are considered convenient and fast techniques for chiral separation. The key and core aspect of separating racemates is to prepare a chiral stationary phase with good stability, enantioseparation ability, and suitability for use in preparing chromatographic columns.…”

Section: Introductionmentioning

confidence: 99%

A chiral emissive porous organic cage used for high-resolution gas chromatography separations

Wang,

Tang

et al. 2024

New J. Chem.

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“…Chirality can also influence pharmacodynamic behavior, a feature that has been demonstrated for R (-)HCQ and S (+)HCQ in the mid-90's ( Ducharme et al, 1994 ; Tett et al, 1994 ). Several methods have been described to obtain the enantiomers of HCQ separately, including direct synthesis using optically resolved reactants ( Blaney et al, 1994 ) and chromatographic separation on a preparative scale ( Wilson et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine

Ballet

Böhme

Brohan

et al. 2022

European Journal of Pharmacology

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A preparative chiral separation of hydroxychloroquine using supercritical fluid chromatography

Abstract: Highlights Robust SFC preparative method for the separation of the enantiomers of Hydroxychloroquine drug repurposing: treatment for Covid-19 caused by SARS-CoV-2 infection

Cited by 11 publications

References 13 publications

Small molecule-mediated insulin hypersecretion induces transient unfolded protein response and loss of function in pancreatic islet beta cells

Small molecule-mediated insulin hypersecretion induces transient unfolded protein response and loss of function in pancreatic islet beta cells

A chiral emissive porous organic cage used for high-resolution gas chromatography separations

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine

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