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Ionic Modulation of Immune Checkpoint Proteins

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Ionic Modulation of Immune Checkpoint Proteins This is a repository copy of Ionic modulation of immune checkpoint proteins. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/175570/ Version: Published Version Conference or Workshop Item: Shandell, Mia Ann orcid.org/0000-0002-7864-758X, Lawrence, Samantha, Brackenbury, Will orcid.org/0000-0001-6882-3351 et al. (1 more author) (2019) Ionic modulation of immune checkpoint proteins. In: Physiology 2019, 07-10 Jul 2019, Aberdeen Conference Centre. Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Ionic modulation of immune Mia A. Shandell 1,2,3 checkpoint proteins Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumour microenvironment on immune , Samantha M. Lawrence , William J. Brackenbury , Dimitris Lagos checkpoint expression and function. We screened effects of ion channel modulating compounds on IDO1 activity. Here, we describeAbstract a mechanistic link between Na /K ATPase inhibition by 1. York Biomedical Research Institute. University of York. York, UK. 2. Department of Biology. University of York. York, UK. cardiac glycosides and activity of indoleamine-2',3'- 3. Hull York Medical School. University of York. York, UK. dioxygenase (IDO1), a well-characterized immune checkpoint. IDO1 catalyses the rate-limiting step of tryptophan catabolism and inhibits the immune response to the tumour by local depletion of tryptophan, an amino acid essential for anabolic functions in cancer and T cells. 1,2 + + IDO1 activity is measured using a kynurenine assay. Day 1 Seed cells Treat with compound Day 2 TNF- Stimulate with 25 ng/mL TNF- and 1000 U/mL IFN- cancer MDA-MB-231 cells. Day 3 A) α Kynurenine, Alamar Blue assays /IFN- Methods kDa HN H 2 55 N A549 γ γ Alamar Blue assay UT stimulates IDO1 expression in lung cancer A549 and breast CO tryptophan 35 2 H α (after removing supernatant in step (1) Add 10 uL Alamar Blue to cells in tissue culture plate. Incubate 4h 37C/5% CO2. Measure absorbance at 570 nm, 600 nm. IDO αγ Kynurenine assay of IDO1 activity 35 MDA-MB-231 (1) Transfer 150 H O Drug/toxin Ion channel 2drugN screen ofround-bottom IDO1 plate. activity 4-aminopyridine +10 UT N-formylkynurenine 50°C/30 min Amiloride CO μ 1,2 1,3 HN L 30% v/v trichloroacetic acid A) Western blot of IDO1 in A549 and MDA-MB-231 cells +/- 25 ng/mL TNF- Benzamil 2 Bupivacaine H O cells. Used 10,000 A549/well and 50,000 MDA-MB-231/well as seeding density in all following experiments. C) Representative kynurenine absorbance standard Cl μ Carvedilol H Cl L supernatant to a curve calculated from kynurenine standards in 0.5M HCl αγ O Results Diclofenac (sodium) EslicarbazepineCelecoxib acetate Flunarizine dihydrochloride Cl 30% v/v H OH Fluoxetine hydrochloride 50C/30 min Cardiac glycosides ouabain and digoxin inhibit kynurenine production Na Memantine hydrochloride + /Ca B) Iberiotoxin 2 2+ O in A549 and MDA-MB-231 cancer cells withIDO1 a modest impact on cell survival. exchanger, ENaC, TRPP3, TRPA1 channels H Nifedipine O K Target(s) 2 N . K NS-1619 v K Tetraethyl-ammonium chloride v 1.5, K ASIC channel+ channels (2) Centrifuge 10 min/3000 rpm/room temperature. 80 Ouabain CO Phenytoin Na Transfer 100 L-typeir Ca2.3 K NH ) v kynurenine2 H (3) +100 , K2P channels A) GAPDH M60 Tetrodotoxin + 2 in acetic acid. Room temperature/10 min m n ( channels, adrenoceptors + (4) Measure absorbance at 492 nm. io e TRAM-34 2+ t L-type and T-type Ca a 150 in IDO1 activity Veratridine K and K μ iv n 40 v L 1.2% w/v Ehrlich’s reagent t e 7 channels μ c r 1-EBIO O L to new plate. a u CarbenoxoloneCapsaicin VGSC v 7 channels n 1 100 y20 K O k Cariporide K v 4.3 channels H A549 - IDO1 assay Charybdotoxin Ca Effect Ehrlich's reagent N ID 1.1 K Inhibitor f L-type NMDACa receptor 2+ o Gabapentin + Inhibitor Glibenclamide channels n 50 0 Loperamide hydrochloride K channels Inhibitor o Ca ti 1.1 K 2+ Inhibitor i Icilin Na ib + + channels Inhibitor h Margatoxin VGSC,/K HERG channels acetic acid A library of 31 compounds targeting ion channels or + in 0 10000 K + 10 min RT Ranolazine ATPase both ) pumps was screened in the kynurenine assay measuring IDO1 channels - all Activator l % 20000 K ro activity in TNF- Ca Inhibitor t . 3.1 K VGSC n 150 30000 Pannexin-1 (GapK junction channel) Inhibitor o cancer (MDA-MB-231) cells. Points (right panel) represent Ca + H 3.1, K c O A549 VGSC channels Inhibitor 2 N 40000 technical replicates and data are normalizedL-type, toT-type their Ca respective TRPV1 channels O 10 K Inhibitor -4 DMSO control, 0.05% or 1% DMSO in PBS. Ca Ca S D) 50000 untreated 1.1, K cell number 2 channels Inhibitor M100 CO C) Inhibitor D 60000 TNF- 2 N n VGCC v/ alpha-2-delta H o MDA-MB-231 TRPA1, KATP1.2, channels K NHE1 Activator % Absorbance: 492 nm ti ( a log(inhibitor) ( α 70000 a α 2+ Inhibitor y v 10 /1000 U/mL IFN- v t i 100 /IFN- /IFN- , HCN channels,TRPM8 channel NMDA, μ-opioid1.3 K receptors Inhibitor i N t -2 80000 untreated iv A549 c ouabain + Inhibitor t 50 a MDA-MB-231 - IDO1 assay Cardiac glycosides decrease activity of immune checkpoint protein B) TNF- g channels Inhibitor c 1 digoxin 90000 γ a * O 80 ) 0.8 stimulated lung cancer (A549)K and breast Inhibitor 1 D v 1.3, K I 100000 a m Activator O f /IFN- n VGSC D o 60 10 Kynurenine standard curve Activator I m 0 110000 2 v 1.6 0 n M) 9 0.6 y = 0.004428x + 0.2388 Inhibitor o g 4 **** ti 40 ( Inhibitor 1% DMSO i 0.6 e R 0.05% DMSO (PBS) ib γ c 2 Inhibitor 100 mM 4-AP **** h stimulation. B) IDO1 assay cell titration for A549n and MDA-MB-231 50 mM amiloride 20 0 0.4 = 0.9982 Inhibitor in 0 a 10 mM benzamil 10 6 b Inhibitor 100 mM bupivacaine 2 s A549 alamar blue t = 4h r 5 mM carvedilol % b o Inhibitor 10 mM celecoxib 0 A 0.4 s Activator *p<0.05 ANOVA, Bonferroni10 mM multiple caps acomparisonsicin - b 3 mM carbenoxo 0.2 lone 0 Inhibitor ) 0.05 mM cariporide 7 A l 0.1 mM ChTx 5 Inhibitor ro 100 mM diclofenac s t 50 mM 1-EBIO G) b Inhibitor n 150 100 mM eslicarbazepine 10 A 0.0 o 10 mM flunarizine -1.5 0.2 c 10 mM fluoxetine 2 mM gabapentin O 10 mM glibenclamide 0 S 0.1 mM IbTx **** 1 mM icilin M 100 1 mM loperamide log(inhibitor) ( me 10 0.0 D 1 mM mantine 0.0005 mM MgTx -1.0 20 % 10 mM nifedipine ouabain E) 10 ( MDA-MB-231 1 mM NS-1619 y 50 mM ouabain digoxin -5 it 50 mM phenytoin v 10 mM ranolazine i 30 mM TEA 40 t 50 0.5 mM TRAM-34 kynurenine ( c 10 mM veratridine 10 a 10 mM TTX IDO1 activitym IC50-0.5 1 M) 10 O 0.6 -4 A) Dose-response curves for ouabain and digoxin treatment of MDA-MB-231 alamar blue t = 4h 60 ID log(inhibitor) ( C) Comparison of IDO1 activity vs. viability as measured by Alamar Blue in A549 cells. D) Dose-response curves 0for ouabain and digoxin treatment of 0 ** 0 ouabain stimulated 10 s 6 1% DMSO 0.0 10 digoxin m 0 0 % D ( BS) b 80 . 5 MSO P Blue in MDA-MB-231 cells. G) Calculated IDO1 activity IC50s from 0.4 -3 C) 100 mM 4-AP A M) 50 mM amiloride - 10 mM benzamil ouabain 0 **** * 7 100 mM bupivacaine 5 5 mM carvedilol s 100 10 mM celecoxib b l) *p<0.05 ANOVA, 1Bonferroni0 mM c multipleapsai ccomparisonsin MDA-MB-231 cells. E) Alamar Blue survival assay after 4h incubation with reagent. F) Comparison of IDO1 activity vs. viability as measured by Alamarm 10 o 2.0 3 mM carbenoxolone A 0.2 r 0.05 mM cariporide digoxin M) -2 t 0.1 mM ChTx n 100 mM diclofenac o 50 mM 1-EBIO /c 100 mM eslicarbazepine r 1.5 10 mM flunarizine to 10 mM fluoxetine 0.0 10 i 2 mM gabapentin ib 10 mM glibenclamide 10 -1 h 0.1 mM IbTx n 1.0 1 mM icilin -2.0 (i 1 mM loperamide **** A549 1 mM memantine Ouabain, but not digoxin, ty 0.0005 mM MgTx li 10 mM nifedipine i 0.5 1 mM NS-1619 b 50 mM ouabain ia 50 mM phenytoin 10 V 10 mM ranolazine downregulates IDO1 expression in -1.5 30 mM TEA 0.0 0.5 mM TRAM-34 10 mM veratridine log(inhibitor) ( 10 mM TTX A549 and MDA-MB-231 cancer cells.
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