High-throughput screening

In this study, we embarked on 2 separate screening campaigns. The first utilized the IFNβ induction assay with the aim of identifying novel modulators

the IFNβ induction pathway. In this screen we were primarily measuring a

reduction in GFP expression, which would result from a test compound inhibiting the IFNβ induction pathway. The second screen, carried out in-house,

utilized the IFN signalling assay and A549/pr(ISRE).GFP.RBV-P cells to identify novel modulators of RBV-P protein function. In this instance, if a test compound were to inhibit RBV-P function, GFP expression would be restored.

2.4.1 Screening compounds and inhibitors

IFN induction and signalling inhibitors BX-795, TPCA-1 and Ruxolitinib (Rux) (Selleck chemicals) were prepared in dimethyl sulfoxide (DMSO) as 10 mM stock. CYT387 (Selleck chemicals) was prepared in DMSO as a 20 mM stock. Unless otherwise stated, the inhibitors were used at 2 µM. Actinomycin D

(AMD) and Cycloheximide (CHX) (Sigma), inhibitors of transcription and translation respectively, were prepared as 10 mg/ml stocks in DMSO and ethanol respectively, and, unless otherwise stated, used at 40 µg/ml.

Compounds constituting the Small Diversity Set of the Drug Discovery Unit (DDU) at the University of Dundee were stored as 10 mM stocks in DMSO and unless otherwise stated, used at 30 µM. Compounds constituting the Maybridge

screening collection were stored as 10 mM stocks in DMSO and unless otherwise stated, used at 11.42 µM. Hit compounds identified during HTS,

which were named StA-IFN-1 to StA-IFN-5 and compounds with high similarity to StA-IFN-1 and StA-IFN-4, and 2 molecules that constitute each half of these hit molecules are detailed below (Table 2.7). Compounds were purchased, prepared as 10 mM stocks in DMSO and used at 10 µM, unless otherwise

Table 2.7: Hit compounds and those with closely related structures

Name Chemical Name CAS Source

StA-IFN-1 4-(1-Acetyl-1H-indol-3-yl)-5-methyl-

2,4-dihydro-3H-pyrazol-3-one 300839-31-0 Chembridge StA-IFN-2 4-{[4-(Thieno[3,2-d]pyrimidin-4-yl)- 1,4-diazepan-1- yl]methyl}benzonitrile 930903-16-4 Enamine StA-IFN-4 2-[(4,5-Dichloro-6-oxo-1(6H)- pyridazinyl)methyl]-8-methyl-4H- pyrido[1,2-a]pyrimidin-4-one 876916-52-8 Enamine

StA-IFN-5 6-Methyl-4-phenyl-N-(pyridin-4-

yl)quinazolin-2-amine 610279-43-1 Mcule StA-IFN-1- 82S 3H-Pyrazol-3-one, 2,4-dihydro-4- (1H-indol-3-yl)-5-methyl- 3133225-48-8 Chembridge StA-IFN-1- SF 3H-Pyrazol-3-one, 2,4-dihydro-5- methyl- 108-26-9 Enamine

StA-IFN-1-LF Ethanone, 1-(1H-indol-1-yl)- 576-15-8 ChemDiv StA-IFN-4- 85S 3(2H) -Pyridazinone, 4,5-dichloro-2- [(6-methylimidazo[1,2-a]pyridin-2- yl)methyl]- 852902-22-8 Enamine StA-IFN-4- SF 3(2H)-Pyridazinone, 4,5-dichloro-2- methyl- 933-76-6 Enamine StA-IFN-4-LF 4H-Pyrido[1,2-a]pyrimidin-4-one, 2,8-dimethyl- 30247-64-4 ChemDiv

2.4.2 Diversity and dose response screening at the Drug Discovery Unit (DDU) University of Dundee

The DDU small diversity set was provided in 384-well Echo plates. A549/pr(IFN).GFP cells were seeded into clear-bottomed black 384-well plates Test compound, at a final concentration of 30 µM (125 nl), was added to

A549/pr(IFN).GFP cells using an Echo 550 liquid handler (Labcyte). Two hours post-compound addition, cells were infected with SeV (40 HA units/ml) for 18 hours. GFP expression was measured with an EnVision plate reader (Perkin Elmer) at excitation/emission 485/535 nm.

To test the potency of putative hits from the small diversity library, each compound was tested in the IFNβ induction reporter assay with and without

SeV infection, and the IFN signalling assay to generate standard ten-point dose response curves. This was achieved through two-fold serial dilutions of test compounds added to seeded A549/pr(IFN).GFP or A549/pr(ISRE).GFP cells using an Echo 550 and overlord3 robotics software.

For all data analysis, the RFU of each well was measured, and normalised to a percentage effect (% effect) of the positive control, calculated as follows.

% Effect = ((RFUUnactivated - RFUTest)/(RFUUnactivated – RFUActivated)) * 100

ActivityBase XE (IDBS) was used for all data processing using % effect, with the utilisation of SARgen (IDBS) and Excel (Microsoft). For determination of potency, 4-parameter logistic fit (Minimum, maximum, hill slope and IC50) was used, being defined in reference to the negative log of the molar value at the point of inflection of a sigmoidal dose-response curve (pIC50). Additionally, all

assay plates were subject to QC analysis. The QC criteria for the acceptance of an assay plate is shown below:

- S/B Ratio ≥2 - CV % <8% - Z’ ≥0.5

2.4.3 In-house HTS using the Maybridge library

A single-point primary screen of 16,000 compounds was carried out in- house using the IFN signalling assay detailed above (2.3.7) with the A549/pr(ISRE).GFP.RBV-P reporter cell line. The Maybridge compound library, loaned by Prof Nicholas Westwood (University of St Andrews), was provided in 50 384-well plates. Compounds were at a concentration of 10 mM dissolved in DMSO and contained in columns 3 to 22. The screen was conducted in 4 batches; 2 batches of 12, and 2 batches of 13 plates. A549/pr(ISRE).GFP.RBV- P (columns 1-22) and A549/pr(ISRE).GFP (columns 23 to 24) cells were seeded in 384-well clear-bottomed, black tissue culture plates (Greiner Bio-one) at 9×104 cells/cm2 and incubated at 37°C with 5% CO2. The following day, cells

were treated with 11.42 µM of test compound using a MiniTrak V Multi Position

Dispenser (Perkin Elmer), which replicates the compound plate in the assay plate, for 2 hours. To activate the IFN signalling pathway, IFN-α in DMEM

supplemented with 10% (v/v) FBS was added to cells in columns 2 to 23 followed by brief centrifugation at 1200 rpm for 1 minute. Following incubation at 37°C with 5% CO2 for 42 hours, cells were fixed with 5% (v/v) Formaldehyde

at room temperature, washed and PBS added to each well. With the exception of test compound transfer, all liquid handling was carried out using a Matrix

WellMate microplate dispenser (Thermo Scientific). Cellular GFP expression was analysed on an Infinite M200 Pro (Tecan) plate reader at excitation/emission 484/518 nm. Additionally, all assay plates were subject to QC analysis by monitoring CV % of A549/pr(ISRE).GFP.RBV-P cells and S/B ratio, CV % and Z’ Factor of A549/pr(ISRE).GFP cells. All data analysis was conducted using Excel (Microsoft) and Prism6 (GraphPad) software.