Huh7 and Huh 7.5 cells transiently transfected with an interferon-stimulated response element (ISRE) -luciferase reporter plasmid were used to identify RIG-I-.
Identification Of Small Molecule Lead Compounds That Activate The RIG-I Pathway To Induce An Anti-Viral Response Nigel Horscroft, Melony Cable, Adele Chambers, Frances Burden, Joel Beltra, Matthias Haddad, Francois Bertelli, Carl Laxton, Richard Bannister, Hannah Perkins, Andy Bell, Manos Perros and Mike Westby Pfizer Global Research and Development, Sandwich, UK
Sendai virus (SV) is known from literature to require RIG-I to induce IFN and has been used for proof of concept experiments. Huh7 and Huh 7.5 cells transiently transfected with an interferon-stimulated response element (ISRE) -luciferase reporter plasmid were used to identify RIG-Idependent signalling.
100
80
80
60 40
40 20
0
0
0.01
0.1
1
10
Compound EC50 (uM) S1-1 0.19 S1-3 0.68 S1-4 0.32 S1-5 0.16
60
20
Scatter Plot
-20 0.001
100
0.01
Dose
Compounds +ve Control Sendai Virus @ 100HA/ml +ve Control IFN @ 100U/ml -ve Control
5000
4000
S1-4
120
100
S1-5 Cytotoxicity
1000
0 1
2
3
4
5
6
7
Cell Line SW480 3LL U2OS Huh7
100
80 80
60 40
60 40
20
20
0
0
0.01
0.1
1
10
100
-20 0.001
0.01
0.1
1
10
8
Run Plate Order
100
Dose
120
120
S2-1
Primary hits SV controls in blue
Primary hit rate: 0.5% Confirmation rate: 7.9%
S2-2
100
100
80
80
Response
Compound EC50 (uM) S2-1 0.32 S2-2 0.49 S2-3 3.5 S2-4 ~10
Response
6. Screening Results
4. Assay Development
60 40
Over 2 million compounds were screened in the Huh7 reporter assay to identify and confirm immune activators. We obtained a primary hit rate of 0.5% with 7.9% confirmation rate. Approximately 300 compounds were selected for counter-screening in Huh7.5 cells resulting in the identification of a number of RIG-I selective hits. Various chemical series were identified and data for representative compounds from 2 series is presented here.
Series 1 Compounds
% IFN Effect
We have worked with GE Healthcare to determine the available options for reagent provision. GE Healthcare have used our Huh7 transient transfection assay with interferon to examine how the assay performs under different conditions: Transfection format (plated cells or cells on beads), transfection reagent (FuGene6 vs JetPEI) and cell source (freshly transfected cells vs frozen transfected cells).
Pluronic acid (detergent) Virus dilution Infection protocol Cell detachment Transfection and infection timing Freezing Stable cell line Luciferase vs b-lactamase
Huh7 Signal
120 100 80 60 40 20 0 S1-1
S1-3
0
0
-20 0.001
0.01
0.1
1
-20 0.001
10
0.01
0.1
120
S2-3
100
100
80
80
60 40
0
0
10
100
S2-4
40 20
1
100
60
20
0.1
10
Dose (uM)
Dose (uM)
120
1
-20 0.01
0.1
1
10
100
Dose (uM)
To date, a small subset of compounds have been tested in the HCV replicon system with mixed results. Given the potential for RIG-I/IPS-1 pathway disruption in replicon cells, potent antiviral activity was not expected, and was not found. A selection of compounds are currently being tested in other anti-viral models.
Compound S1-1 S2-3 S2-4
Replicon EC50 (uM) 1a 1b >40 >10 0.69 1.18 6.2 3.9
Huh7 CC50 (uM) >10 9.1 >19
Discussion
Huh7.5 Signal
S1-4 S1-5 Compounds at 9uM
40 20
-20 0.01
Parameters Investigated
60
20
Dose (uM)
Cell number Cell recovery period Virus titre FCS Optimem vs media Automation RIG-I overexpression Singleton vs compression
CC50 6.56nM 94.7nM >100mM >320mM
The dose-response curves for the Series 1 compounds (above) tested were characteristic of toxic or cytostatic compounds. Cytotoxicity data on compound S1-5 revealed a cell-type dependent toxicity. The lack of toxicity in Huh7 cells suggests that this compound is cytostatic in the screening and confirmation assays used. Series 2 dose-response curves (below) resembled the shallow curves seen with IFN treatment of replicon cells and toxicity was only found with a few compounds at high concentrations.
2000
To confirm that signalling from SV infection was RIGI specific we designed a complementation assay using plasmids expressing wild-type (WT) and dominant negative (dn) forms of RIG-I. In Huh7 cells, the co-transfection of dn-RIG-I blocked SV-induced signalling. Conversely, in Huh7.5 the co-transfection of WT RIG-I restored SV-induced signalling. This assay will be important to confirm on target activity of any hits identified in HTS.
10
S1-5
100
-20 0.001
3000
3. Target Confirmation
1
Dose (uM)
Dose
The NFkB reporter gave very high background levels of luciferase that was not upregulated on infection with SV. The ISRE reporter, on the other hand, was strongly upregulated in Huh7 but not Huh7.5 cells.
0.1
120
6000
Activation of RIG-I can lead to signalling via both NFkB (NFkB reporter) and IRF3 (ISRE reporter). We tested both reporters in Huh7 and Huh7.5 cells.
S1-3
100
-20 0.001
2. Reporter Selection
120
S1-1
Response
Assay Design
Huh 7.5
120
Response
In order to identify activators of RIG-I, we have established a cell-based assay suitable for HTS that exploits the differential response of Huh7 and Huh7.5 cells. We have explored numerous parameters during primary assay development including the use of frozen cells vs. cultured cells, stable cell lines vs. transiently transfected cell lines and reporter plasmids with NFkB promoters vs. ISRE promoters. We have also developed secondary assays that allow us to characterise the mode of action of any hits through the use of a dominant negative RIG-I construct. We expect to have a screening cascade in place and a primary assay transferred to HTS by the end of the year.
Graph showing effect of viral inoculum and time on reporter activity as measured in Huh7 or Huh7.5 cells transfected with an ISRE-luc plasmid. At 18hrs post-infection SV strongly induces a signal in Huh7 cells but not Huh7.5 cells. Virus titre does not appear to be important.
Response
The initial sensing of infection by some RNA viruses is mediated by the cytoplasmic retinoic acid inducible gene I (RIG-I), via a Toll-like receptor (TLR) independent signalling pathway. This results in the induction of type I interferon and the innate antiviral response. Interferon treatment forms the cornerstone of current therapy for hepatitis C virus (HCV) infection and methods to induce interferon in HCV patients by TLR 3, 7 or 9 agonism is being aggressively pursued by Pfizer and other pharmaceutical companies. Huh7.5 cells are a tissue culture adapted strain of Huh7 cells (a human hepatoma cell line) that contain a loss-offunction point mutation in the RIG-I gene. They were initially characterised after HCV replicon containing Huh7 cells were “cured” by IFN treatment. It has been shown that HCV replicon cell lines are more readily established in Huh7.5 cells than Huh7 cells, and this has been linked to the loss of RIG-I function.
Developing a robust assay was extremely challenging. The main problem was the large amount of variability in the maximum signal. This could be due to the use of Sendai virus as the activator because the use of IFN as the activator gives an acceptable Z’. However, IFN signals via a different pathway and without a tool compound we could not predict how an active small molecule would work in this assay. The final assay used our stable reporter cell line with both SV and IFN used to determine the max signal. Z’ was ≥0.4 with SV and ≥0.6 with IFN.
Response
Huh 7
1. Assay Concept
RIG-I selective compounds were further profiled in dose-response assays with the ISRE reporter cell line, and EC50 values were determined.
Response
5. Assay Statistics
Response
Background
Results
SV
IFN
Selectivity screening was performed at 9uM in stably transfected Huh7 cells and transiently transfected Huh7.5 cells. Results were normalized to percentage of IFN signalling in the same cell lines (100IU/ml), Across all the compounds identified as RIG-I selective none signalled to the same extent as IFN. However, most were within the same signalling range seen with Sendai virus.
RIG-I represents a novel target for the interferon-mediated induction of an anti-viral state. It has an important role in sensing viral infection and has a specific role in detecting HCV. We have designed a screening cascade to allow us to identify and confirm the mechanism of action of small molecule agonists of RIG-I. Developing a robust primary assay in stably transfected Huh7 cells proved challenging, with the issue of assay variability requiring extensive trouble-shooting. However, we succeeded in screening a large smallmolecule library and identified a number of hits that can be grouped in to a number of structurally related series. Early results are encouraging but there is still a significant amount of work to perform to complete the hit-to-lead process and identify a compound with suitable physicochemical properties to progress along the drug discovery pathway.
References Sumpter et. al. J Virol. 2005 Mar;79(5):2689-99 Li et. al. J Biol Chem. 2005 Apr 29;280(17):16739-47 Loo et. al. Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):6001-6
Pfizer Global Research & Development Sandwich Kent CT13 9NJ
Disclosure: All authors are employees of Pfizer Ltd.
