Researchers at the University of Illinois at Chicago last month entered into the final year of a three-year, $750,000 grant from the National Institute of Allergy and Infectious Diseases to implement, optimize, and validate a fluorescence-resonance energy transfer-based NS3 protease hepatitis C infection assay that is amenable to high-throughput screening.
The proposed assay would have several advantages over currently used high-throughput approaches to HCV antiviral drug discovery because it recapitulates the entire viral life cycle and does not restrict screening to any predetermined viral targets.
The assay could potentially provide novel leads for drug discovery efforts and identify new compounds with unique mechanisms of action that could be used to study HCV infection.
“We just started our third year, and it was during that third year that we planned to do a pilot screen with a [compound] library, so we have the NCI diversity set,” Susan Uprichard, an assistant professor in the departments of medicine and microbiology and immunology at the University of Illinois at Chicago, told CBA News this week. The amount awarded for FY 2008 was $232,000.
“We have evidence that this is a very good HTS system, but until you use it to screen a reasonably sized library and demonstrate that proof-of-principle, you are not going to get a lot of people interested,” Uprichard said.
She added that she and her colleagues plan to begin this pilot screen as soon as they return from the 15th International Symposium on HCV and Related Viruses, to be held Oct. 5-9 in San Antonio.
The assay uses as a reporter NS3, a peptide encoded by the HCV virus. It also uses a commercially available FRET probe made by Anaspec. The UIC scientists use a peptide that on one side has a 5-FAM, and absorbs energy at approximately 480 nm and fluoresces at about 510-520 nm. The quencher used is QXL 520.
The amount of substrate that gets cleaved when added to the cell is proportional to the amount of NS3 present. For its part, the amount of NS3 is proportional to the robustness of the infection in the presence of the inhibitor of interest. So if little fluorescence is seen, there is not a lot of NS3 and the compound added to the well-inhibited HCV infection. If much fluorescence is observed, the compound did not inhibit the infection.
“[O]ver the next six days, the infection, amplification of the viral RNA, and spread through the culture … depends on every aspect of the viral life cycle…”
“We have shown that viral proteins, viral RNA, and viral titers parallel each other,” said Uprichard, explaining that the viral proteins can be used to determine how far the infection has progressed or how high the level of infection is. The NS3 protease FRET assay is very linear with the amount of NS3 protein and the reproducibility is good, Uprichard added.
Uprichard and her colleagues wanted to test for elements that inhibited any aspect of the viral life cycle. “We decided to do a six-day assay, although with HTS, cell-based assays are typically somewhat problematic in that you get a lot of variability due to the cell culture,” she said.
The assay is a six-day assay because the scientists infected the cells at a very low multiplicity of infection, and then they immediately began treatment. “So over the next six days, the infection, amplification of the viral RNA, and spread through the culture … depends on every aspect of the viral life cycle — entry, replication, assembly, secretion, and entry into living cells,” said Uprichard.
Uprichard said that she and her colleagues felt that they could find antiviral compounds that target any aspect of the viral life cycle. “This is an academic lab, so the exciting thing is that we might find inhibitors that actually block a part of the life cycle we were not even aware of before,” she said. As an academic lab, they would use these compounds as tools to probe the viral life cycle and learn more about it.
Identified hits would go to medicinal chemists and “people who would actually do the pharma end of things,” Uprichard said.
Most researchers use Huh-7 cells for their HCV infection system. However, they use them in their growing state, because as a hepatoma cell line, Huh-7 cells replicate once every 24 hours.
Evidence in the literature exists that in vitro, the state of growth of the host cell actually impacts how HCV is replicating. So when a cell is actively replicating in an S phase, the virus is replicating quite well. But then when the cells become confluent and they go into a more stationary phase, the virus levels go down.
“That was an issue we wanted to avoid,” said Uprichard, because in a high-throughput cell-based assay, every time one sees cells, one does not know if they are going to be in the same growth state. “We have used a slightly modified tissue culture method where we induce the Huh-7 cells to go into G0. So we actually get a non-dividing synchronized culture,” said Uprichard.
If an HCV infection is done under these conditions, the viral titer increases and then reaches a steady state, which makes for a very stable system. “I think that is why we have had such good luck getting such reproducible results, and getting Z′ factors that are acceptable,” Uprichard said.
She also said that she and her group have actually done small screens where they have used viral entry inhibitors, as well as translation and replication inhibitors, and shown that regardless of the stage in the viral life cycle that the inhibitor is acting, it can be detected in their high-throughput assay system.
Next Top Model
For a long time, the problem with hepatitis C has been that there have not been experimental models to really study the virus. That dearth of models has restricted drug discovery. “No system existed to screen for inhibitors that inhibited the viral lifecycle regardless of target,” said Uprichard.
Back in 2005, Uprichard and her colleagues, as well as two groups, one from the US, Germany, and Japan, and the other from the Howard Hughes Medical Institute, the Rockefeller University, and the Scripps Research Institute, published within the same week what Uprichard described as three parallel papers that described the first HCV in vitro infection system.
“This was the first time that viral clone of HCV was found that was infectious in vitro. You could transfect in the viral RNA, the cells would then go through the whole viral replication process, and secrete infectious virus,” said Uprichard.
“What we have done with this grant is we have taken the infection system, and we have downscaled it to a 96-well format, and we have actually created an assay that we can use to look [at HCV infection] in a high-throughput manner,” she said.