Introduction                                                                                                                    Click to Enlarge

Apath’s R&D efforts are focused on finding novel therapeutics for RNA viruses including hepatitis C virus (HCV) and respiratory syncytial virus (RSV).  Our antiviral drug-discovery program is based on a state-of-the-art combination of high-throughput screening, cell culture techniques, and molecular virology.  The cornerstone of our screening platform is the use of viral replicons.  Replicons are subgenomic, self-replicating RNA molecules that contain all the nucleotide sequences required for RNA replication, transcription, and translation, but are not themselves infectious.  Apath has developed a broad-based proprietary antiviral screening platform based on multiple viral replicons.

Apath’s approach to antiviral drug discovery differs significantly from the standard target-based discovery methodology employed by most pharmaceutical companies.  In target-based drug screening, an assay is developed for a particular enzymatic pathway necessary for viral replication (for example, a viral protease).  Large compound libraries are then screened, typically in a “high throughput” format, toward the goal of finding inhibitors of the chosen target (i.e. “hits”).  The hits generated from this process are then further qualified in various secondary assays, including cell-based assays, where the mechanism of action and potential suitability of lead drug candidates are studied in greater detail.

Another approach to finding active compounds is to use cell-based assays in the screening process.  Although less commonly employed than target-based screening, cell-based primary screening has played an important role in drug discovery. Generally there are two types of cell-based assays utilized in library screening:

• Phenotypic endpoint assays. In this approach a cell-based assay is employed in which the effect of a compound is measured using a simple phenotypic endpoint such as cell death or inhibition of cell division. Anti-bacterial screening and anti-cancer screening have been performed using this approach.

• Cell-based, single-target assays. In this approach genetically engineered cells are used in which a single target is placed into the context of the cell to make the assay more physiologically relevant.  A reporter gene that is linked to a single biochemical pathway (e.g. a signal transduction pathway) is an example of this approach.

In the field of antiviral drug discovery both cell-based approaches have been employed.  Classically, many groups have searched for antiviral activity using cell-based plaque reduction assays (PRA), reduction of cytopathic effects (CPE) or reduction of viral antigen expression.  These approaches are cumbersome and not amenable to high throughput. There have also been some recent descriptions of cell-based single-target approaches to identify antiviral compounds.  For example, several groups have reported the use of cells engineered to contain a viral protease which activates expression of an easily measurable reporter protein. 

Apath replicon-based screening program

Apath’s approach to antiviral drug discovery differs significantly from both standard target-based and cell-based discovery.  Apath has developed a broad-based proprietary antiviral screening platform based on multiple viral replicons. Apath has significant expertise in molecular virology and viral genetics and specifically in the manipulation of viral replicons.  We have assembled a program in which we have proprietary know-how and intellectual property in this field.  We are in the process of developing the capability to design, construct, and utilize replicons of virtually any RNA virus.

In the past, research into potential therapeutics for HCV has been hampered by the inability to grow the virus in culture and by the lack of small animal models in which potential therapeutic compounds could be evaluated.  Several significant developments have improved this situation.  First, an infectious cDNA for HCV was generated in 1996 by Kolykhalov et al. from the Rice lab.  This set the stage for developing replication systems for HCV.  Second, in 1999 a subgenomic, noncytopathic HCV replicon was isolated by Lohman et al., followed by the isolation of a replicon that exhibited a higher efficiency of transfection by the Blight et al. in 2000.  These replicons were the first useful models of HCV replication in cell culture.  Apath is utilizing the HCV replicon as a primary screening tool to identify inhibitors of HCV replication and we are extending the replicon-based approach to antiviral drug discovery to other medically important RNA viruses.

The subgenomic replicon represents the only robust HCV replication system in cell culture. For other viruses that are able to be grown in cell culture, subgenomic replicons represent a critical tool for antiviral screening that enables us to avoid problems associated with using infectious virus.  Whereas this is useful for any viral pathogen, it is particularly important for BL-3 or BL-4 pathogens.  For several positive-strand RNA viruses such as Yellow fever virus (YFV), Dengue virus (DV) and West Nile virus (WNV), infectious cDNA clones are available and subgenomic replicons can be constructed in a manner very similar to that of HCV.  Using replicons of prototypical positive-strand viruses such as Sindbis virus and Yellow fever virus, Apath has shown that the replicon-based screening approach that we are using for HCV is adaptable to other positive-strand RNA viruses.

Apath has also successfully demonstrated that the replicon concept can be applied to medically important negative-strand RNA viruses.  For many negative-strand viruses important advances in reverse genetic systems have identified the critical cis-acting elements and the trans-acting factors required for viral genome replication.  We have designed a proprietary system based on ‘minigenomes’ that are replicated in trans by viral replication proteins.  A key step in the development of this platform was achieved when the company successfully developed a subgenomic or ‘minigenome’-based screening platform for respiratory syncytial virus (RSV).  This RSV ‘replicon’ assay has been used successfully by Apath, along with the HCV replicon, in screening libraries of small molecules.

Advantages of replicon-based screening:

Replicon-based screening combines the advantages of both types of cell-based screening and avoids several of the disadvantages of each.

1. It avoids the use of infectious material which is a major problem of screening with virus because it complicates efforts to increase screening throughput.

2. Since it operates as a model of viral replication, the replicon assay is not target specific…it can determine potential inhibitors of any of the multiple viral and host cell pathways necessary for viral replication.  For any given number of compounds screened, this serves to significantly increase “screening density” relative to single-target assays.

3. Because it is not target specific, replicon screening is unbiased in its approach…the specific target does not need to be known at the outset of the process.  The potential exists that the use of replicons to screen for inhibitors will result in the discovery of viral pathways that are not presently known.

4. Replicon screening is pre-validated…it quantitatively measures a process (inhibition of viral RNA replication) that is directly relevant to viral pathogenesis.

5. Because it is cell-based, in addition to measuring antiviral effects, replicon-based screening also provides an early measure of compound toxicity.

6. Replicon-based screening allows for the possibility of identifying antiviral compounds that have broad-spectrum antiviral activity.

Multi-virus screening

Apath’s proprietary edge in the application of replicon-based screening for antiviral compounds has been further enhanced by a new technology that enables us to screen for antiviral activity against multiple viruses in the same assay.  Apath has filed a U.S. patent application covering this invention.  The essence of this technology is to pool multiple cell lines each of which contains a specific viral replicon.  An antiviral effect can be tested against multiple subgenomic viral replication systems and the relative efficacy of the candidate antiviral agent can be determined on each viral replication system.  Apath intends to develop and implement this multi-virus assay as an integral element in our screening strategy going forward.  In addition to improving screening productivity for the two principal viruses targeted by the company (HCV and RSV), implementation of the multi-virus assay will allow Apath to add other RNA viruses to the company’s drug screening and discovery program at very low incremental cost.  An additional advantage to this approach is that we will obtain specificity information about ‘hits’ that will improve our prioritization criteria as well as promote our efforts to identify broad-spectrum antiviral agents.

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