The Search for Promising Viral Targets in Drug Discovery

In the past decade we have seen advances in laboratory automation and in assays used to assess biological endpoints. At the Southern Research Institute (Birmingham, AL), these new technologies have greatly enhanced its high-throughput screening (HTS) capabilities for viral targets in cell-, protein-, and enzyme-based assays. These technologies can be leveraged to rapidly screen large compound libraries to discover new antivirals for previously unrecognized, emerging pathogens. There has been a dramatic increase in emerging pathogens, which impact public health and can result in serious economic impact at regional or global levels.1,2 Emerging viral illnesses present a number of unique challenges in drug discovery efforts, including limited reagents, access to viral strains, containment, and availability of trained scientists. In the last decade, resources have become available for the development of facilities to study these pathogens, and importantly, for the development of therapeutics for the treatment of the diseases they inflict. This article presents the special issues that must be considered in the creation of a drug discovery program for these types of viral pathogens, with examples from the Southern Research Institute drug discovery research program in Biosafety Level 2 (BSL2) and Biosafety Level 3 (BSL3) containment.

Special considerations for pathogens

The National Institutes of Health (NIH, Bethesda, MD) and Centers for Disease Control (CDC, Atlanta, GA) have three categories for emerging and biodefense-related pathogens: A, B, and C.3,4 These pathogens are grouped by the ease with which they can be spread and the severity of the disease they cause. For example, many of the hemorrhagic fever diseases, such as the Ebola and Machupo viruses, and the bacterium Bacillus anthracis (anthrax), are placed in Category A, because they are thought to have the highest risk and impact in spread and illness. Nipah virus, influenza, severe acute respiratory distress syndrome coronavirus (SARS CoV), and hantaviruses are grouped as Category C agents, and are considered emerging infectious disease threats. In addition to the basic research efforts needed to understand the life cycles of these pathogens and their effect on human or wildlife hosts, it is essential that we begin to develop therapeutics for the treatment of the diseases they inflict. The vast majority of these agents require specialized containment laboratories such as BSL3 or 4.5

Several important matters must be considered when establishing a facility dedicated to the discovery and development of new therapeutics, i.e., the requirement for BSL3 or 4 laboratory facilities, regulatory issues (e.g., Select Agent Rule,6 GLP compliance, and CDC and USDA permits), personal protective equipment (PPE), and containment of equipment as necessary. In addition, the processes for flow of work among the various laboratories must be thoroughly reviewed to ensure high-quality results. The Southern Research Institute’s overall approach to the implementation of HTS for BSL2 and 3 viral pathogens7,8 is outlined below.

Phase 1: Assay design and development

Figure 1 - Steps undertaken through hit validation. The four phases, with various steps, from assay design and development through hit validation.

The first HTS assay implemented at Southern Research was for the SARS CoV.9 The luminescent-based assay measures the inhibition of SARS CoV-induced cytopathic effect (CPE) in Vero E6 cells. The following section describes the various steps undertaken through hit validation (Figure 1). The first step in the process was a risk assessment of the pathogen with respect to its transmissibility and the type of containment required within the BSL3. Hence, prior to the development of the screen, decisions were made on the type of PPE to be worn by laboratory workers (Figure 2) and type of containment required for equipment that might aerosolize virus. The coronaviruses (order Nidovirales—family Coronaviridae—genus Coronavirus) are members of a family of positive-sense RNA viruses that replicate in the cytoplasm of animal host cells. They are a diverse group of large, enveloped viruses that infect many mammalian and avian species, causing upper respiratory, gastrointestinal, hepatic, and central nervous system diseases. It is generally accepted that SARS CoV is transmitted by close person-to-person contact through coughing or sneezing, which deposits respiratory droplets on the mucous membranes of the mouth, nose, or eyes of persons nearby. Additionally, transmission can occur through contact with surfaces contaminated with the infectious droplets.

Figure 2 - A scientist at Southern Research conducts in vitro efficacy testing of a vaccine candidate to prevent high path Avian influenza (HPAI) infections. This work is being conducted in the company’s BSL3 laboratory in Birmingham, using both hand screening and robotic screening measures.

Because SARS CoV is airborne and has the ability to be transmitted from person to person, it was decided that those handling the virus would wear positive air-pressure respirators (PAPRs), full-length Tyvek® suits (DuPont Co., Wilmington, DE) with a wraparound gown over the scrubs, double shoe covers, and double latex gloves (Figure 2). Workers dressed in the wraparound gown and additional layer of gloves and shoe covers before entering the HTS laboratory within the BSL3, and removed them when exiting the HTS laboratory. The liquid handling equipment that dispenses the virus into drugged plates was placed in a biosafety cabinet (BSC). However, the plate reader for reading the endpoints was kept outside. The plate reader and its plate stacker will not fit in a BSC, and while there is inherent risk of plates dropping or becoming jammed in the reader, it was decided that the PPE and BSL3 would act as sufficient barriers to any possible accidental exposure.

Once the protective barriers were in place, the next step in the establishment of the screen was to define the optimal flow of work (Figure 3). While it would be advantageous to add chemical compounds to the screen after virus addition, the problems associated with maintaining compound libraries in the BSL3 would be formidable. In addition, because of BSL3 regulations, compounds cannot leave the facility without undergoing a decontamination procedure, which could alter the integrity of the sample. It was agreed that storage and aliquoting of compounds would take place 24 hr after the addition of cells to multiple-well plates. All of the processes, procedures, and safety considerations were submitted to the Institutional Biosafety Committee for review.

Figure 3 - Optimal flow of work.

For the BSL2 virus HTS such as for influenza, the majority of the safety guidelines above continue to be followed. The major difference lies solely in the PPE. In the BSL2 laboratory, gloves, laboratory coats, and safety goggles are worn. All of the equipment safety barriers are the same as described for BSL3.

Once approval was met, the actual development and optimization of the assay and evaluation of the flow of work outlined was undertaken. Optimization of the assay requires judicious attention to every detail, such as the choice of consumables, cell age, growth conditions, and liquid handler. For example, using an eight-channel peristaltic pump, Vero E6 cells were plated in black, clear-bottom, 96-well plates at a density of 10,000 cells/well in a 50-μL assay medium. Careful attention needs to be paid to controlling the humidity of the CO2 incubator, and some brands appear to do this better than others. The next day, 25 μL of compounds were added to the cells using a flexible liquid handling system in a biosafety cabinet. In this case, the BSC was used to protect the cells from the environment, i.e., keep them in an aseptic environment. In the Southern Research work flow, plates are drugged and then hand-carried into the BSL3 in groups of 50 plates per run. Using more than 50 plates is difficult in the BSL3 environment simply because of movement logistics. In the BSL2 HTS virology laboratory, however, up to 125 plates can be moved effectively, since the constraints of BSL3 are not present. Plating of the compounds requires about twice as much time as plating of the virus; thus the flow of work depends on the equipment chosen for this activity. In all instances, compounds are plated prior to virus addition.

After incubation for three days, a luminescent cell viability reagent is added that measures cellular adenosine triphosphate (ATP) levels to each well using a laboratory automation workstation benchmark system. Recently, the laboratory switched to a system with a smaller footprint. Smaller footprints in equipment are of particular advantage in BSL3, where space is at a premium. For 96-well assay plates, plates are shaken on a compact benchtop plate shaker, which has a small footprint as well. For 384-well plates, the plates are not rocked. Luminescence is measured using a plate reader. An Ethernet interface allows direct uploading of the data into the database and analyses using an integrated data management software application outside of the BSL3. Each plate contains negative and positive controls that are used to fail or pass plates that have Z-values of less than 0.5.9

Phase 2: Assay validation

As part of the validation for the viral HTS assay, small compound libraries are used to determine the reproducibility of the hits and the hit rate. A “hit” is identified as an active compound that either activates or inhibits the assay signal above a defined threshold value from the sample mean signal. A hit for this assay was considered to be any compound exhibiting a % CPE inhibition of >50%. The hit rate can vary depending on the virus.9,10 While the library in this study was not validated against a high and low drug concentration, subsequent studies with other viruses have shown it to be an important step at this stage of assay validation. For example, in the validation effort of the assay to discover small molecules that inhibit the cytopathic effect exerted by influenza viruses, the laboratory’s high-throughput cell-based assay was used to screen 16,000 compounds from a commercially available compound library at a low and high concentration. 10 The hit rate for the compounds screened at low concentration was 0.02% compared to 0.38% at the high concentration, for an approximately 19-fold increase in the hit rate. For the SARS CoV screen, hit rates of 0.01%9 were observed.

A comparison of the screening results from the same set of compounds assayed on different days is the most effective way to determine the reproducibility of the assay. If one relies solely on the control wells to assess the assay, one may develop a false sense of confidence in its capabilities. The liquid handling steps used for the control wells, which are usually on either end of the plate, often differ from the steps used for the unknowns in the compound library. Also, the positive control compounds are usually limited to one or two mechanisms of action. Reliance only on data from these compounds may inadvertently bias one against compounds operating by different mechanisms.

While two libraries comprising known biologically active compounds were used for the initial validation, another rationale for choosing the validation library is that one can select a subset of compounds representing the chemical diversity of the larger compound library. By doing this, the hit rate at a proposed screening concentration can be estimated, allowing one to adjust the concentration up or down for the large screen. A benefit to choosing the validation set in this way is that when the large library is screened, the validation compounds are scattered throughout the large screen. This provides an independent third assay of these compounds that can be used to assess the quality of the large run.

Another advantage of choosing a large compound validation set relates to troubleshooting the automation. Typically, running a few plates at a time is insufficient to identify problems that are likely to occur when handling a large number of plates in one day. The optimized validation set of Southern Research is 10,000 compounds, or about 10% of the large library.

Phases 3 and 4: Compound library screen and hit validation

At this stage, the general algorithm for HTS antiviral screening employs the validated primary cell-based assay for determining single-dose efficacy with one or more of the laboratory’s large libraries. This type of approach has been used successfully to screen nearly two million compounds. This is followed by a second, similar cell-based screen for toxicity evaluation of the compound, which showed 50% or better activity and dose response (DR) confirmation. Those compounds with robust SI values are confirmed in secondary assays (such as a plaque assay). As an example of the large compound library screen, 100,000 compounds were screened in duplicate from the 100,000-compound library against SARS CoV.9 The hit rate for this library (compounds that inhibited CPE by >50%) in a single-dose (SD) format was determined to be approximately 0.8%.

To screen selected hits from the SD assay, compounds were confirmed in a DR format. Every HTS assay has a false positive rate, and hits from the primary SD screen are only considered potential positives until that activity is confirmed in a second DR screen, as mentioned above. Hence, the SD screen identifies a smaller subset of effective lead compounds (i.e., hits), which are then run through one or several secondary assays to define the compound dose response and/or toxicity. The accuracy of the primary assay is paramount for identifying hits, while simultaneously reducing the number of compounds funneled through the secondary assays and greatly decreasing the amount of time and resources required to evaluate lead antiviral compounds. The criterion for determining compound activity is based on its selective index (SI50). The compounds with an SI50 value of <4 are classified as not active, SI50 = 4–9 as slightly active, SI50 = 10–49 as moderately active, and SI50 >50 as highly active. Following confirmation, structure–activity relationships are defined using a cheminformatic approach to identify other similar molecules that might be available commercially. Additional parallel chemical synthesis may be done to generate a group of structurally related compounds, which are reevaluated with the aforementioned DR assays and further characterized for mechanism of action if promising.

Next, an assay is used to determine the point in the SARS CoV life cycle that the DR hits inhibited. In effect, this simple screen allows one to ascertain if the inhibition of the compound was early (entry) versus late (replication). The laboratory’s biochemical assays also exploit the high sensitivity of fluorescence detection, or use proximity assay formats with ultralow background interference.

The development of a panel of biochemical assays is important for the identification of the mode of action for each lead candidate. These assays target vital components of the virus life cycle that are necessary for infection, disruption of host–cell mRNA translation, transcription, and virus budding. The targets are often unique to the virus, and similar activities are not found in the host such as for influenza, which has neuraminidase, M2 proton channel, and NS1A RNA binding activities.


The activities described above give an introduction to the first phase of activity essential to drug discovery, and this is clearly just the beginning. Once a hit has been verified using a combination of cell-based and biochemical methods, we can move forward to determine the toxicity and efficacy of the drug in an animal model. It is after this stage that we will gain valuable insight into the suitability of the compound class for lead optimization medicinal chemistry and follow-up preclinical toxicology testing. It is hoped that recent advances in infrastructure, new assays, and HTS procedures will lead to the discovery of new antiviral drugs for emerging and reemerging pathogens, those of which we are aware and those that are yet to come.


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Dr. Jonsson is Program Leader, Emerging Infectious Diseases Research Program; Ms. White is Manager, High Throughput Screening Center and Enzymology Laboratory; Dr. Noah is Research Biologist; Dr. Severson is Research Biologist; and Dr. Heil is Associate Research Biologist, Emerging Infectious Diseases Research Program, Southern Research Institute, 2000 9th Ave. S., Birmingham, AL 35205, U.S.A.; tel.: 205-581-2681; fax: 205-581-2093; e-mail: [email protected].