Current drug discovery efforts have primarily focused on identifying agents that tackle specific preselected cellular targets.1
However, in most cases a single drug does not correct all of the aberrantly functioning pathways in a disease to produce an effective treatment. Drugs directed at an individual target often have limited efficacy and poor safety profiles due to various factors, including compensatory changes in cellular networks upon drug stimulation,3
and off-target activities.6
The use of drug combinations which act on multiple targets has been shown to be a more effective treatment strategy and is being used more frequently.7
This approach has been supported by successful clinical applications to treat various diseases, such as AIDS, cancer, and atherosclerosis.9
Often, the studies used high dosages of individual drugs to ensure treatment efficacy.12
Unfortunately, the high doses necessary to provide efficacy often come with toxic side effects. Therefore, treatments with a drug combination at the lowest optimal dosages will achieve the goal of high efficacy and low toxicity, resulting in the most desirable drug cocktail.13
However, identifying the combination of effective drug molecules, and determining the proper dosage of each constitute is a challenging task. For example, even a small number of different drugs (six drugs) each tested at a few concentrations (seven dosages) results in 76
= 117,649 combinations. Screening all 117,649 combinations for the most desirable combination is an enormous task in terms of labor and time. Furthermore, another problem with combination medicine is that the highly efficacious drug combination may include drug(s) that are toxic or have side effects.
Viral infections have stood out as an interesting candidate for combination drug therapy. HIV, hepatitis C virus, and influenza infections have been shown to be effectively treated by combinations of antiviral drugs. The pathogenesis of viral infections is caused by a coordinated reprogramming of cellular pathways and protein complexes by viral factors to favor the replication and spread of the virus. Within these pathways and protein complexes, single targets have been found that upon drug manipulation can disrupt viral replication. However, intervention against a single drug target usually results in the selection of escape mutants that are ineffectively suppressed by the single drug. The preferred method is to target multiple viral pathways simultaneously, so that the drugs target distinct steps of viral replication to more effectively block replication and limit the likelihood that a multiple drug-resistant mutant will arise.
Herpes simplex virus-1 (HSV-1) is one of the most pervasive infections worldwide, causing genital, skin, and eye infections in millions of people.15
Common treatments for HSV-1, including virus-specific drugs such as acyclovir, are effective but exhibit limited long-term efficacy due to the development of drug resistant strains. Thus, more effective therapeutic methods are needed to combat the increasing spread of drug-resistant HSV-1. Based on an intensive literature search, six drugs associated with antiviral gene regulation, viral proliferation, cell growth, and cell death were selected in our experiments as candidates for establishing a new combination drug therapy. First, the standard HSV-1 antiviral drug acyclovir,17
which is effective for the treatment of most herpes virus infections, acts as a chain terminator of DNA polymerase in virus infected cells. Acyclovir is also an effective control to measure efficacy. The second drug we included was ribavirin,18
which has well established antiviral activity against RNA virus infections such as poliovirus and hepatitis C virus but the mechanism for antiviral activity against DNA viruses, such as HSV-1, remains unknown. Next, we included three cellular produced interferons (IFNs), IFN-α,19
that have potent antiviral effects through the induction of cellular innate immune pathways. Finally, we also included tumor necrosis factor (TNF)-α,22
a cellular protein that induces activation of nuclear factor kappa B (NF-κB) and cellular death pathways. Each of these compounds can potentially block HSV-1 replication by modulating distinct viral or cellular protein complexes and pathways, and thus represent distinct potential therapies. Therefore, a combination of these drugs should be a highly efficacious drug therapy.
Instead of testing all possible combinations of these drugs at different dosages by a high-throughput screen, an experimental feedback system control (FSC) approach can identify optimal drug combinations by testing approximately 0.1% or less of all possible combinations.23
Previously, we focused only on rapid searching for highly efficacious drug combinations.23
Here, we have successfully applied the FSC approach in our experiments to search for drug combinations that have high antiviral efficacy and then applied FSC in cascade to lower the doses of a toxic drug (ribavirin) for the treatment of HSV-1 using an in vitro infection model.