Molecular Biology and Virology

EIDD has capabilities for the analysis of activity and toxicity of compounds.

Antiviral Efficacy Assays

  • Cell-based activity assays with BSL-2 live viruses allow the testing of both nucleoside and non-nucleoside based compounds in order to assay general antiviral activity.
  • Cell-based activity assays with viral replicons allow for the testing of nucleoside based compounds since they adequately represent intracellular genome replication machinery without danger of being exposed to dangerous viruses since they do not produce infectious viral particles.
  • Extracellular enzymatic assays to allow for the assessment of affinity of specific compounds for specific targets and pathways. This is particularly useful for the assessment of nucleoside based compounds in polymerase assays since the active triphosphate forms can be directly assessed without the need for cell entry and anabolism which complicate data interpretation.

Cytotoxicity of Compounds

  • Cytotoxicity assays have been established in the same cell lines used for replicon systems.
  • An expanded panel of cell lines is used to determine the therapeutic index of the drugs.
  • As mitochondrial toxicity has been associated with several nucleoside analogues, we also have the ability to determine the effects of our compounds on mitochondrial DNA content, lactate production and human DNA polymerases.

Intracellular EC50 and EC90 Levels of Parent Compounds and Active Metabolites

In conjunction with our Bioanalytical Chemistry group, the intracellular level of parent compounds and active metabolites, such as nucleoside triphosphates, can be measured to EC50 and EC90 values, corresponding to that of the replicon inhibition.

Drug-drug Interaction studies

The convenience and ease of use of replicon system provides unique opportunity for biological measurement of drug interaction. Synergism, additivity or antagonism of drugs can be quickly and accurately assessed using McSynergy II tests. Mechanisms of observed synergy/antagonism can be further investigated in conjunction with our Analytical Chemistry.

Characterization of drug resistant mutants

Viral infections are characterized by selection of mutants resistant to the action of antivirals. The viruses acquire one or more mutations in the genome that result in decreased affinity of the antiviral drugs to their targets. Analysis of such mutations is an integral part of an antiviral drug development process. Since nucleoside/nucleotide-based drugs target the replication complex fully represented in viral replicon systems, we select such mutants by treating the replicons starting with sub-optimal concentrations of the drugs and gradually increasing the concentration. The selected drug-resistant replicons are then analyzed for mutations in the replicon’s genome. The identified resistance mutations are, in turn, re-engineered into original replicons to provide secondary screening/analysis systems to identify complimentary drugs active against the viral mutants.