Optimization of a positive validation assay based on chemical denaturation experiments

Abstract

Viral infectious diseases have been considered as extremely potential risks throughout human history. Vaccination or viral-directed antivirals have been proved ineffective as long-term treatments against highly mutable pathogens like RNA virus, including VIH, Marburg or Ebola. In the last decade, several studies have laid on the table the feasible targeting of human machinery recruited during viral infection as a way of disrupting viral cycle. The aim of this work was to validate the binding to the human ESCRT domain Tsg101-UEV of a non-peptidic ligand identified by in vitro massive campaigns, NIH-11. The binding was validated measuring the degree of stabilization of Tsg101-UEV in a chemical denaturation assay followed with intrinsic fluorescence measurements. The results showed a clear, but weak binding to Tsg101- UEV with a Kd on the micromolar range. The binding constant estimated here constitutes a starting point in the further thermodynamic explanation of this interaction, which will lead to an affinity optimization to turn the candidate into an effective broad-spectrum antiviral.