Resumen de Establishment of a fully automatized microfluidic platform for the screening and characterization of novel Hepatitis B virus capsid assembly modulators
In its current form, drug discovery faces major challenges due to the constant erosion of earnings per drug resulting from a reduction in new FDA approvements combined with the steadily rising development costs. In order to reduce the development costs, multiple enabling technologies have been developed. Among them the integrated microfluidic screening platforms with feedback mechanism showed promising results by accelerating the hit-to-lead development cycle from several weeks required for conventional synthesis, shipping and screening to a few hours. In order to obtain suitable equipment, several modular devices were obtained, tested, and finally connected into a single system. The harmonization and automation were performed using workflows written in Microfluidic Automation Tool (MAT). Finally, evaluation protocols were written for the fluorescence and reflection data in KNIME, allowing the calculation of Z’-factor, standard deviations, dilution curves, and robust half‐maximal effective concentrations (EC50 values) (Chapter III). Hepatitis B virus (HBV) core protein (HBc) was selected as primary target due to the continuing demand for a functional cure to help address the economic and social challenges imposed by the chronic HBV disease. An existing HBc assembly assay was adapted to the microfluidic setup, using inflow dilution with convection dominated Taylor Aris Dispersion (TAD), rotate-split-andrecombine mixing, and fluorescence readout. Based on high-resolution dose response curves, EC50s were calculated at unexplored early kinetics of under 1 min. Furthermore, CAM-induced HBc assembly kinetics were measured for the first time at residence times down to 5 s post-mixing. The kinetic data permitted rapid categorization of CAMs according to their mode of actions (Chapter IV). Lastly, droplet microfluidics were used to eleminate further TAD mediated dilution had post mixing. Early results in droplets showed that mixing of HBc and reagents prior to encapsidation is necessary to prevent HBc's adsorption to the droplet-droplet interface. Finally, the system was complemented with a liquid handling system (LHS) as an interface between the microfluidics and 96 microtiter wells. This upgrade allows construction of a semi-automated synthesis/ screening system representing a novel approach to reduce cycle times and costs in early-stage drug discovery.
