Rapid screening approaches to identify and confirm lysosomotropic agents among ECCS class 2 basic drugs

Lysosomes are acidic organelles that function as the digestive system of the cell. Weak basic small molecule compounds can diffuse into lysosomes and become protonated and trapped in a phenomenon known as lysosomal sequestration or trapping. Lysosomal trapping can influence unbound drug concentrations at the site of action, and consequently, drug responses. Drug accumulation in lysosomes may also impair phospholipid metabolism, leading to a severe side effect called drug-induced phospholipidosis. Current methods used to evaluate lysosomotropism are either indirect (high-throughput, but low sensitivity) or direct (sensitive but time- and labor-intensive). Moreover, no validated in-silico lysosomotropism prediction tools are publicly available. In this study, we developed a rapid fluorescence-based platform to screen for lysosomotropism and quantify lysosomal trapping propensity (IC50) of compounds in a standardized way. We also apply this method to various compound libraries including BBB (Blood-Brain Barrier) and protein degrader/PROTAC (Proteolysis-Targeting Chimera) compounds. This platform confirmed basic ECCS (Extended Clearance Classification System) class 2 compounds as lysosomotropic, and other ECCS class compounds as non-lysosomotropic. Collectively, this study provides measurable and efficient tools (in-silico & in-vitro) to identify and measure lysosomotropism, which can be applied by preclinical programs to decipher causes of intracellular accumulation.