Substrate specificity and in vivo efficacy of engineered cocaine esterases toward cocaine and its toxic metabolites.

Cocaine abuse remains a significant global public health challenge, yet no specifically approved pharmacotherapies are currently available. While enzyme-based strategies offer promise, an effective treatment must address not only cocaine but also its major active and/or toxic metabolites-benzoylecgonine, norcocaine, and cocaethylene, several of which exhibit greater toxicity than cocaine itself. Here, we report for the first time that two engineered cocaine esterases, E196-301 and BZEase2, are capable of hydrolyzing cocaine and its three toxic metabolites. By integrating molecular modeling with comprehensive in vitro kinetic analysis and in vivo efficacy studies, we demonstrate that these enzymes possess distinct yet complementary substrate selectivity: E196-301 efficiently catalyzes the hydrolysis and clearance of cocaine, norcocaine, and cocaethylene, whereas BZEase2 preferentially degrades benzoylecgonine. Both enzymes conferred significant therapeutic benefits in rodent models of acute cocaine-metabolite exposure and cocaine-alcohol co-administration, though neither alone achieved complete detoxification against cocaine and its toxic metabolites under the tested conditions. Their complementary substrate profiles suggest combining E196-301 and BZEase2 or engineering next-generation broad-spectrum hydrolases may offer a rational strategy for comprehensive detoxification across the full spectrum of cocaine-related toxins. This work establishes a critical foundation for enzyme-based therapy targeting cocaine's multifaceted toxicology.