Abstract
Therapeutic treatment of cocaine toxicity or addiction is a grand medical challenge. As a promising therapeutic strategy for treatment of cocaine toxicity and addiction to develop a highly efficient cocaine hydrolase (CocH) capable of accelerating cocaine metabolism to produce physiologically/biologically inactive metabolites, our previously designed A199S/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE), known as cocaine hydrolase-1 (CocH1), possesses the desirably high catalytic activity against cocaine. The C-terminus of CocH1, truncated after amino acid #529, was fused to human serum albumin (HSA) to extend the biological half-life. The C-terminal HSA-fused CocH1 (CocH1-HSA), known as Albu-CocH1, Albu-CocH, AlbuBChE, Albu-BChE, or TV-1380 in literature, has shown favorable preclinical and clinical profiles. However, the actual therapeutic value of TV-1380 for cocaine addiction treatment is still limited by the short half-life. In this study, we designed and tested a new type of HSA-fused CocH1 proteins, i.e., N-terminal HSA-fused CocH1, with or without a linker between the HSA and CocH1 domains. It has been demonstrated that the catalytic activity of these new fusion proteins against cocaine is similar to that of TV-1380. However, HSA-CocH1 (without a linker) has a significantly longer biological half-life (t1/2 = 14 ± 2 h) compared to the corresponding C-terminal HSA-fused CocH1, i.e., CocH1-HSA (TV-1380 with t1/2 = 5-8 h), in rats. Further, the N-terminal HSA-fused CocH1 proteins with a linker have further prolonged biological half-lives: t1/2 = 17 ± 2 h for both HSA-EAAAK-CocH1 and HSA-PAPAP-CocH1, and t1/2 = 18 ± 3 h for HSA-(PAPAP)2-CocH1. These N-terminal HSA-fused CocH1 proteins may serve as more promising protein drug candidates for cocaine addiction treatment.