Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells.

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Reengineering of Albumin-Fused Cocaine Hydrolase CocH1 (TV-1380) to Prolong Its Biological Half-Life.

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.

Dimerization of human butyrylcholinesterase expressed in bacterium for development of a thermally stable bioscavenger of organophosphorus compounds.

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.

Computational design, functional analysis and antigenic epitope estimation of a novel hybrid of 12 peptides of hirudin and reteplase.

Cardiovascular and cerebrovascular diseases are leading causes of morbidity and mortality for human beings, and thrombosis is the major risk factor. Thrombolytic therapy has been testified to be the most effective approach to cure thrombosis-related diseases. In clinical treatment, we often adopt a combination therapeutic regimen of both thrombolytic and anticoagulant agents to prevent the recurrence of thrombosis. Thus, a novel hybrid (HV12p-rPA) comprised of the C-terminal 12 residues of hirudin-PA (HV12p) and reteplase (rPA) was designed. The three-dimensional structure of this hybrid was mimicked based on homology modeling and refined with dynamics simulation by utilizing Amber12.0 software. The function of the hybrid was analyzed by structure comparison and the root mean square deviation (RMSD) of Cα atoms between the hybrid and native rPA was calculated. The results showed that HV12p, which was located in the N-terminus of the hybrid, was far from the rPA segment of the hybrid and had no influence on the conformational stability of the rPA domain. The RMSD of Cα atoms of these superimposed proteins was about 40Å, implying that the hybrid had a similar spatial conformation to that of native rPA. Additionally, the antigenic epitopes of the hybrid were predicted by estimations of Hopp-Wood hydrophilicity, Janin accessibility, Zimmermane-Simha polarity, Bhaskaran-Ponnuswamy flexibility, as well as secondary structure analysis and Kolaskar-Tongaonkar antigenicity prediction. The results showed that the most likely antigenic determinants were located at or near regions 148-152, 257-262 and 321-330.