Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine

Abstract

Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. human butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native human BChE has a low catalytic activity against cocaine. We recently designed and discovered a BChE mutant (A199S/F227A/S287G/A328W/Y332G) with a high catalytic activity (kcat = 5700 min−1, KM = 3.1 μM) specifically for cocaine, and the mutant was proven effective in protecting mice from acute cocaine toxicity of a lethal dose of cocaine (180 mg/kg, LD100). Further characterization in animal models requires establishment of a high-efficiency stable cell line for the BChE mutant production in a relatively larger scale. It has been extremely challenging to develop a high-efficiency stable cell line expressing BChE or its mutant. In the present study, we successfully developed a stable cell line efficiently expressing the BChE mutant by using a lentivirus-based repeated-transduction method. The scale-up protein production enabled us to determine for the first time the in vivo catalytic activity and the biological half-life of this high-activity mutant of human BChE in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant (administrated to mice 1 min prior to cocaine) can quickly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Oligomerization and Catalytic Parameters of Human UDP-Glucuronosyltransferase 1A10: Expression and Characterization of the Recombinant Protein

“…Cell lines stably overexpressing human UGT1A10 were generated using a lentivirus-based method described in our previous report (Xue et al, 2013). Briefly, the human UGT1A10-6xHis gene was first synthesized by Genscript Corporation (Piscataway, NJ) based on the published sequence in GenBank (NM_019075.2) and inserted in the pCSC-SP-PW vector, lentivirus plasmid.…”

Long-acting cocaine hydrolase for addiction therapy

“…The control curves in Fig. 3 reflect the overall effects of all possible cocaine-elimination pathways (25)(26)(27). In the control rats, the average concentration of cocaine at the first time point (2 min) was ∼7.4 μM, whereas the average concentration of benzoic acid was ∼0.2 μM.…”

“…Further, we note that the enzyme fusion with Fc (or Fc mutant) does not necessarily prolong the biological t 1/2 of an enzyme. In fact, we also tested fusing a thermally stable mutant of bacterial cocaine esterase (CocE) (24) with Fc(M3) and found no significant improvement in the biological t 1/2 (within the measurement errors), with t 1/2 still being only within a few minutes (SI Appendix, To characterize further the most promising CocH3-Fc form, CocH3-Fc(M3), we developed a stable cell line using a lentiviral vector (25) for large-scale production of CocH3-Fc(M3). The purified CocH3-Fc(M3) is indeed a dimer, as expected (SI Appendix, Fig.…”

“…Large-Scale Protein Expression and Purification. A lentivirus-based method described in our previous report (25) was performed to generate a highly efficient stable cell line expressing CocH3-Fc(M3). Briefly, to package the lentivirus particles carrying the gene of CocH3-Fc(M3), lentivirus was produced by cotransfecting pCSC-CocH3-Fc(M3) plasmid with the packaging vectors (pMDLg/pRRE and pRSV-Rev) and envelope plasmid (pCMV-VSV-G) into HEK-293FT cells by lipofection.…”

“…The chromatographic analysis was carried out on a Gemini 5-μm C18 column (Phenomenex), using a mobile phase at a flow rate of 1 mL/min. The (−)-cocaine fluorescence was monitored at 315 nm with excitation at 230 nm (25,34), and benzoic acid absorbance was monitored at 230 nm. The chromatographic system used comprised a Waters 1525 binary HPLC pump, a 717 plus Autosampler, a 2487 dual λ absorbance detector, and a 2475 multi λ fluorescence detector (Waters).…”