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Thermal stabilizing mutants of cocaine esterase
Author(s) -
Narasimhan Diwahar,
Ko Meichuan,
Nance Mark,
Macdonald Joanne,
Landry Don,
Woods James,
Zhan ChangGuo,
Sunahara Roger
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.657.1
Subject(s) - pharmacology , cocaine abuse , allosteric regulation , enzyme , addiction , dopamine , chemistry , transporter , dopamine transporter , esterase , medicine , biochemistry , psychiatry , gene
Despite advances in pharmacotherapeutics that target the dopamine transporter the development of therapeutics that combat cocaine abuse and overdose have been less fruitful. More classical approaches to therapeutic development against cocaine abuse and overdose have inherent challenges in that competitive and allosteric inhibitors of cocaine binding to the transporter exhibit similar behavioral effects of cocaine: inhibition of dopamine uptake. We have recently reported the use of cocaine esterase as a protective therapy against cocaine‐induce lethality. The acceleration of enzyme‐mediated digestion of systemic cocaine by exogenously added cocaine esterase represents a significant paradigm shift in cocaine abuse therapy. Here we report the design and generation of significantly more stable enzyme preparations using computational approaches. We provided evidence from both in vitro and in vivo studies that the modified enzyme displays a prolonged half‐life (up to 30‐fold) and improved thermostability than the wild‐type enzyme. Moreover we have obtained x‐ray crystallographic evidence that provide a structural rationale for the improved enzyme stability. The improved enzyme stability will have a profound influence on the potential of this agent to challenge cocaine‐induced lethality and cocaine addiction in humans.