Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

T. Thisted, Z. Biesova, C. Walmacq, E. Stone, M. Rodnick-Smith, S.S. Ahmed, S.K. Horrigan, B. Van Engelen, C. Reed, M.W. Kalnik*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Background Smoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction. Results This paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2- to 19-fold enhanced catalytic activity compared to wt at 10 mu M nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen's access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity. Conclusions Systematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme's immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.
Original languageEnglish
Article number56
Number of pages16
JournalBMC Biotechnology
Volume19
Issue number1
DOIs
Publication statusPublished - 2 Aug 2019

Keywords

  • addiction
  • blood
  • brain reward systems
  • degradation
  • disease
  • drug design
  • enzyme
  • enzyme catalysis
  • immunization
  • metabolism
  • motivation
  • nicotine
  • nicotine-degrading enzyme
  • noncontingent nicotine
  • opponent-process theory
  • protein engineering
  • rats
  • reinforcement
  • smoking cessation
  • smoking withdrawal dynamics
  • Nicotine-degrading enzyme
  • RATS
  • NONCONTINGENT NICOTINE
  • Degradation
  • Smoking cessation
  • IMMUNIZATION
  • Enzyme catalysis
  • BRAIN REWARD SYSTEMS
  • Drug design
  • BLOOD
  • Protein engineering
  • Enzyme
  • SMOKING WITHDRAWAL DYNAMICS
  • Metabolism
  • MOTIVATION
  • OPPONENT-PROCESS THEORY
  • Addiction
  • DISEASE
  • REINFORCEMENT
  • Nicotine

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