Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinoprop...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2022-03, Vol.298 (3), p.101689-101689, Article 101689
Main Authors: Kirley, Terence L., Norman, Andrew B., Greis, Kenneth D.
Format: Article
Language:English
Subjects:
addiction therapy
Antibodies, Monoclonal, Humanized - immunology
cocaine
Cocaine - immunology
Cocaine - metabolism
Immunoglobulin Fab Fragments - chemistry
Immunoglobulin Fab Fragments - immunology
Immunoglobulin Fab Fragments - metabolism
ligand affinity
monoclonal antibody
oxidation
Oxidation-Reduction
tryptophan
Tryptophan - chemistry
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000–10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.
ISSN:0021-9258
1083-351X
DOI:10.1016/j.jbc.2022.101689