Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb.
Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb.
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DOI：
10.1016/j.jbc.2022.101689
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Greis KD
中科院分区：
文献类型：
--
作者：
Kirley TL;Norman AB;Greis KD
可卡因成瘾仍然是一个严重的问题，缺乏有效的药物治疗。因此，我们开发了一种高亲和力的抗可卡因单克隆抗体（mAb）， h2E2，用于治疗可卡因使用障碍。我们发现选择性色氨酸（Trp）被2,2 ' -偶氮（2-氨基丙烷）二盐酸（AAPH）氧化导致可卡因失去与该单抗的高亲和力结合。新开发的使用过量蛋氨酸（Met）来保护mAb的Met残基免受AAPH氧化的影响，并没有实质性地减弱氧化对可卡因结合的影响，但大大减少了mAb中Met残基的修饰。使用可卡因和可卡因的两种代谢物，可卡因和苯甲酰茶碱，也以纳米摩尔亲和力结合h2E2单抗，观察到氧化后配体亲和力的类似大幅下降（5000 - 10,000倍）。结合亲和力的降低伴随着色氨酸荧光的下降约50%，mAb 310至370 nm吸光度的增加与色氨酸氧化形式的存在一致。最后，对对照和aaph氧化mAb衍生的肽的质谱分析表明，过量的游离met确实有效地保护mAb满足的残基免受氧化，并且aaph氧化的mAb重链Trp33和轻链Trp91残基对可卡因结合很重要，这与最近获得的含有结合苯甲酰ecgonine的h2E2 Fab片段晶体结构一致。因此，抗可卡因h2E2单抗在临床给药前免受色氨酸氧化的保护对于其在治疗可卡因使用障碍方面的治疗用途至关重要。
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.
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影响因子：
2.7
作者：
Kirley TL;Greis KD;Norman AB
通讯作者：
Norman AB
影响因子：
4.8
作者：
Kirley TL;Norman AB
通讯作者：
Norman AB
影响因子：
2.2
作者：
Kirley, Terence L.;Norman, Andrew B.;Wetzel, Hanna N.
通讯作者：
Wetzel, Hanna N.
影响因子：
--
作者：
Wong, Jensen J. H.;Wright, Sara K.;Katayama, Derrick S.
通讯作者：
Katayama, Derrick S.
DOI：
10.1016/j.bbrc.2017.04.117
发表时间：
2017-06-03
影响因子：
3.1
作者：
Wetzel HN;Webster RP;Saeed FO;Kirley TL;Ball WJ;Norman AB
通讯作者：
Norman AB