Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
Department of Physiology and Biophysics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
PLoS One. 2013 Dec 5;8(12):e82690. doi: 10.1371/journal.pone.0082690. eCollection 2013.
Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K(D) = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy"). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni(2+). Two of the histidine residues of each C-terminal His-tag interact with Ni(2+) in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.
疫苗和单克隆抗体（mAb）治疗（+）-甲基苯丙胺（METH）滥用分别处于临床前后期和早期临床试验阶段。这些免疫疗法作为药代动力学拮抗剂，将 METH 及其代谢物从大脑中的作用部位隔离出来，并减少药物的奖赏和毒性作用。这些免疫疗法策略的一个关键方面是了解产生对 METH 具有高亲和力和特异性的抗体的重要细微分子相互作用。我们之前确定了与 METH 和 (+) 立体异构 3,4-亚甲二氧基甲基苯丙胺（MDMA，或“摇头丸”）复合物的高亲和力抗 METH 治疗性单链抗体片段（scFv6H4，K(D) = 10 nM）的晶体结构。在这里，我们报告了 scFv6H4 在同源三聚体未结合（apo）形式（2.60Å）的晶体结构，以及与两种活性代谢物；（+）苯丙胺（AMP，2.38Å）和（+）-4-羟基 METH（p-OH-METH，2.33Å）复合物的单体形式。apo 结构在晶体晶格中形成三聚体，导致形成具有三倍对称的分子间复合β-片层。我们还能够对 His 标签与 Ni(2+) 的配位进行结构表征。每个 C 末端 His 标签的两个组氨酸残基以八面体几何形状与 Ni(2+)相互作用。在 apo 状态下，scFv6H4 的 CDR 环形成结合口袋的开放构象。配体结合后，CDR 环采用封闭构象，几乎完全包裹药物。这里报道的结构信息阐明了在抗甲基苯丙胺滥用免疫治疗中重要的关键分子相互作用。
