Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

Abstract

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, KD = 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 Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ 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.

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

“…For this study, two different scFv constructs were used scFv6H4 and scFv7F9, with the primary differences being in their sequences of the three complimentarity-determining regions (CDRs). [ 43 ] As can be seen in Fig 3 , scFv6H4 was consistently detected at roughly 2.5-3x higher concentrations than scFv7F9, yet in Fig 5A and 5B the disposition of METH in the brains and serum samples of the mice are similar for the two scFvs. Considering this, AAV-scFv6H4 was likely in higher concentrations due to its ability to form multimers with itself and thus have a longer half-life.…”

Searching for a Reliable Density Functional for Molecule–Environment Interactions, Found B97M-V/def2-mTZVP

“…Specifically, we have examined additional complexes of aspirin and complexes for d -amphetamine, ibuprofen, and paracetamol, which are also commonly used small molecular drugs. The PDB crystal structures that we use for extracting the additional model complexes are 3IAZ, 4NSB, and 6MQF for aspirin; 4LAR and 4XP9 for d -amphetamine; 2BXG, 2PWS, 2WD9, 3P6H, 5JQB, and 6OCI for ibuprofen; and 1TYL, 2DPZ, 2OCU, 4A9J, 4CUT, and 4YJI for paracetamol. We can see that the r 2 SCAN-3c method shows significant deviations (MAD = 21.9 kJ mol –1 ).…”

Impact of methamphetamine on infection and immunity

“…(Roll et al, 2006 ) Currently, no specific medications exist that counteract the effects of METH or that prolong abstinence from the abuse of METH by an addict. However, novel anti-METH immunotherapies, primarily in the form of monoclonal antibodies and lipid-based vaccines, are in early clinical trial phases and act as pharmacokinetic antagonists, isolating METH and its metabolites from vulnerable areas in the brain and minimizing the toxic effects of the drug (Peterson et al, 2013 ; Rüedi-Bettschen et al, 2013 ; Collins et al, 2014 ; Hambuchen et al, 2014 ).…”