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Generation of high affinity anti‐methamphetamine antibodies using structure‐based engineering
Author(s) -
Thakkar Shraddha,
NanawareKharade Nisha,
Gonzalez Guillermo,
Celikel Reha,
Peterson Eric C.,
Varughese Kottayil I.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.895.8
Subject(s) - meth , mutant , chemistry , binding site , protein engineering , antibody , mutation , stereochemistry , biochemistry , enzyme , biology , gene , genetics , monomer , organic chemistry , acrylate , polymer
Methamphetamine (METH) abuse is a threat in the US and worldwide, with no FDA approved treatments available. Anti‐METH IgGs and its smaller variant, single chain fragments (scFvs) have shown efficacy in preclinical studies. The goal of study is to improve efficacy of a therapeutic single chain fragment (scFv6H4) by increasing its affinity for METH. Here we describe our efforts to design higher affinity antibody mutants based on the crystal structure of scFv6H4, in complex with METH. Using the structural information, we made single amino acid mutations in the binding pocket of scFv6H4 to alter the hydrophobicity of the binding site. The new mutants; scFv‐S93T, ‐I37M, and ‐Y34M were cloned, expressed in yeast and tested for affinity by saturation binding techniques. Two of the mutant scFv showed enhanced binding affinity for METH; scFvI37M by 25% and scFvS93T by 150%. We have also determined the crystal structure of the highest‐affinity mutant scFvS93T in complex with METH. This structure shows that, as we hypothesized, the substitution of a Ser residue by a Thr clearly facilitated the removal of a water molecule from the binding cavity. Interestingly, this mutation increased the hydrophobicity of the binding pocket as a result of expulsion of water molecule, and hence dramatically improved the binding of METH in the cavity. NIDA R01 DA026423 (ECP)