175 Structure-based engineering to generate high-affinity immunotherapy for the drug of abuse |
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| Authors: | Shraddha Thakkar Nisha Nanaware-Kharade Guillermo Gonzalez III Reha Celikel Eric Peterson |
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| Institution: | University of Arkansas for Medical Sciences , Little Rock , AR , 72205 , USA |
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| Abstract: | Methamphetamine (METH) abuse is a major threat in the USA and worldwide without any FDA approved medications. Anti-METH antibody antagonists block or slow the rate of METH entry into the brain and have shown efficacy in preclinical studies (Peterson, Laurenzana, Atchley, Hendrickson, & Owens, 2008 Peterson, E. C., Laurenzana, E. M., Atchley, W. T., Hendrickson, H. P. and Owens, S. M. 2008. Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine. Journal of Pharmacology and Experimental Therapeutics, 08: 124–133. Google Scholar]).?A key determinant of the antibody’s efficacy is its affinity for METH and we attempted to enhance the efficacy by designing mutations to alter the shape or the electrostatic character of the binding pocket. Towards this goal, we developed a single chain anti-METH antibody fragment (scFv6H4) from a parent IgG (1). The crystal structure of scFv-6H4 in complex with METH was determined (Celikel, Peterson, Owens, & Varughese, 2009 Celikel, R., Peterson, E. C., Owens, S. M. and Varughese, K. I. 2009. Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine. Protein Science, 09: 2336–2345. Google Scholar]). Based on its elucidated binding interactions, we designed point mutations in the binding pocket to improve its affinity for METH and amphetamine (AMP), the active metabolite of METH. The mutants, scFv-S93T,-I37?M and -Y34?M were cloned, expressed in yeast and tested for affinity against METH and AMP. Two mutants showed enhanced binding affinity for METH: scFv-I37?M by 1.3-fold and scFv-S93T by 2.6-fold. Additionally, all the mutants showed increase in affinity for AMP: scFv-I37?M by 56-fold, scFv-S93T by 17-fold and scFvY34?M by 5-fold. Crystal structure for one of the high-affinity mutant, scFv-S93T, in complex with METH was determined (Figure 1). Binding pocket of the mutant is more hydrophobic in comparison with the wild type. ScFv-6H4 binds METH in a deep pocket containing two water molecules. The substitution of a serine residue by a threonine leads to the expulsion of a water molecule (Figure 2), relieving some unfavorable contacts between the hydrocarbon atoms of METH and the water molecule and increasing the affinity to sub-nanomolar range. Therefore, the present study shows that efficacy could be enhanced by altering the hydrophobicity or the shape of the binding pocket. | |
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