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Inhibition of cocaine binding to the human dopamine transporter by a single chain anti-idiotypic antibody: its cloning, expression, and functional properties.

ABSTRACT: Conventional drug development for treatment of cocaine addiction is greatly hindered by the extreme difficulty in designing a selective cocaine antagonist. We employed anti-idiotypic (anti-Id) antibodies to generate cocaine antagonists. The purpose of this study was to investigate the feasibility of this alternative approach. Herein, we describe the molecular cloning, bacterial expression, and functional properties of an anti-Id monoclonal antibody (mAb), designated K2-3f, which possesses an internal image of cocaine within its variable regions. The heavy and light chain variable domains of K2-3f were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and a single chain antibody variable fragment (scFv) was assembled for expression in Escherichia coli. The scFv bound to the human dopamine transporter (hDAT) with moderate affinity (K(a)=5.3 x 10(6) M(-1)) and excellent mimicry of the cocaine molecule completely inhibited cocaine binding at a molar concentration closely resembling in vivo conditions while allowing approximately 90% of equimolar dopamine uptake. Our data suggest that the use of anti-Id antibody as a template for generation of a cocaine antagonist is a promising approach well worth pursuing. If this strategy is successful, it could be applied to potential ligand-receptor interactions in the treatment of other diseases.

SUBMITTER: Ho M

PROVIDER: S-EPMC3295240 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Inhibition of cocaine binding to the human dopamine transporter by a single chain anti-idiotypic antibody: its cloning, expression, and functional properties.

Ho Mitchell M Segre Mariangela M

Biochimica et biophysica acta 20030701 3

Conventional drug development for treatment of cocaine addiction is greatly hindered by the extreme difficulty in designing a selective cocaine antagonist. We employed anti-idiotypic (anti-Id) antibodies to generate cocaine antagonists. The purpose of this study was to investigate the feasibility of this alternative approach. Herein, we describe the molecular cloning, bacterial expression, and functional properties of an anti-Id monoclonal antibody (mAb), designated K2-3f, which possesses an int ...[more]

PMID: 12878327

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Project description:The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

Dataset Information

Inhibition of cocaine binding to the human dopamine transporter by a single chain anti-idiotypic antibody: its cloning, expression, and functional properties.

Publications

Inhibition of cocaine binding to the human dopamine transporter by a single chain anti-idiotypic antibody: its cloning, expression, and functional properties.

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