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Designed ankyrin repeat proteins as scaffolds for multivalent recognition.

ABSTRACT: Ankyrin repeat (AR) proteins are composed of tandem repeats of a basic structural motif of ca. 33 amino acid residues that form a ?-turn followed by two antiparallel ?-helices. Multiple repeats stack together in a modular fashion to form a scaffold that is ideally suited for the presentation of multiple functional groups and/or recognition elements. Here we describe a biosynthetic strategy that takes advantage of the modular nature of these proteins to generate multivalent ligands that are both chemically homogeneous and structurally well-defined. Glycosylated AR proteins cluster the tetrameric lectin concanavalin A (Con A) at a rate that is comparable to the rate of Con A aggregation mediated by globular protein conjugates and variable density linear polymers. Thus, AR proteins define a new class of multivalent ligand scaffolds that have significant potential application in the study and control of a variety of multivalent interactions.

SUBMITTER: Hollenbeck JJ

PROVIDER: S-EPMC3398510 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Designed ankyrin repeat proteins as scaffolds for multivalent recognition.

Hollenbeck Jessica J JJ Danner Derek J DJ Landgren Rachel M RM Rainbolt Thomas K TK Roberts Danielle S DS

Biomacromolecules 20120621 7

Ankyrin repeat (AR) proteins are composed of tandem repeats of a basic structural motif of ca. 33 amino acid residues that form a β-turn followed by two antiparallel α-helices. Multiple repeats stack together in a modular fashion to form a scaffold that is ideally suited for the presentation of multiple functional groups and/or recognition elements. Here we describe a biosynthetic strategy that takes advantage of the modular nature of these proteins to generate multivalent ligands that are both ...[more]

PMID: 22681396

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Project description:Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g-1 (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g-1; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

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Designed ankyrin repeat proteins as scaffolds for multivalent recognition.

Publications

Designed ankyrin repeat proteins as scaffolds for multivalent recognition.

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