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Designed Ankyrin Repeat Proteins: A New Class of Viral Entry Inhibitors.

ABSTRACT: Designed ankyrin repeat proteins (DARPins) are engineered proteins comprising consensus designed ankyrin repeats as scaffold. Tightly packed repeats form a continuous hydrophobic core and a large groove-like solvent-accessible surface that creates a binding surface. DARPin domains recognizing a target of interest with high specificity and affinity can be generated using a synthetic combinatorial library and in vitro selection methods. They can be linked together in a single molecule to build multispecific and multifunctional proteins without affecting expression or function. The modular architecture of DARPins offers unprecedented possibilities of design and opens avenues for innovative antiviral strategies.

SUBMITTER: Walser M

PROVIDER: S-EPMC9611651 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Designed Ankyrin Repeat Proteins: A New Class of Viral Entry Inhibitors.

Walser Marcel M Mayor Jennifer J Rothenberger Sylvia S

Viruses 20221012 10

Designed ankyrin repeat proteins (DARPins) are engineered proteins comprising consensus designed ankyrin repeats as scaffold. Tightly packed repeats form a continuous hydrophobic core and a large groove-like solvent-accessible surface that creates a binding surface. DARPin domains recognizing a target of interest with high specificity and affinity can be generated using a synthetic combinatorial library and in vitro selection methods. They can be linked together in a single molecule to build mul ...[more]

PMID: 36298797

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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.

Dataset Information

Designed Ankyrin Repeat Proteins: A New Class of Viral Entry Inhibitors.

Publications

Designed Ankyrin Repeat Proteins: A New Class of Viral Entry Inhibitors.

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