Dataset Information

Identification of C-6 as a New Site for Linker Conjugation to the Taccalonolide Microtubule Stabilizers.

ABSTRACT: The taccalonolides are a class of microtubule stabilizers that circumvent clinically relevant forms of drug resistance due to their unique mechanism of microtubule stabilization imparted by the covalent binding of the C-22-C-23 epoxide moiety to tubulin. A taccalonolide (8) with a fluorescein group attached with a linker at C-6 was generated, and biochemical and cell-based assays showed that it bound directly to tubulin and stabilized microtubules. This pharmacological probe has allowed, for the first time, a direct visualization of a taccalonolide binding to microtubules, verifying their cellular binding site. This C-6-modified taccalonolide showed potency comparable to the untagged compound in biochemical experiments; however, its potency was lower in cellular assays, presumably due to decreased cellular permeability. These studies provide a valuable tool to facilitate the further understanding of taccalonolide pharmacology and demonstrate that C-6 is a promising site for a linker to be added to this novel class of microtubule stabilizers for targeted drug delivery.

SUBMITTER: Du L

PROVIDER: S-EPMC6952213 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Identification of C-6 as a New Site for Linker Conjugation to the Taccalonolide Microtubule Stabilizers.

Du Lin L Risinger April L AL Yee Samantha S SS Ola Antonius R B ARB Zammiello Cynthia L CL Cichewicz Robert H RH Mooberry Susan L SL

Journal of natural products 20190225 3

The taccalonolides are a class of microtubule stabilizers that circumvent clinically relevant forms of drug resistance due to their unique mechanism of microtubule stabilization imparted by the covalent binding of the C-22-C-23 epoxide moiety to tubulin. A taccalonolide (8) with a fluorescein group attached with a linker at C-6 was generated, and biochemical and cell-based assays showed that it bound directly to tubulin and stabilized microtubules. This pharmacological probe has allowed, for the ...[more]

PMID: 30799622

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Project description:Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

Dataset Information

Identification of C-6 as a New Site for Linker Conjugation to the Taccalonolide Microtubule Stabilizers.

Publications

Identification of C-6 as a New Site for Linker Conjugation to the Taccalonolide Microtubule Stabilizers.

Similar Datasets

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