Dataset Information

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

ABSTRACT: Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

SUBMITTER: Ozay EI

PROVIDER: S-EPMC5167783 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Publications

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Ozay E Ilker EI Gonzalez-Perez Gabriela G Torres Joe A JA Vijayaraghavan Jyothi J Lawlor Rebecca R Sherman Heather L HL Garrigan Daniel T DT Burnside Amy S AS Osborne Barbara A BA Tew Gregory N GN Minter Lisa M LM

Molecular therapy : the journal of the American Society of Gene Therapy 20160916 12

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase C ...[more]

PMID: 27633441

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Project description:Protein-protein interactions (PPIs) are ubiquitous in biological systems and often misregulated in disease. As such, specific PPI modulators are desirable to unravel complex PPI pathways and expand the number of druggable targets available for therapeutic intervention. However, the large size and relative flatness of PPI interfaces make them challenging molecular targets. This Account describes our systematic approach using secondary and tertiary protein domain mimics (PDMs) to specifically modulate PPIs. Our strategy focuses on mimicry of regular secondary and tertiary structure elements from one of the PPI partners to inspire rational PDM design. We have compiled three databases (HIPPDB, SIPPDB, and DIPPDB) of secondary and tertiary structures at PPI interfaces to guide our designs and better understand the energetics of PPI secondary and tertiary structures. Our efforts have focused on three of the most common secondary and tertiary structures: α-helices, β-strands, and helix dimers (e.g., coiled coils). To mimic α-helices, we designed the hydrogen bond surrogate (HBS) as an isosteric PDM and the oligooxopiperazine helix mimetic (OHM) as a topographical PDM. The nucleus of the HBS approach is a peptide macrocycle in which the N-terminal i, i + 4 main-chain hydrogen bond is replaced with a covalent carbon-carbon bond. In mimicking a main-chain hydrogen bond, the HBS approach stabilizes the α-helical conformation while leaving all helical faces available for functionalization to tune binding affinity and specificity. The OHM approach, in contrast, envisions a tetrapeptide to mimic one face of a two-turn helix. We anticipated that placement of ethylene bridges between adjacent amides constrains the tetrapeptide backbone to mimic the i, i + 4, and i + 7 side chains on one face of an α-helix. For β-strands, we developed triazolamers, a topographical PDM where the peptide bonds are replaced by triazoles. The triazoles simultaneously stabilize the extended, zigzag conformation of β-strands and transform an otherwise ideal protease substrate into a stable molecule by replacement of the peptide bonds. We turned to a salt bridge surrogate (SBS) approach as a means for stabilizing very short helix dimers. As with the HBS approach, the SBS strategy replaces a noncovalent interaction with a covalent bond. Specifically, we used a bis-triazole linkage that mimics a salt bridge interaction to drive helix association and folding. Using this approach, we were able to stabilize helix dimers that are less than half of the length required to form a coiled coil from two independent strands. In addition to demonstrating the stabilization of desired structures, we have also shown that our designed PDMs specifically modulate target PPIs in vitro and in vivo. Examples of PPIs successfully targeted include HIF1α/p300, p53/MDM2, Bcl-xL/Bak, Ras/Sos, and HIV gp41. The PPI databases and designed PDMs created in these studies will aid development of a versatile set of molecules to probe complex PPI functions and, potentially, PPI-based therapeutics.

Dataset Information

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Publications

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

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