Dataset Information

Protein Footprinting and X-ray Crystallography Reveal the Interaction of PD-L1 and a Macrocyclic Peptide.

ABSTRACT: Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.

SUBMITTER: Niu B

PROVIDER: S-EPMC7485629 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Protein Footprinting and X-ray Crystallography Reveal the Interaction of PD-L1 and a Macrocyclic Peptide.

Niu Ben B Appleby Todd C TC Wang Ruth R Morar Mariya M Voight Johannes J Villaseñor Armando G AG Clancy Sheila S Wise Sarah S Belzile Jean-Philippe JP Papalia Giuseppe G Wong Melanie M Brendza Katherine M KM Lad Latesh L Gross Michael L ML

Biochemistry 20191231 4

Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, h ...[more]

PMID: 31841311

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Project description:BackgroundPD-1/PD-L1 immune checkpoint inhibitors are currently the most commonly utilized agents in clinical practice, which elicit an immunostimulatory response to combat malignancies. However, all these inhibitors are currently administered via injection using antibody-based therapies, while there is a growing need for oral alternatives.MethodsThis study has developed and synthesized exosome-wrapped gold-peptide nanocomplexes with low immunogenicity, which can target PD-L1 and activate antitumor immunity in vivo through oral absorption. The SuperPDL1exo was characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and gel silver staining. The transmembrane ability of SuperPDL1exo was evaluated by flow cytometry and immunofluorescence. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay. ELISA experiments were conducted to detect serum and tissue inflammatory factors, as well as serum biochemical indicators. Tissue sections were stained with H&E for the evaluation of the safety of SuperPDL1exo. An MC38 colon cancer model was established in immunocompetent C56BL/6 mice to evaluate the effects of SuperPDL1exo on tumor growth in vivo. Immunohistochemistry (IHC) staining was performed to detect cytotoxicity factors such as perforin and granzymes.ResultsFirst, SuperPDL1 was successfully synthesized, and milk exosome membranes were encapsulated through ultrasound, repeated freeze-thaw cycles, and extrusion, resulting in the synthesis of SuperPDL1exo. Multiple characterization results confirmed the successful synthesis of SuperPDL1exo nanoparticles. Furthermore, our data demonstrated that SuperPDL1exo exhibited excellent colloidal stability and superior cell transmembrane ability. In vitro and in vivo experiments revealed that SuperPDL1exo did not cause damage to multiple systemic organs, demonstrating its good biocompatibility. Finally, in the MC38 colon cancer mouse model, it was discovered that SuperPDL1exo could inhibit the progression of colon cancer, and this tumor-suppressive effect was mediated through the activation of tumor-specific cytotoxic T lymphocyte (CTL)-related immune responses.ConclusionThis study has successfully designed and synthesized an oral nanotherapeutic, SuperPDL1exo, which demonstrates small particle size, excellent colloidal stability, transmembrane ability in tumor cells, and biocompatibility. In vivo experiments have shown that it effectively activates T-cell immunity and exerts antitumor effects.

Dataset Information

Protein Footprinting and X-ray Crystallography Reveal the Interaction of PD-L1 and a Macrocyclic Peptide.

Publications

Protein Footprinting and X-ray Crystallography Reveal the Interaction of PD-L1 and a Macrocyclic Peptide.

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