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Tumor Necrosis Factor-α Trimer Disassembly and Inactivation via Peptide-Small Molecule Synergy.

ABSTRACT: Aberrant signaling by tumor necrosis factor-α (TNFα) is associated with inflammatory diseases that can be treated with engineered proteins that inhibit binding of this cytokine to cell-surface receptors. Despite these clinical successes, there is considerable interest in the development of smaller antagonists of TNFα-receptor interactions. We describe a new 29-residue α/β-peptide, a molecule that contains three β-amino acid residues and three α-aminoisobutryic acid (Aib) residues, that displays potent inhibition of TNFα binding to TNFα receptor 1 (TNFR1) and rescues cells from TNFα-induced death. The complement of nonproteinogenic residues renders this α/β-peptide highly resistant to proteolysis, relative to all-α analogues. The mechanism of inhibitory action of the new 29-mer involves disruption of the trimeric TNFα quaternary structure, which prevents productive binding to TNFα receptors. Unexpectedly, we discovered that peptide-induced trimer disruption can be promoted by structurally diverse small molecules, including a detergent commonly used during selection procedures. The discovery of this synergistic effect provides a new context for understanding previous reports on peptidic antagonists of TNFα-receptor interactions and suggests new avenues for future efforts to block signaling via proteins with an active form that is oligomeric, including other members of the TNFα family.

SUBMITTER: Checco JW

PROVIDER: S-EPMC7654703 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOne-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-? (TNF-?).MethodsMice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-?, were tested for their effects on breast cancer cell proliferation.ResultsWe produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-?, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-?.ConclusionsNovel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-? provide evidence for a complex interplay between HER2 and TNF-? signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

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Tumor Necrosis Factor-α Trimer Disassembly and Inactivation via Peptide-Small Molecule Synergy.

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