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Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide.

Luzi S, Kondo Y, Bernard E, Stadler LK, Vaysburd M, Winter G, Holliger P - Protein Eng. Des. Sel. (2015)

Bottom Line: Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging.The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNFα cytokine into dimers and monomers.The discovery of M21 underlines the potential of semi-synthetic bicyclic peptides as ligands for the discovery of cryptic epitopes, some of which are poorly accessible to antibodies.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

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Mass spectrometry (MS), analytical ultracentrifugation (AUC) and light-scattering experiments (MALS). (A) LCT ESI-TOF MS: TNF-α (10 μM) without (blue) or with excess M21h (100 µM) incubated for (A) 4 weeks (RT) or (B) 1 h (4°C, PBS) (C) AUC: 10 µM TNFα with (red line) or without (blue line) unlabeled bicyclic peptide (M21h) and cy5-labeled bicycle (cy5-M21h) incubated for 42 h at RT before centrifugation at 45 krpm and sedimentation and sedimentation (D) SEC-MALS: TNFα (10 µM) with or without M21h pre-incubated for different times at RT.
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GZU055F3: Mass spectrometry (MS), analytical ultracentrifugation (AUC) and light-scattering experiments (MALS). (A) LCT ESI-TOF MS: TNF-α (10 μM) without (blue) or with excess M21h (100 µM) incubated for (A) 4 weeks (RT) or (B) 1 h (4°C, PBS) (C) AUC: 10 µM TNFα with (red line) or without (blue line) unlabeled bicyclic peptide (M21h) and cy5-labeled bicycle (cy5-M21h) incubated for 42 h at RT before centrifugation at 45 krpm and sedimentation and sedimentation (D) SEC-MALS: TNFα (10 µM) with or without M21h pre-incubated for different times at RT.

Mentions: In order to investigate the nature of the time-dependent effect on TNFα inhibition by M21, we analyzed M21h peptide-TNFα complexes using liquid chromatography electrospray ionization time-of-flight mass spectrometry (LCT ESI-TOF MS), analytical ultracentrifugation (AUC) and size-exclusion chromatography multi-angle static light-scattering (SEC-MALS). MS spectra of TNFα showed a clear peak at around 52,508 Da corresponding to the trimeric form and stable over a long timeframe (4 weeks at 4°C). However, after incubation with bicyclic peptide M21h two peaks of 36,463 and 37,145 Da form, corresponding to a TNFα dimer with and without bound bicyclic peptide (Fig. 3A). After longer pre-incubation, TNFα trimers are converted quantitatively into dimers and monomers by M21h (Fig. 3B).Fig. 3.


Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide.

Luzi S, Kondo Y, Bernard E, Stadler LK, Vaysburd M, Winter G, Holliger P - Protein Eng. Des. Sel. (2015)

Mass spectrometry (MS), analytical ultracentrifugation (AUC) and light-scattering experiments (MALS). (A) LCT ESI-TOF MS: TNF-α (10 μM) without (blue) or with excess M21h (100 µM) incubated for (A) 4 weeks (RT) or (B) 1 h (4°C, PBS) (C) AUC: 10 µM TNFα with (red line) or without (blue line) unlabeled bicyclic peptide (M21h) and cy5-labeled bicycle (cy5-M21h) incubated for 42 h at RT before centrifugation at 45 krpm and sedimentation and sedimentation (D) SEC-MALS: TNFα (10 µM) with or without M21h pre-incubated for different times at RT.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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GZU055F3: Mass spectrometry (MS), analytical ultracentrifugation (AUC) and light-scattering experiments (MALS). (A) LCT ESI-TOF MS: TNF-α (10 μM) without (blue) or with excess M21h (100 µM) incubated for (A) 4 weeks (RT) or (B) 1 h (4°C, PBS) (C) AUC: 10 µM TNFα with (red line) or without (blue line) unlabeled bicyclic peptide (M21h) and cy5-labeled bicycle (cy5-M21h) incubated for 42 h at RT before centrifugation at 45 krpm and sedimentation and sedimentation (D) SEC-MALS: TNFα (10 µM) with or without M21h pre-incubated for different times at RT.
Mentions: In order to investigate the nature of the time-dependent effect on TNFα inhibition by M21, we analyzed M21h peptide-TNFα complexes using liquid chromatography electrospray ionization time-of-flight mass spectrometry (LCT ESI-TOF MS), analytical ultracentrifugation (AUC) and size-exclusion chromatography multi-angle static light-scattering (SEC-MALS). MS spectra of TNFα showed a clear peak at around 52,508 Da corresponding to the trimeric form and stable over a long timeframe (4 weeks at 4°C). However, after incubation with bicyclic peptide M21h two peaks of 36,463 and 37,145 Da form, corresponding to a TNFα dimer with and without bound bicyclic peptide (Fig. 3A). After longer pre-incubation, TNFα trimers are converted quantitatively into dimers and monomers by M21h (Fig. 3B).Fig. 3.

Bottom Line: Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging.The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNFα cytokine into dimers and monomers.The discovery of M21 underlines the potential of semi-synthetic bicyclic peptides as ligands for the discovery of cryptic epitopes, some of which are poorly accessible to antibodies.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

Show MeSH
Related in: MedlinePlus