N‐locking stabilization of covalent helical peptides: Application to Bfl‐1 antagonists

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N‐lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N‐locked covalent BH3 peptides that were designed to selectiv...

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Published in:	Chemical biology & drug design Vol. 95; no. 4; pp. 412 - 426
Main Authors:	Baggio, Carlo, Udompholkul, Parima, Gambini, Luca, Jossart, Jennifer, Salem, Ahmed F., Håkansson, Maria, Perry, J. Jefferson P., Pellecchia, Maurizio
Format:	Journal Article
Language:	English
Published:	England 01-04-2020
Subjects:	Amino Acid Sequence Apoptosis Regulatory Proteins - chemistry Apoptosis Regulatory Proteins - pharmacokinetics Bcl‐2 family proteins Bfl‐1 covalent inhibitors Crystallization Forkhead Transcription Factors - chemistry Forkhead Transcription Factors - metabolism Humans Models, Molecular Nerve Tissue Proteins - chemistry Nerve Tissue Proteins - metabolism N‐locked peptide Peptide Fragments - chemistry Peptide Fragments - metabolism PPIs protein Protein Binding Protein Conformation protein interactions Proto-Oncogene Proteins - chemistry Proto-Oncogene Proteins - metabolism stapled alpha helix protein interactions covalent inhibitors stapled alpha helix N-locked peptide protein Bcl-2 family proteins Bfl-1 PPIs
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Summary:	Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N‐lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N‐locked covalent BH3 peptides that were designed to selectively target the anti‐apoptotic protein Bfl‐1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl‐1 and cellular activity. The crystal structure of the complex between such N‐locked covalent peptide and Bfl‐1 provided insights on the geometry of the N‐locking strategy and of the covalent bond between the agent and Bfl‐1. We report on a simple yet very effective as a valuable complementary method, termed covalent N‐locking, to hydrocarbon‐ and/or lactam‐based alpha helix stapling, for the design of helix‐based covalent protein–protein interaction antagonists. As an application, we derived and characterized, using biochemical assays, NMR and X‐ray crystallography, a covalent N‐locked BH3 peptide targeting the anti‐apoptotic protein Bfl‐1.
Bibliography:	Author contributions: MP directed research, designed and conceived the compounds listed, coordinated testing of the molecules, interpreted data and results, and wrote the manuscript with the help of CB and other authors. MP also performed modeling studies and prepared several of the Figures with the help of CB. In addition, CB expressed and purified proteins, synthesized and purified several of the peptides, carried out the CD measurements, conducted NMR studies including all data analysis and resonance assignments, and ran the SDS gel electrophoresis experiments. PU performed the DELFIA assays reported in Fig 3A and Table 1. LG helped with the synthesis and purification of selected agents. AFS performed the cell based apoptosis assays reported in Fig 5. MH performed crystallization studies and prepared Fig 4B. JJ and JPP and in collaboration with MP, analyzed X-ray structural data and helped prepare Fig 4.
ISSN:	1747-0277 1747-0285
DOI:	10.1111/cbdd.13661