Fig 3. Comparative characterizations of N-locked covalent peptides targeting Bfl-1.

A) Chemical structure of 138C5 (Table 1). Biochemical dose response curves representing the ability of each tested agent to displace the binding of a reference BH3 peptide from Bfl-1 or Bcl-xL (2 h incubation, see methods) are also reported. The curves represent the dose response curves obtained with the BIM peptide (black), with a linear covalent Bfl-1 antagonist 135P1 (blue), and with its N-locked corresponding agent, 138C5 (red) (Table 1 and structure shown above). Data were collected in duplicates and experiments were repeated twice, all other experimental details for the DELFIA assay are reported in the methods section. B) SDS gel electrophoresis were performed incubating either hBfl-1-ΔTM or hBcl-xL-ΔTM (10 μM) with the indicated covalent agents (each at 100 μM concentration). Gel shifts can be clearly appreciated when the Bfl-1 is exposed to the covalent agents, while no shifts are observed when the compounds are exposed to Bcl-xL. No DTT was present in the protein buffer. C) Superposition of the 1D ¹H NMR spectra of the linear covalent agent 135P1 (blue) and its corresponding N-locking equivalent, 138C5 (red) (Table 1). The spectra were recorded at 700 MHz ¹H frequency with equimolar samples of each peptide (500 μM) at 15 °C in water. The ³J^αHN coupling constant for residue Ile 2 of the N-locked agent is also reported. D) 2D [¹H,¹H] NOESY and CD spectra of the 138C5 recorded with a 500 μM peptide sample in water at 15 °C. A number sequential NOEs are clearly observed. NOEs between the amide proton of residue 1 (Dap) and the H^γ1,2 of Glu 4 are highlighted (confirming the cyclic structure) as well as the NOEs between the amide proton of Ile2 and H^β1,2 of Dap1.