Fluorine-Thiol Displacement Stapling on the Disordered α_B of pKID Domain Increases Its Helicity and Affinity to KIX

Abstract

The development of high-affinity ligands specifically targeting intrinsically disordered protein interactions has remained challenging due to the lack of well-defined binding pockets and shallow binding surfaces commonly found at their interfaces. Here, we employed our fluorine-thiol displacement reaction (FTDR) peptide-stapling platform to synthesize a library of peptide-based ligands derived from the α_B-helix of the disordered pKID to target its binding partner KIX. Our library revealed that helical formation and affinity to KIX is highly favored when the α_B peptide was stapled at sites corresponding to Arg¹³⁵ and Ser¹⁴², further supporting the hypothesis that stabilization of α_B significantly influences the overall binding affinity of pKID to KIX. We also found that the highest binding peptide, α_B-RSpS, may form secondary contacts at the MLL site on KIX in addition to binding at the primary pKID site. Lastly, no binding to KIX was observed for any α_B-stapled peptide that lacked the conserved helix-flanking prolines Pro¹³² and Pro¹⁴⁶. Conserved helix-flanking prolines have previously been shown to modulate the binding affinities of other disordered domains in other proteins including MLL and p53. However, to our knowledge this is the first evidence within α_B of pKID.

Key words

stapled peptides - fluorine-thiol displacement reaction (FTDR) - pKID - phosphorylation - KIX - intrinsically disordered proteins (IDPs)

Publication History

Received: 15 June 2024

Accepted after revision: 03 September 2024

Accepted Manuscript online:
03 September 2024

Article published online:
27 September 2024

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• 38 General Procedures Peptide Synthesis All peptides were synthesized using a CS136X (CSBio) automated peptide synthesizer on rink amide resin. For each coupling step, 3 equiv of Fmoc-protected amino acid, 3 equiv of HBTU, and 6 equiv of DIPEA were added to resin suspended in DMF. Synthesis and incorporation of the Fmoc-protected fluoroacetamide amino acid building block (X_L) used for stapling was followed as previously reported.¹⁹,²³ Phosphorylated α_B peptides were synthesized using Fmoc-Ser[PO(OBzl)OH]-OH (Bachem) and coupled in the same fashion as described above. For N-terminal FITC labeling of the peptides, an additional Fmoc-β-Ala residue was coupled, subsequently deprotected, and resin-bound peptides were reacted with 3 equiv of FITC and 6 equiv of DIPEA in DMF for 15 h. Peptides acetylated at the N-terminal were capped using 50 equiv of acetic anhydride and 50 equiv of DIPEA in DMF for 1 h. Peptides were cleaved from resin with 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane for 3 h at room temperature. The cleaved peptide solution was filtered from the resin, evaporated under nitrogen, precipitated with cold diethyl ether, and pelleted through centrifugation. Crude peptide pellets were resuspended in 25% acetonitrile in water and purified using a Waters RP-HPLC equipped with a semipreparative C18 column (10 mL/min flow rate, solvent A: water/0.1% TFA, solvent B: acetonitrile/0.1% TFA, 10–60% acetonitrile gradient). Collected fractions were lyophilized and the purity were confirmed with an Agilent 1260 LC–MS. Fluorine-Thiol Displacement Reaction Peptide Stapling All α_B peptides were stapled using the FTDR procedure as previously reported.¹⁹ In general, 250 mM dithiol linker (1,3-benzenedimethanthiol for i,i+4 stapling and 1,4-benzenedimethanethiol for i,i+7 stapling) in DMF was premixed with 250 mM sodium hydroxide at room temperature for 20 min to ensure complete deprotonation of the dithiol linkers. After 20 min, 50 mM of unstapled peptide in water was added and the pH was adjusted to 9.5–10. The reaction mixture was incubated at 37 °C until full conversion to the stapled product was confirmed by LC–MS. Reactions were quenched with acetic acid, stapled peptides were extracted with 25% acetonitrile in water and purified by HPLC as previously described.