Abstract
Inhibiting the histone H3–ASF1 (anti‐silencing function 1) protein–protein interaction (PPI) represents a potential approach for treating numerous cancers. As an α‐helix‐mediated PPI, constraining the key histone H3 helix (residues 118–135) is a strategy through which chemical probes might be elaborated to test this hypothesis. In this work, variant H3118–135 peptides bearing pentenylglycine residues at the i and i+4 positions were constrained by olefin metathesis. Biophysical analyses revealed that promotion of a bioactive helical conformation depends on the position at which the constraint is introduced, but that the potency of binding towards ASF1 is unaffected by the constraint and instead that enthalpy–entropy compensation occurs.
Highlights
Histone chaperone antisilencing function 1 (ASF1) is a highly conserved histone chaperone involved in both Histone H3-H4 handling and buffering. [34,35,36,37,38] It has been shown to play a key role in development and progression of some cancers, represents a potential target for chemical probe and drug discovery.[39,40,41]
The ASF1 protein comprises a conserved N-terminal domain of 156 amino acids, which is essential for its function in vivo, and a divergent unstructured C-terminal domain, which is not considered necessary for function.[37, 43]
The histone H3 binding site is located in the concave face of ASF1 (Fig. 1b) and involves strands 3, 4, and 6-9.[37, 43,44] The main interactions occur through the Cterminal helix of H3, where the key residues Leu126 and Ile130 form a hydrophobic clamp with the hydrophobic region of ASF1
Summary
W e used S-pentenylglycine rather than S-pentenylalanine as the former is easier to synthesize and demonstrates comparable behavior in biophysical analyses.[27] The sites to incorporate the mono-alkenyl substituted amino acids within the peptide sequence were selected taking into account: (i) the requirement to appropriately position the non-natural amino acids so as to constrain in a manner that promotes a helical conformation (i.e. i and i+4 positions); (ii) the need to position the hydrocarbon bridge so as not to sterically occlude “wild-type” interactions necessary for recognition. It is perhaps unsurprising that constraining the peptide between residues 120 and 124 did not promote a helical conformation in H3118-135(St120-124) given the observation from the H3/ASF1 NMR structure that the H3 helix is Binding of the peptides to ASF1 was assessed using isothermal titration calorimetry (Fig. 3, Table 1).
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