T NIH-PA Author ManuscriptRESULTSModelling /-Puma:Mcl-1 interactions Our previous research making use of
T NIH-PA Author ManuscriptRESULTSModelling /-Puma:Mcl-1 interactions Our preceding studies utilizing /-peptides primarily based on the Puma BH3 domain involved an backbone pattern. Upon adoption of an -helix-like conformation, this pattern gives rise to a “stripe” of residues along the helix axis [4c]. There are seven techniques in which this pattern could be imposed on a given helical amino acid sequence, and we found that the placement on the residues within the Puma K-Ras Inhibitor Molecular Weight sequence strongly influences pro-survival protein binding [4c]. Comparable trends have been subsequently observed with Bim BH3-based Calcium Channel Inhibitor Gene ID foldamers [4b]. The Puma-based foldamers that displayed high affinity for pro-survival proteins bound selectively (100-fold) to Bcl-xL more than Mcl-1. The most beneficial of those molecules, 1 (Fig. 1A), was shown to bind tightly to Bcl-2 and Bcl-w as well; however, 1 exhibited only weak affinity for Mcl-1. Making use of the structure with the 1:Bcl-xL complicated (PDB: 2YJ1), we created a model of 1 bound to Mcl-1 with the aim of designing Puma-based /-peptides that display improved affinity for Mcl-1. This model complicated was generated by superimposing the structure of Bcl-xL in complex with 1 with all the structure of Mcl-1 in complex with -Puma (PDB: 2ROC) [6b], removing Bcl-xL and -Puma, and then minimizing the remaining 1:Mcl-1 complicated. Inspection in the model recommended many adjustments towards the /-peptide that could potentially improve affinity. 1) Replacement of Arg3 of 1 with Glu. We previously observed that changing of Arg3 of 1 to Ala leads to enhanced Mcl-1 affinity, most likely because of removal of a possible steric clash and/or electrostatic repulsion with all the side-chain of His223 [5c]. This putative unfavorable interaction is reflected within the calculated model by a movement of His223 away from the Arg3 side-chain (Supp Fig. 1A). The binding of 1 to Mcl-1 was also enhanced by changing Arg229 and His233 of Mcl-1 to Ala [5c]. We therefore proposed that replacing Arg3 on 1 with Glu could engage a favourable electrostatic interaction with Arg229, as shown inside the model (Supp. Fig. 1B), or alternatively mimic the interaction among 1 and Bcl-xL in this area, forming a hydrogen bond among Arg3 on 1 and Glu129 on Bcl-xL (this residue is analogous to His223 in Mcl-1). two) Filling a smaller hydrophobic pocket adjacent to Gly6 of 1. We proposed that this pocket could accommodate a D-alanine residue, resulting in favourable contacts with Mcl-1 (Supp Figs 1C,D). three) Replacement of Leu9 having a residue bearing a bigger side-chain. Our Mcl-1+/-peptide model revealed a hydrophobic pocket beneath Leu9, that is also observed in some X-ray crystal structures of BH3 peptides bound to Mcl-1 [13]. Accordingly, we predicted that lengthening this side chain on the /-peptide would improve affinity for Mcl-1. Modeling predicted that a norleucine side-chain (n-butyl) would have minimal impact on affinity (Supp. Fig. 1E), but that extension to an n-pentyl side-chain would completely fill the pocket (Supp. Fig. 1F) and most likely impart higher affinity. Binding affinities of modified /-Puma foldamers Variants of 1 primarily based on the designs described above have been synthesised (Fig. 1A) and tested in competition binding assays employing surface plasmon resonance (Figs. 1B,C). /-Peptide two, in which Arg3 was replaced with Glu, had a 15-fold decrease IC50 for Mcl-1 relative to 1, while 3, in which Gly6 was replaced with D-Ala, had a 10-fold achieve in affinity compared to 1. Replacing Leu9 with norleucine (four) had no impact on affinity for Mcl-1, w.
