Fluences pro-survival protein binding [4c]. Comparable trends have been subsequently observed with Bim BH3-based foldamers [4b]. The Puma-based foldamers that displayed higher affinity for pro-survival proteins bound selectively (100-fold) to Bcl-xL more than Mcl-1. The most beneficial of these molecules, 1 (Fig. 1A), was shown to bind tightly to Bcl-2 and Bcl-w as well; on the other hand, 1 exhibited only weak affinity for Mcl-1. Working with the structure of the 1:Bcl-xL complex (PDB: 2YJ1), we produced a model of 1 bound to Mcl-1 with all the aim of designing Puma-based /-peptides that show elevated affinity for Mcl-1. This model complex was generated by superimposing the structure of Bcl-xL in complicated with 1 with all the structure of Mcl-1 in complicated with -Puma (PDB: 2ROC) [6b], removing Bcl-xL and -Puma, and after that minimizing the remaining 1:Mcl-1 complicated. Inspection in the model recommended numerous changes for the /-peptide that could potentially enhance affinity. 1) Replacement of Arg3 of 1 with Glu. We previously observed that changing of Arg3 of 1 to Ala results in enhanced Mcl-1 affinity, in all probability as a consequence of removal of a possible steric clash and/or electrostatic repulsion together with the side-chain of His223 [5c]. This putative unfavorable interaction is reflected in 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 for that reason proposed that replacing Arg3 on 1 with Glu could engage a favourable electrostatic interaction with Arg229, as shown in the model (Supp. Fig. 1B), or alternatively mimic the interaction amongst 1 and Bcl-xL in this region, forming a hydrogen bond among Arg3 on 1 and Glu129 on Bcl-xL (this residue is analogous to His223 in Mcl-1). 2) Filling a modest 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 boost affinity for Mcl-1.A 1120 MedChemExpress Modeling predicted that a norleucine side-chain (n-butyl) would have minimal influence on affinity (Supp.Trypsin Description Fig.PMID:23522542 1E), but that extension to an n-pentyl side-chain would totally fill the pocket (Supp. Fig. 1F) and most likely impart higher affinity. Binding affinities of modified /-Puma foldamers Variants of 1 determined by the styles described above have been synthesised (Fig. 1A) and tested in competitors binding assays working with surface plasmon resonance (Figs. 1B,C). /-Peptide 2, in which Arg3 was replaced with Glu, had a 15-fold decrease IC50 for Mcl-1 relative to 1, whilst 3, in which Gly6 was replaced with D-Ala, had a 10-fold achieve in affinity compared to 1. Replacing Leu9 with norleucine (4) had no effect on affinity for Mcl-1, when replacing Leu9 with homonorleucine (pentyl side-chain), which we designate HL (five), enhanced affinity by approximately 4-fold. The behaviour of 4 and 5 is constant together with the modelbased predictions. Combinations with the effective substitutions resulted in additional increasesChembiochem. Author manuscript; offered in PMC 2014 September 02.Smith et al.Pagein affinity. The Arg3Glu plus Gly6D-Ala combination (six) binds to Mcl-1 55-fold much more tightly than does /-peptide 1.