Rational design of a helical peptide inhibitor targeting c-Myb–KIX interaction

Abstract

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb–KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb–KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb–KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein–protein interactions.

The free energy folding penalty accompanying binding of intrinsically disordered α‐helical motifs

“…This translates into a roughly 2–3 times stronger IDP‐target affinity. Interestingly, the same magnitude of stabilization has been observed recently for the c‐Myc‐KIX peptide inhibitor, which has been stabilized using several conservative K to R substitutions 26 . In this modified variant IDP helicity increased by around 10% leading to the affinity increase of −0.5 kcal/mol.…”

“…Interestingly, the same magnitude of stabilization has been observed recently for the c‐Myc‐KIX peptide inhibitor, which has been stabilized using several conservative K to R substitutions. 26 In this modified variant IDP helicity increased by around 10% leading to the affinity increase of −0.5 kcal/mol. A similar, around −0.2 to −0.3 kcal/mol stabilization per 10% helicity increase have been also reported for other systems.…”

“… 16 , 20 , 23 , 25 On the other hand, substitutions with the helix promoting residues, such as alanine, leucine or arginine can increase IDP helicity, reduce the folding penalty and consequently increase the IDP‐target affinity. 16 , 23 , 25 , 26 , 27 Stabilization of helical binding motifs, particularly using the hydrocarbon stapling, has proven to be an efficient strategy for the rational design of peptide inhibitors of IDP‐target interactions (reviewed in Walensky and Bird 28 ). In this strategy, a covalent bond (hydrocarbon staple) is introduced between two modified residues on the same side of helix (usually between residues i and i + 4), which effectively stabilizes helical structure.…”

Design and Biochemical Characterization of Peptidic Inhibitors of the Myb/p300 Interaction

“…A tyrosine at position-301 increases potency by about ninefold (29), indicating that the presence of an aromatic moiety and/ or of a hydroxyl group, which could form a H-bond, is favorable for the interaction with p300 KIX . Finally, we observe a 38-fold gain in potency using the large aliphatic homo-βcyclohexyl-alanine (30). Altogether, the results obtained with a limited number of mutations at position-301 show that, despite the properties of the p300 KIX surface in the contact region with Leu301 Myb , it is possible to significantly improve the potency of 3.…”

“…For several years, it has been known that the dysregulation of Myb can lead to various cancers and, more particularly, those of hematopoietic origin. − The interaction between Myb, via its transactivation domain, and the KIX domain of p300 has also been shown to be important for tumorigenesis. − This suggests that preventing the Myb/p300 interaction could be a strategy to develop new anticancer drugs. , To date, several tool molecules that block the interaction between Myb and the KIX domain of p300 have been described. − These initial findings are encouraging because they show that it is possible to inhibit the Myb/p300 interaction; however, more potent compounds need to be developed to successfully treat cancer patients. Since the region of Myb that binds to p300 KIX is intrinsically disordered, these molecules should be designed so that they bind to p300 KIX , the only well-folded component of this interaction.…”