Rational Design of Proteolytically Stable, Cell-Permeable Peptide-Based Selective Mcl-1 Inhibitors

Abstract

Direct chemical modifications of helical peptides have provided a simple and effective means to ‘translate’ the bioactive helical peptides into potential therapeutics targeting intracellular protein-protein interactions. Previously, we have shown that the distance-matching bisaryl cross-linkers can reinforce peptide helices containing two cysteines at the i.i+7 positions and confer cell permeability to the cross-linked peptides. In this work, we report the first crystal structure of a biphenyl cross-linked Noxa peptide in complex with its target Mcl-1 at a 2.0 Å resolution. Guided by this structure, we remodeled the surface of this cross-linked peptide through side chain substitution and N-methylation, and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells. The success of this structure-based design of Mcl-1 inhibitors underscores the value of synergistic use of multifaceted modifications in developing peptide-based therapeutics.

Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery

“…However, peptide-based drugs generally suffer from short half-lives due to proteolytic degradation and fast renal clearance, rendering higher doses and frequent injections necessary, which negatively affects patient compliance (2). To improve their pharmacological properties, peptides have been chemically modified by conformational restriction (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) to increase potency and reduce proteolysis, and also by lipidation (14)(15)(16), polymer conjugation (17)(18)(19)(20)(21)(22)(23), and protein fusion (24)(25)(26) to decrease renal clearance. Although these latter conjugates can have enhanced circulatory half-lives, they often suffer from reduced potency and, as a result, require injection of relatively large quantities of the modified peptides.…”

Identifying Immunogenic CD4+ T-Cell Epitopes of Myeloid Cell Leukemia 1 Using Overlapping 20-mer Peptides Spanning the Whole Protein

“…Recently, it has been demonstrated that downregulation of Mcl-1 by various pharmacological agents or genetic approaches dramatically increases ABT-737 lethality in various malignant cell types [4]. Different strategies for targeting Mcl-1 include (i) small interfering RNA [5] (ii) small-molecule inhibitors [6] and (iii) peptide inhibitors [7]. In recent years, therapeutic vaccination with synthetic peptides derived from anti-apoptotic proteins such as Mcl-1 has emerged as a promising strategy against hematological cancers.…”

Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing simple aryl and vinylaryl cross-linkers

“…7 More recently, other covalent linkages generated through olefin metathesis, 8 1,3-dipolar cycloaddition reactions, 9,10 and cysteine alkylation 11,12 have renewed the interest in developing the side chain cross-linked peptides (“stapled peptides”) as potential drug candidates for the various biological targets. 13–15 For example, we reported a simple side chain cross-linking chemistry based on the distance-matching biaryl cross-linkers, 12 and successfully employed this strategy in designing the cell-permeable and proteolytically stable stapled Noxa BH3 peptides as potent Mcl-1 inhibitors, 16 the stapled peptide-based dual inhibitors of Mdm2/Mdmx, 17 and a dual-active stapled peptide that binds to the C-terminal domain of HIV capsid protein as well as the envelop glycoprotein gp120 and inhibits HIV-1 virus entry and assembly. 18 …”

“…Several parameters such as helicity, amphipathicity, charge, and hydrophobicity have been shown to be important for peptide cell permeability. 12,16,17,19,20 While the amino acid residues are crucial to the physicochemical properties of a stapled peptide including its cell permeability, the effect of the cross-linker structure has not been systematically investigated, particularly with regard to the length and flexibility of the cross-linkers. It has been shown that the rigid cross-linkers exhibited stronger effect in stabilizing helical conformation than the flexible ones.…”

“…In our previous studies, we designed a pair of rigid distance-matching cross-linkers, 12 namely, 4,4′-bisbromomethyl-biphenyl (Bph) and 6,6′-bis-bromomethyl-[3,3′] bipyridine (Bpy) and showed that the attachment of Bph and Bpy to the cysteines located at i , i +7 positions of the NoxaB-(75–93)-C75A peptide dramatically increased the biological activity and cellular uptake of the Noxa BH3 peptides. 16 To probe the effect of cross-linker structure on physicochemical properties of the stapled Noxa BH3 peptides, here we synthesized a series of simple aryl and vinylaryl cross-linkers with varying length, rigidity, and hydrophobicity. The effect of cross-linker structure on stapled peptide conformation was investigated by circular dichroism.…”