Stapled Peptides Inhibitors: A New Window for Target Drug Discovery

Abstract

Protein-protein interaction (PPI) is a hot topic in clinical research as protein networking has a major impact in human disease. Such PPIs are potential drugs targets, leading to the need to inhibit/block specific PPIs. While small molecule inhibitors have had some success and reached clinical trials, they have generally failed to address the flat and large nature of PPI surfaces. As a result, larger biologics were developed for PPI surfaces and they have successfully targeted PPIs located outside the cell. However, biologics have low bioavailability and cannot reach intracellular targets. A novel class -hydrocarbon-stapled α-helical peptides that are synthetic mini-proteins locked into their bioactive structure through site-specific introduction of a chemical linker- has shown promise. Stapled peptides show an ability to inhibit intracellular PPIs that previously have been intractable with traditional small molecule or biologics, suggesting that they offer a novel therapeutic modality. In this review, we highlight what stapling adds to natural-mimicking peptides, describe the revolution of synthetic chemistry techniques and how current drug discovery approaches have been adapted to stabilize active peptide conformations, including ring-closing metathesis (RCM), lactamisation, cycloadditions and reversible reactions. We provide an overview on the available stapled peptide high-resolution structures in the protein data bank, with four selected structures discussed in details due to remarkable interactions of their staple with the target surface. We believe that stapled peptides are promising drug candidates and open the doors for peptide therapeutics to reach currently "undruggable" space.

Keywords: Drug discovery; Inhibitor; PPI; Stapled peptide; Synthetic chemistry.

Graphical abstract

Fig. 1

The three classes of targeted medicines. The traditional small- molecules inhibitors were the first class discovered to inhibit different PPIs surfaces with MW of <500 Da and high bioavailability. Most of the biologics, the second class of PPI targeted molecules, have a MW of >5000 Da (eg. antibodies and growth hormones) aimed to overcome a broad range of diseases. Stapled α-helix peptides as a class address this gap in MW between small molecules and biologics, aiming to combine the oral bioavailability of small-molecules with the high specificity of biologics toward the target protein.

Fig. 2

Statistical representation of therapeutic peptides until March 2017. The numbers are indicated in percentage at each category with a total number of 484 medicinal peptides that were produced with development activity regulatory approval from major pharmaceutical markets as, the United States, Europe, and Japan. From these peptides 12% were approved, while 32% are in clinical trials and further classified as phases I, II, III and pre-registered. The highest percentage (54%; “Discontinued”) category encompasses peptides terminated before approval. The lowest percentage 2% is the “Withdraw” category that refers to previously approved peptides that are no longer available in the market [18].

Fig. 3

Ruthenium-catalyzed ring-closing metathesis (RCM) reaction for peptides stapling was a) published for the first time by Verdine and Schafmeister in 2000 by engaging α,α- disubstituted non-natural amino acids harboring all-hydrocarbon tethers [19]. Their work was a continuation of b) Blackwell and Grubbs work in 1998 [24]; who performed ruthenium-catalyzed olefin metathesis for macrocyclisation of synthetic peptides using a pair of O-allylserine residues in a metathesis reaction.

Fig. 4

Workflow of all hydrocarbon-stapled peptides generated for biological investigation. Computational designation of the peptides including in-situ mutagenesis to screen all possibilities based on previous reported structures, followed by in vitro biochemical, structural, and functional studies compromising peptides binding affinities measurements toward the target protein interface utilizing biophysical assays and crystallization trials. Potent binder peptides will be further tested for their cellular uptake and permeability using live confocal microscopy. Lastly, successful peptides are subjected to a broad spectrum of cellular and in vivo analyses, using mouse models of the studied disease.

Fig. 5

a) The common stapling insertion positions for α-helix peptides. Combinations of two non-natural amino acids S5, R5, S8 and R8 are used for different positions of stapling the hydrocarbon linker. Employing S5/S5 at position i,i + 4 is the most common stapling position on the same face of helix turn. For i,i + 7 position, two combinations could be applied either S8/R5 or S5/R8. Synthetic chemistry evolved to introduced i,i + 3 and i,i + 11 as new possible positions for stapling in addition to double-stapling. b) The structures of the four designed amino acids used to introduce all-hydrocarbon staples into peptides. All possess an α-methyl group (Me) and an α-alkenyl group, but with opposite stereochemical configuration and different length at the alkenyl chain.

Fig. 6

RCM or ring closing metathesis reaction for synthesis of the all-hydrocarbon stapled peptide reported by Schafmeister et al. 2000, which increase peptides helicity as found by circular dichroism (CD) [19].

Fig. 7

A Lactamisation study that was conducted by Fairlie and co-workers on penta and hexa-peptides in order to optimize lactam stapling between Orn/Lys and Asp/Glu residues. It wasn't the first study for lactam optimization; however, the group was abled to systematically and quantitatively found the shortest peptide with retained helicity in water as judged by CD [39].

Fig. 8

Optimized CuAAC-stapled peptide was successfully developed to inhibit the BCL9 oncogenic interaction. After screening different stapling length, Wang and co-workers concluded that five units of methylene was optimal stapled peptide for BLC9 inhibition [43].

Fig. 9

Schultz and co-workers described an i,i + 7 stapling methodology using disulphide bridges between D and L amino acids bearing thiol-side chains. The amino acids were connected with acetamidomethyl (Acm) protecting groups, deprotected and then oxidised with iodine to give a disulphide stapled peptide. CD spectra of disulphide stapled peptides exhibited a high level of α-helicity in comparison to the Acm-protected precursors that were significantly less helical [45].

Fig. 10

Thioether stapling method was reported by Brunel and Dawson in 2005. They demonstrated the reaction of Cys thiol and alpha-bromo amide groups to report a i,i + 3 thioether stapled peptide that inhibited HIV fusion using the gp41 epitopes as template for peptide synthesis [49].

Fig. 11

Alignment of the SAH-p53-8 peptide (yellow, PDB 3V3B) and the native p53 peptide (cyan, PDB 1YCR). The MDM2 molecule is shown in surface representation. SAH-p53-8 peptide mimics the three pharmacophore residues (Phe19, Leu26, Trp23) in the binding site in a similar manner to the native p53. The residues outside the Phe19- Leu26 regions are not visible, indicating conformational flexibility in the bound state. Moreover, the whole helix of stapled peptide moves by ~1 Å and is rotated by 18°, allowing the Trp23 indole ring to form a hydrogen bond with MDM2 Leu54 (green line). Interestingly, Leu26 orientates itself in a distinct manner to that of the native p53 ^Leu26, (moving by 2.7 Å toward the N-terminus of the peptide) and the side chain is flipped by approximately 180° to fill the same pocket space. This feature is not found in any other reported structure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 12

A closer view of the SAH-p53-8 stapled peptide in a “closed” conformation state. The MDM2 molecule is shown in surface representation, the peptide (yellow) and the staple (orange) in sticks. A hydrogen bond is formed between the indole nitrogen atom of the peptide helix and the carbonyl oxygen of Leu54 of MDM2 (green line). This H-bound is protected from solvent competition by the staple that lied directly over Met50-Lys64 helix (the rim of p53 binding site). In addition, the staple intimately interacts with the protein surface and forms an extended hydrophobic interface with Leu54, Phe55, Gly58, and Met62 of Mdm2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 13

The E1-MDM2 complex high-resolution structure at 1.9 Å. a) top view of E1 stapled peptide (magenta, PDB 5AFG) aligned with the native peptide p53 (cyan, PDB 1YCR), revealing the typical mode of binding within the MDM2 hydrophobic pocket (grey surface) - placing the triad residues responsible for binding (P2he19, Trp23, Leu26) in the correct orientation to engage the MDM2 hotspots. The staple is found in anti regioisomer form and interacts with protein surface a similar mode as b) previously reported hydrocarbon SAH-p53-8 stapled peptide (PDB 3V3B), in that the stapled form four hydrophobic interactions with MDM2 surface residues (Leu54, Phe55, Gln59 and Met62, lime green), in which Phe55 is the most critical residue. The superimposition of the triazole-stapled E1 peptide with the correlated hydrocarbon-stapled p53 peptide (yellow, PDB 3V3B) suggests that both staples engage the same area that is located at the rim of the p53 binding pocket, on the Met50–Lys64 helix. The E1 stapled peptide is also shown in 2D for clarity (right). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 14

The crystal structure of MCL-1 SAHB_D stapled peptide (slate helix) binding to the MCL-1ΔNΔC (light pink surface) interface at the canonical BH3-binding groove, solved at 2.3 Å resolution (PDB 3MK8). The peptide makes several hydrophobic interactions, including the hydrophobic residues Leu213, Val216, Gly217, and Val220 of MCL-1 SAHB_D making direct contact with a hydrophobic cleft at the surface of MCL- 1ΔNΔC (hot pink). The hydrophobic interaction are reinforced by a salt bridge between MCL-1 SAHB_D Asp218 and MCL-1ΔNΔC Arg263 (blue) and these residues also contribute to a hydrogen bond cluster that includes MCL-1ΔNΔC Asp256 and Asn260 (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 15

The hydrocarbon staple of MCL-1 SAHB_D peptide with an alkene functionality in the cis conformation (yellow stick) makes distinct hydrophobic contacts with the MCL-1ΔNΔC binding site border (light pink surface). A methyl group of the α,α-dimethyl functionality engages with a groove consisting of MCL-1ΔNΔC Gly262, Phe318, and Phe319 residues (raspberry sticks). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 16

The crystal structures of the SP1 ERβ (PDB 1YJD) and SP2 ERα (PDB 2YJA) complexes at 1.9 and 1.8 Å resolution, respectively. Van der Waal interactions (yellow dash lines) were found between both staples of SP1 and SP2 peptides and the hydrophobic residues on the surface of the co-activator binding site Val307, Ile310 and Leu490. ERβ and ERα proteins are shown in surface representation, while the staple of the both peptides and the interacting residues are in sticks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 17

Comparison analysis of a) SP1 (lime green helix) and b) SP2 (aquamarine helix) stapled peptides in relation to previously reported NR co-activator peptide 2 (light magenta helix, PDB 2QGT). a) SP1 stapled peptide exhibited a quarter turn with respect to the co-activator peptide locating the hydrophobic staple to the recognition site position. While b) SP2 rotates differently and packs tighter than the coactivator peptide 2 does. Additionally, two residues in the receptor site (Asp538 and Ile358) induce conformational changes bridging the staple of SP2 helix to the C-terminus site of the recognition motif that is rotated by 100° toward the other side of the protein IL__LL contact site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 18

Crystal structures of successful small molecules inhibiting drug-target PPIs that have entered clinical trials. a) ABT-737 (pink sticks) binds to BCL-xL (PDB 2YXJ) with nanomolar binding affinity, b) Nutlin-2 (green sticks), is one of the first identified potent MDM2–p53 inhibitors and is shown bound to the N-terminal domain of MDM2 (PDB 1RV1) and c) geldanamycin (GM) (light blue stick) in complex with Hsp90 (PDB 1YET), considered the first Hsp90 inhibitor to enter clinical trials. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)