The a-helix is a ubiquitous structure within proteins that is characterized by a rotation of the backbone with 3.6 amino acids within each turn. Defined torsion angles of the a-helix backbone, with phi (NH—Ca) being −57.8° and psi (Ca—C=O) being −47.0°, have been well defined using X-ray crystal structures.10 This turn creates three separate faces of the a-helix (Figure 1.2). An interesting feature of this secondary structure was first elucidated by Isabella Karle and colleagues where they determined an unstructured peptide could transform into an a-helix upon conformational nucleation.11 This allows for
only a portion of the a-helix to be synthetically constrained which ultimately leads to a fully structured a-helix.
Understanding the natural helical structure of peptides within the binding site is the first step in identifying potential utility of the cross-link motif. If the peptide does not have a natural propensity to fold into an a-helix when it is in the active state, an installed crosslink may not achieve enhanced affinity. It has been identified that robust ring-closing metathesis reactions with full conversion typically confers a high degree of helicity.12,13 Certain amino acids have less propensity to fold into the a-helical secondary structure.14
The bulky character of b-branched residues (threonine, valine, isoleucine) often reduces helicity. A variety of studies have assessed crosslinked helical peptides for an optimal
structural design. To perturb side chain interactions as little as possible, crosslinking motifs have been added to only one face of an a-helix, usually away from the binding side and solvent exposed. Synthetic constraints are placed at an initial position (i) and either 3 or 4 residues away to encompass one helical turn. To expand over two helical turns, i, i+7 positions are utilized. These sites are somewhat near in space, spanning 6.7-10 Å in distance, Figure 1.3. Recently, ‘stitched’ peptides have been identified as having even more stability.15 Two hydrocarbon constraints are placed adjacently, e.g. i,i+4+7. These ‘stitched’ peptides have shown significant thermal stability as well as cell penetration capabilities.
Stereochemistry of the amino acids used in appending constraints can greatly affect the reactivity, depending on the linkage size and the number of helical turns constrained (i, i+3,4 vs. i, i+7). For an i, i+3 linkage, DiLi+3 stereochemistry leads to a much higher %
product conversion, whereas for i, i+4, the same stereochemistry with DiDi+4 or LiLi+4 has
more than 98% conversion in a model RNAseA peptide.12 A follow up study further investigated how stereochemistry affected a stapled BH3 peptide, and identified LiLi+4 to
have marked enhancement of not only helicity, but also in vivo reduction of leukemic cell growth.16 The p53 helical stapled peptide mimics have been identified to incorporate DiLi+7
stereochemistry (SAH p53-5) leading to 20% helicity, cell permeability, and 0.80 ± 0.05 nM.17 There are several structural changes that can substantially affect the chemistry, as
well as the stability, helicity, and permeability of the constructs. Identifying an optimal structural design can influence the effectiveness of the inhibitors, and should be prudently assessed before moving forward.
1.5 Stability
Identifying the extent of a-helicity achieved with added structural constraints is the first step in achieving potent inhibitors. Circular Dichroism (CD) allows for an assessment of the average a-helical content within the peptides. A generic a-helix displays a double- well minimum at 208 and 222 nm. The degree of helicity utilizes the molar ellipticity at 222 nm, since there is overlap of other structural signatures near 208 nm, such as a random coil. The percent helicity for shorter peptides is the ratio of [q]222/max[q]222 where
max[q]
222 = (−40 000 X [1 − k/n]), k = 2.5, n = number of residues.18
1.6 Solubility
Achieving highly soluble crosslinked peptide inhibitors can be an issue. With the introduction of a highly hydrophobic cross-linker such as the all-hydrocarbon ‘staples’,
i+3
i+4 i+7
Figure 1.3 Calculated distance between the Cβ of amino acid residues i (green) to i + 3
(magenta), 4 (blue), and 7 (red) amino acids are 5.7 Å, 6.0 Å, and, 10.2 Å respectively. Distances were calculated on the CREB peptide (PDB: 1KDX).
caution needs to be taken towards aggregation. Often synthetic crosslinks are appended onto a face of the helix away from the binding site, typically solvent exposed. The lack of polar substituents may lead to aggregation and inactivation by serum.19–21 Furthermore, diminished solubility can cause disruptions to precise measurements to fully characterize the properties of potential drug candidates. The problematic nature warrants consideration of more polar crosslinking motifs.
1.7 Protease Resistance
Unstructured peptides rapidly undergo proteolytic degradation, but with the addition of a synthetic constraint, they can withstand proteolysis. Trypsin is a serine protease enzyme that identifies peptides with a catalytic triad made up of histidine, aspartate and serine, which recognize and cleave the amide bond on the c-terminal side of lysine and arginine residues. A synthetic crosslink disallows the peptide to unfold into an extended strand, and fit into the binding pocket, thus making it difficult for the protease to cleave the scissile bond. The constrained peptides confer resistance similar to that of small molecule inhibitors, and dramatically increases the stability versus the unconstrained peptide.18
1.8 Intracellular Uptake
It is still unclear as to what dictates cross-linked peptide’s membrane permeability. Typically, unfolded peptides do not readily pass through the membrane, except for a few highly basic peptides, termed cell penetrating peptides (CPPs) (Figure 1.4). These include TAT, penetratin, and octa-arginine. While it is not known exactly what makes these arginine rich peptides pass through, we do know the guanidino functional group induces
interactions with the cell surface. Sulfated glycans and other phosphates and carboxylates that have anionic character are present at the cell surface. The guanidino group on arginines can form hydrogen bonds with these molecules to confer an increase in hydrophobicity leading to cell penetration.
Based on the punctate pattern seen on microscopy images, stapled peptides can be taken up actively through endocytosis.22,23 Export of the peptides from pinosomes into the intracellular matrix has also been observed.22,24 However, not all stapled peptides confer intracellular uptake.17,19,25–27 The overall charge, hydrophobicity, and a-helical structure are all believed to affect cellular penetration. Increasing the net charge of the peptide often leads to enhanced permeability, as well as changing the location of the cross-link.18,19,28,29
Kfold
disordered peptide α-helix 1 2 3 Cell Protease Degradation Nucleus
Figure 1.4 Process by which a disordered protein folds into a stable a-helix (1), crosses into the cell membrane (2), and is finally degraded by proteases (3).
A variety of clever methods have been employed to add rigidity, stability, and affinity to a-helical peptidomimetics, including stapled peptides, hydrogen bond surrogates, lactam linkers, and thioether bridges, Figure 1.5.
1.9 Disulfide Bridge
Naturally occurring disulfide motifs inspired the first studies of stabilizing a- helices into locked, rigid structures with disulfide tethers (Figure 1.5).31,32 The work of
NH R NH O O R NH R O NH NH O NH R O NH NH O O R NH O NH NH O NH R O S S NH NH O O R NH R O NH NH O NH R O NH O NH NH O O R NH R O NH NH O NH R O Lactam bridge Disulfide bridge Hydrocarbon staple H O R NH R O NH N O NH R O
Hydrogen Bond Surrogate (HBS) α-helix Thioether bridge NH NH O O R NH R O NH NH O NH R O S S
Figure 1.5 a-Helical peptidomimetics, including stapled peptides, hydrogen bond surrogates, lactam linkers, disulfides, and thioether bridges. Image adapted from Azzarito et al.30
helicity compared to reduced, uncyclic analogs. Furthermore, the L,L enantiomers had increased helicity versus the L,D enantiomers. Few disulfide bridging techniques have been published recently, potentially due to a major drawback of intracellular disulfide reductions. Reducing environments can reverse disulfide bonds back to the unconstrained peptide, limiting their applicability towards intracellular targets. Cystathionine thioethers, where one sulfur atom is replaced by a methylene group has become an alternative strategy that has maintained some structure and activity while eliminating the disulfide bond susceptible to reduction.33 This strategy has been evaluated on Compstatin, currently in clinical trials for macular degeneration.34 Compstatin contains a susceptible disulfide bond that was exchanged to a cystathionine thioether. Activity was maintained while increasing biological stability.
1.10 Lactam Bridge
Incorporation of a lactam cross-link to stabilize helical mimetics has been researched by several including Fairlie, Osapay, and Taylor.35–37 Lactamization on one side of an amphiphilic helix showed increased helicity that potentially stabilizes the peptide and increases binding affinity (Figure 1.5).36–38 In work done by Osapay et al., a condensation reaction involving a lysine residue and an oxime resin-bound aspartic acid at the i, i+4 positions created a lactam crosslink that showed 69% a-helicity via CD. Fairlie has also shown peptides with increased helicity as well as increased binding affinity.35
1.11 Stapled Peptides
The pioneering work of Grubbs and Blackwell propelled the area of peptide mimetics forward (Table 1.1). Using a ruthenium catalyzed ring closing metathesis
reaction (for which Grubbs received the Nobel Prize in Chemistry for), they cross-linked short peptides on resin supports. By introducing allyl-glycine or O-allyl serine amino acids, they formed a new C-C bond between the two alkene moieties to afford the macrocycle (Figure 1.5).13 This work inspired Verdine and colleagues to use the RCM reaction to afford an all-hydrocarbon crosslinked a-helix peptide.12 Incorporation of a-methyl,a-
alkenyl glycines to afford a crosslink over one or two a-helical turns were assessed for the optimal stereochemistry and linker length. This method of ‘Stapling Peptides’ has been used on a variety of PPIs by numerous laboratories, Table 1.1. Recently, Aileron Therapeutics published a potent stapled peptide inhibitor ATSP-7041 that targets the oncogenic proteins MDM2 and MDMX, Figure 1.6.39 With high a-helicity, protease stability, and increased cell penetration coupled with favorable pharmacokinetic properties and in vivo stability, they highlight the capability of stapled peptides developing as pre- clinical drug candidates.
N H O Glu-Tyr-NH O NH Ala-Gln Leu-Thr-Phe O H N O N H Ser-Ala-Ala-NH2 O ATSP 7041
Figure 1.6 Peptide inhibitor from Aileron Therapeutics targeting MDM2 (Ki = 0.9 nM) and MDMX (Ki