Hydrocarbon Stapled Peptides as Modulators of Biological Function

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Hydrocarbon Stapled Peptides as Modulators of Biological Function Philipp M. Cromm,†,‡ Jochen Spiegel,†,‡ and Tom N. Grossmann*,†,‡,§ †

Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany Technical University Dortmund, Department of Chemistry and Chemical Biology, Otto-Hahn-Str. 6, 44227 Dortmund, Germany § Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, 44227 Dortmund, Germany ‡

ABSTRACT: Peptide-based drug discovery has experienced a significant upturn within the past decade since the introduction of chemical modifications and unnatural amino acids has allowed for overcoming some of the drawbacks associated with peptide therapeutics. Strengthened by such features, modified peptides become capable of occupying a niche that emerges between the two major classes of today’s therapeuticssmall molecules (5000 Da). Stabilized α-helices have proven particularly successful at impairing disease-relevant PPIs previously considered “undruggable.” Among those, hydrocarbon stapled α-helical peptides have emerged as a novel class of potential peptide therapeutics. This review provides a comprehensive overview of the development and applications of hydrocarbon stapled peptides discussing the benefits and limitations of this technique.

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therapeutics experience a renaissance with six new peptide drugs being approved in 2012.7 Distinct chemical modifications provide the opportunity to stabilize bioactive peptide conformations, thereby implementing more drug-like properties and enhanced target affinity. Conformational constraints can be implemented by the incorporation of unnatural amino acids and/or by marcocylization, thereby modulating bioactivity and bioavailabilty of the peptide.8 Following this approach, relatively large structures (up to 60 amino acids) are generated by the grafting of bioactive peptide epitopes onto rigid mini-proteins such as cysteine-knot scaffolds.9,10 The chemical stabilization of isolated secondary structures such as α-helices, β-sheets, or turns yields smaller peptide structures.11,12 In particular, the stabilization of α-helices has proven useful for the design of bioactive peptidederived molecues.13−17 α-Helices are highly abundant and involved in many PPIs, thereby resembling potential inhibitors of numerous PPIs.18,19 Many strategies have been developed to stabilize α-helical conformations involving α-methylation,20 Ncapping,21,22 and side-chain-to-side-chain cross-linking.23−25 Inspired by the use of ring-closing metathesis for a crosslinking of two O-allyl homoserine side chains introduced by Blackwell and Grubbs,25 Verdine and co-workers designed an all-hydrocarbon cross-link that combines side-chain-to-sidechain tethers with the helix inducing properties of α-methylated amino acids.26 Hydrocarbon stapled α-helical peptides (from now on stapled peptides) contain two α,α-disubstituted unnatural building blocks that are cross-linked by an all-

ver the past century, the precise understanding of disease relevant cellular processes and molecular insights into interactions between bioactive molecules and their targets fostered tremendous progress in the development of therapeutic agents. Most of these agents belong to the two main drug classes: small molecules and biologics. Small molecular compounds hold the potential to efficiently penetrate into cells and prove highly effective if the target protein carries a hydrophobic pocket suitable for binding.1 However, extended and shallow protein surfaces are hardly accessible with small molecules. Over the past three decades, a new class of therapeutics has emerged, the so-called biologics. This class encompasses engineered antibodies, growth factors, and other protein based bioactive molecules which are typically over 5000 Da in size. Biologics can be evolved to bind almost every given protein target with high affinity and selectivitybut their use is restricted to extracellular applications, and they do not show oral availability. As a consequence, a number of challenging intracellular targets are not approachable for small molecules and biologics and have been therefore termed “undruggable”.2−4 Mainly, this accounts for proteins that are involved in protein−protein interactions (PPI),5 such as the highly interesting oncogenic targets c-Myc and K-Ras.4,6 In principle, peptides hold the potential to bridge the gap between small molecules and biologics. They possess excellent surface recognition properties and minimal toxicity. However, in most cases peptides suffer from proteolytic instability and low cell permeability. These limitations are mainly associated with the flexible conformation that short peptides adopt when free in solution. This flexible nature also affects target affinity, due to entropic penalties caused by the restriction in conformational freedom associated with binding. Nevertheless, peptide-based © XXXX American Chemical Society

Received: December 15, 2014 Accepted: March 23, 2015

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DOI: 10.1021/cb501020r ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. Stapled peptide architectures and building blocks. (a) Schematic representation of stapled peptide synthesis and architecture. Incorporation of α,α-disubstituted amino acids into the peptide sequence followed by ring closing metathesis yields stapled peptides with various architectures, e.g.: [i, i + 3], [i, i + 4], [i, i + 7] and [i, i + 4, i + 4 + 7] (stitched peptides). (b) Structure of the α-methyl, α-alkenyl building blocks used for peptide stapling and bis-pentenylglycine (B5) for stitched peptides.

hydrocarbon tether.26,27 After having introduced the general concept of peptide stapling in 2000, Verdine and co-workers reported the design of BCL-2 protein-derived stapled peptides which target an intracellular PPI and show in vivo activity.28 This ground breaking study demonstrated the potential of stapled peptides for targeting proteins previously considered to be undruggable. Among the various approaches toward conformational stabilization of α-helical peptides, hydrocarbon peptide stapling has proven particularly successful, providing numerous examples of extra- and intracellular PPI inhibitors.29,30 In this review, we give a comprehensive overview on the stabilization of α-helices using the hydrocarbon stapling technique, not considering other successful approaches such as lactam cross-links, hydrogen-bond surrogates,31 or α/βpeptides.32 For a broader overview, the reader is directed to more general reviews on PPI inhibitors5,11,12,33 or on techniques for the stabilization of α-helices.13−19 In the following, we first elaborate design principles and properties of stapled peptides before discussing their application in a target-based setup.

positions i, i + 4, and i + 4 + 7 to provide two adjacent hydrocarbon staples (Figure 1a).36 The synthesis of stapled peptides requires α-methyl, α-alkenyl amino acids (Figure 1b) which are incorporated during solid phase peptide synthesis following standard coupling protocols.37 The unnatural building blocks are termed Sn or Rn, depending on their absolute configuration (S or R) and the number of carbon atoms (n) in their olefinic side chain. Macrocycle formation is accomplished on solid support using ring-closing olefin metathesis. This synthetic strategy allows the introduction of various N-terminal modifications such as acetylation and fluorescence or affinity labeling. Linker length and absolute configuration of the introduced α-methyl, α-alkenyl amino acids have been optimized to ensure maximal helix stabilization. The installation of i, i + 3 and i, i+7 staples requires incorporation of an R-configured building block at position i and an S-configured one at position i + n.26,27 For the i, i + 7 architecture, a cross-link containing 11 carbon atoms is applied (R8 + S5). The i, i + 3 staple features two different versions with a linker length bearing either six or eight carbon atoms.34 Thus, far, the most widely used architecture involves the i, i + 4 crosslink. In this setup, an eight carbon cross-link derived from two S-configured building blocks (S5 + S5) is used.26 Notably, its closest analogue containing the two enantiomeric R5 building blocks (R5 + R5) shows reduced helix stabilization as well as reduced cellular uptake.38 Long peptides allow the introduction of two individual staples,39 while the staples are preferably separated by more than four or seven amino acids to prevent cross-reactions. The recently reported stitched peptides were

2. DESIGN AND PROPERTIES OF STAPLED PEPTIDES Synthesis and design of stapled peptides have been optimized in various studies. Hence, stapled peptides comprise a variety of different architectures involving cross-linking at peptide positions i, i + 327,34 and i, i + 426 both bridging one helical turn and at position i, i + 726,35 bridging two helical turns (Figure 1a). Recently, stitched peptides were reported which, in their most stabilizing architecture, facilitate cross-linking at B


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particles occurs after protease cleavage of the Gag polyprotein into matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains. The pol gene encodes the viral enzymes essential for HIV-1 replication like reverse transcriptase (RT) and integrase (IN), while the env gene, encodes proteins of the viral envelope (Env). Although there is an increasing number of anti-HIV therapeutics on the market and in clinical trials, eradication of a chronic HIV-1 infection still remains ellusive.49 Gag Modulators. Several strategies were evolved to interfere with HIV-1 infection. One possibility involves the inhibition of virus particle assembly by impairing capsid formation. Several stapled peptides have been developed to inhibit correct capsid assembly of both immature- and mature-like particles in vitro.50−53 These peptides were either derived from phage display, like the capsid inhibitor (CAI)54 (Figure 2) or originate

designed to further improve the stabilizing effect of the hydrocarbon cross-link. In these peptides, the introduction of bis-pentenylglycine (B5, Figure 1b) allows the formation of a dual hydrocarbon staple emerging from a single attachment point and creating a spiro-bicyclic ring junction (Figure 1a).36 Stitched peptides (S5 + B5 + S8) cover three helical turns with an N-terminal i, i + 4 followed by an i, i + 7 staple. The most controversially discussed attribute of stapled peptides is their ability to penetrate cells.40−43 In general, achieving cell penetration of peptides is a tremendously challenging task, and a lot of effort has been put into the identification of appropriate strategies.44 So far, strict rules for the implementation of cell permeability have not been revealed, but investigation of cellular uptake of more than 200 stapled peptides revealed that cell penetration is mainly depending on staple type, position, and the formal charge of the peptide.40 In many cases, stapled peptides exceed the cell penetrating properties of unmodified peptides. Additionally, an overall positive charge between +1 and +7 promotes cellular uptake. However, the incorporation of positive charges to increase cell penetration should be performed carefully as highly charged peptides exhibit the tendency to disrupt cell membranes.43 Experimental data suggest that most stapled peptides penetrate cells via a clathrin- and caveolin-independent endocytoic pathway.40 This ATP-dependent endocytosis is partly mediated by the interaction with anionic cell surface proteoglycans which is aided by a positive overall charge of the peptide. Notably, the introduction of a hydrocarbon staple alone does not ensure cell penetration. Stapling supports cellular uptake but often needs to go in hand with distinct sequence optimization to yield cell penetrating stapled peptides.45,46 In most cases, stapled peptides are designed based on crystal structures of protein−protein complexes. Usually, noninteracting residues are selected for the incorporation of the unnatural building blocks. However, the prediction of most stabilizing architectures and optimal sites for staple incorporation is complicated. For a small test set of peptides, computational studies were able to match experimental results,35 but these elaborated calculations have not been used as a standard technique. As a result, different staple architectures and positions have to be examined to ensure optimal helix stabilization and target affinity. The most successfully applied architectures so far involve i, i + 4 and i, i + 7 staples. Notably, stitched peptides proved to be exceptionally stable toward thermal and chemical denaturation when compared to classic stapled peptides also exceeding their cell penetration properties.36,40 However, these promising first results still have to prove general applicability.

Figure 2. Inhibitors of CA-CTD dimerization. CAI peptide (gray) bound to a hydrophobic groove on CA-CTD (left, PDB 2BUO). Hydrophobic interacting residues are displayed explicitly and are highlighted in the sequence. Superimposition of CAI with stapled peptide NYAD-13 (wheat, PDB 2L6E) reveals that NYAD-13 mimics the CAI helix upon binding (right). The hydrocarbon staple (red) does not interact with the protein and points toward the solvent.

from the C-terminal domain of CA (CA-CTD), which was reported to disrupt HIV-1 particle formation, as well. The most advanced stapled peptide NYAD-1 (12-mer; i, i + 4) was designed based on the crystal structure of CAI55 with the intention to overcome the low cell permeability and protease stability of the precursor peptide. NMR analyses revealed that the binding site of NYAD-1 and CAI on CA-CTD are almost identical. In addition, effective cell penetration and colocalization of NYAD-1 with the Gag polyprotein was shown. NYAD-1 proved to disrupt the formation of immature- and mature-like particles in vitro and in cell based assays and shows low micromolar potency (half-maximum inhibitory concentration (IC50) = 4−15 μM) toward several primary HIV-1 isolates without having any effect on the equine lentivirus EIAV. The more water-soluble derivative NYAD-13 was used to determine an NMR structure in complex with the monomeric form of CACTD representing the first published structure of a stapled peptide in complex with a protein (Figure 2).56 The interaction between NYAD-13 and CA-CTD is mediated by hydrophobic contacts, whereas the hydrocarbon cross-link sits on the solvent exposed site of the peptide showing no interaction with the protein (Figure 2). Another set of NYAD-family peptides containing an i, i + 7 staple reveals dual activity in HIV-1 assembly as well as HIV-1 entry.51 The affinity of these peptides (Kd = 2.5−10 μM) toward the CA-CTD is in the range of NYAD-1, and they impair mature-like particle formation in vitro. Surprisingly, these i, i + 7 stapled peptides decrease virus infectivity by impairing virus entry into the cell, as well. This

3. INFECTIOUS DISEASES Within the past decade, stapled peptides have been successfully applied to various targets related to infectious diseases. A number of pathogenic microorganisms tend to develop resistance, resulting in high tolerance against existing therapeutics, thereby generating a constant need for novel anti-infective agents. Antiviral Strategies. HIV-1. Human immunodeficiency virus type 1 (HIV-1) as the cause of acquired immunodeficiency syndrome (AIDS) is one of the prime targets for antiinfectious research. The HIV genome holds three genes (gag, pol, and env) coding for proteins that are considered promising drug targets. The gag gene encodes the Gag polyprotein which forms the immature virus particle.47,48 Maturation of virus C


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development as most of them target the microbial cell membrane.72 They form amphipathic α-helices which can permeabilize the microbial membrane, leading to leakage of cytoplasmic components and cell death. Hydrocarbon stapling was used to improve protease resistance and helicity of the AMPs lasioglossin III (LL-III) and melectin (MEP)70 as well as truncated versions of esculentin-2EM (E2EM).69 Introduction of staples into LL-III and MEP resulted in peptides with increased α-helicity and protease resistance but significantly promoted hemolytic activity without increasing their antimicrobial potency.70 The i, i + 4 hydrocarbon stapling converts E2EM15W−S1, a 15 amino acid analogue of the 36 amino acid parent peptide E2EM, into an α-helical biologically active AMP against Bacillus subtilis and Staphylococcus aureus with highly promoted protease resistance.69 A major cause of multidrugresistance of pathogenic microorganisms are multidrug efflux systems (MES) clearing applied drugs from the cytosol. Several efforts were taken to inhibit these MES by blocking the binding site.73 One subfamily, the small multidrug resistance proteins (SMRs), forms a four helix bundle in the membrane environment that requires oligomerization to exert its function. On the basis of the minimal dimerization sequence,74 stapled peptides were designed to disrupt this oligomerization, thereby impairing efflux pump function.71 They show specific efflux inhibition and resensitize the bacteria to the applied toxin (ethidium bromide) without being cytotoxic for mammalian blood cells. Malaria. A unique structural feature of Plasmodium falciparum represents the interaction between the C-terminal tail of myosin A (myoA) and the myoA tail interacting protein (MTIP). The helical interaction motive of myoA binds a channel-like cavity between two MTIP domains.75 myoA is relocalized via MITP and drives ATP hydrolysis, thereby promoting overall motility and the invasion into the host red blood cells.76,77 For this specific setup encompassing a fully buried α-helix, FRET measurements and cocrystallization were used to compare hydrogen-bond surrogates, stapled peptides, and hydrocarbon cross-linked peptides lacking α-methylation (using pentenyl glycine instead of pentenyl alanine).78 It was observed that the introduction of a pentenyl glycine-based cross-link or an N-terminal hydrogen-bond surrogate is favored over classic peptide stapling. The benefit in this setup results from less steric hindrance and improved reproduction of native side chain interactions.78 For these peptides, only binding data was reported, so far.

effect has been traced back to additional binding to the V3 loop of gp120, which is encoded on the env gene. However, the molecular basis for the dual targeting ability of these i, i + 7 stapled peptides compared to the i, i + 4 ones still remains elusive. Env Modulators. FDA approved drug Enfuvirtide, a 36amino acid peptide, is an HIV-1 fusion inhibitor that blocks virus entry in humans. It disrupts the formation of a six-helix bundle of the env glycoprotein gp41 by mimicking the heptad repeat2 (HR2).57 As Enfuvirtide is an unmodified peptide it suffers from the general drawbacks, such as lack of oral availability and severe proteolytic degradation.58,59 The introduction of two isolated i, i + 4 staples at the termini of Enfuvirtide and of T649v, another peptidic HIV-1 fusion inhibitor derived from gp41, improves the bioavailability of these peptides dramatically.39 Enhanced antiviral activity, and an 82- and 62-fold prolonged half-life toward chymotrypsin and pepsin digestion, respectively, were observed for SAHgp41(626−662)(A,B). Notably, the detection of low concentrations of SAH-gp41(626−662)(A,B) in the blood of mice after oral administration indicates the possibility of oral delivery for stapled peptides and should be investigated in more detail. In the pursuit of developing anti-HIV-1 vaccines, a fragment of the membrane-proximal external region (MPER) of gp41 was stapled to bind to several anti-HIV antibodies.60 As the introduction of two staples, i, i + 4 (C8) and i, i + 3 (C6) renders the peptide resistant toward proteolytic degradation, stapled peptides might be used as chemically stabilized antigens for vaccine development, in the future. Pol Modulators. Besides surface proteins like gp41, the main targets of antiretroviral drugs are RT and IN. Cell permeable stapled peptides inhibiting IN were developed either based on known peptide inhibitors61−63 or derived from the HIV-1 Vpr protein.64,65 After optimization, the i, i + 4 stapled peptide NLH6 was identified as the most potent inhibitor of the two processing events catalyzed by IN (3′-processing, IC50 = 9 μM; strand transfer, IC50 = 6 μM).64 For Vpr-derived peptides, cellular uptake was achieved either via addition of octa-arginine or peptide stapling. Interestingly, the addition of octa-arginine promotes cytotoxicity which was not observed for the stapled analogue.65 Hepatitis C and RSV Infection. Impairing with virus membrane fusion using stapled peptides has also been used to interfere with hepatitis C virus (HCV) and respiratory syncytial virus (RSV) infections.66,67 SAHH-5, a 14 amino acid i, i + 7 stapled peptide derived from an extracellular loop of DC81, shows good inhibitory activity against different HCV subtypes in cell-based assays (half-maximum effective concentration (EC50) = 17−39 μM) without being cytotoxic at tested concentrations.67 T118, a 35 amino acid stretch originating from the C-terminal heptad domain of RSV, was identified as an inhibitor of RSV infection, however suffering from low protease stability.68 The introduction of two i, i + 7 hydrocarbon staples into the sequence of T118 yielded a low nanomolar inhibitor of RSV infection in vitro and in vivo.66 Compared to the introduction of a single staple, the use of two hydrocarbon cross-links yielded improved protease stability and highly efficient reduction of nasal RSV infection when administered intratracheally as peptide coated nanoparticles. Antimicrobial Strategies. Different approaches have been applied to tune the properties of antimicrobial peptides (AMPs).69−71 AMPs are produced by various organisms as an effective defense and seem to be less prone to resistance

4. INHIBITION OF THE P53−MDM2/MDMX INTERACTION The interaction between the tumor suppressor p53 and its negative regulators MDM2 and MDMX is an attractive target in anticancer strategies serving as a model system for many approaches that aim at the conformational stabilization of αhelical peptides. The p53 protein is a transcription factor that plays a crucial role in guarding the cell in response to DNA damage, oncogenic activation, and other cellular stress. In many tumors, overexpression of negative regulators of p53 leads to reduced levels of functional p53 protein.79 The two major negative regulators are E3 ubiquitin protein ligase MDM2 (also known as HDM2) and its structural homologue MDMX (also known as MDM4 and HDM4/HDMX). While MDM2 is able to bind to p53, targeting it directly for proteasomal degradation, MDMX is missing ubiquitin ligase activity sequestering p53 for degradation via heterodimerization with MDM2.80,81 The D


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ACS Chemical Biology finding that the transactivation domain 1 of p53 mediates binding to the N-terminus of MDM2 through a short α-helical peptide in which three key hydrophobic residues (F19, W23, and L26) constitute hot spots for MDM2 binding82 (Figure 3a

helical conformation and additional hydrophobic interactions between the hydrocarbon staple and the MDM2 protein surface explaining the considerable gain in affinity compared to the wild-type sequence. Inspired by these initial studies, a number of second generation i, i + 7-stapled peptides were developed based on phage-display derived MDM2/MDMX-binding peptides aiming at an improvement of affinity and pharmacological properties. Starting from two high affinity linear peptides94,95 a set of stapled derivatives with varying affinity and specificity for MDM2 and MDMX were generated. Substitution of W7 for L-6-chloro tryptophan (Figure 3c) resulted in peptide sMTide02A with an approximately 9-fold higher affinity for MDM2 but drastically attenuated affinity for MDMX (Figure 3a).96 Interestingly, cell-based experiments indicate that the two stapled peptides sMTide-02 and sMTide-02A induce stronger p53 reporter gene activity combined with reduced general cytotoxicity.96 In comparison with these stapled peptides, nutlin-3A appears to be more sensitive to resistance mutations in MDM2. While different point mutations of MDM2 considerably disrupt nutlin-3A affinity, the binding of stapled peptides is only moderately affected. A possible explanation is the larger plasticity of the stapled peptide and their greater number of interaction points with the protein surface.97,98 Aileron Therapeutics developed the so far most advanced peptide inhibitor based on the phage display derived linear peptide pDI.99 Optimized i, i + 7-stapled peptide ATSP-7041 binds both MDM2 (Kd = 0.9 nM) and MDMX (Kd = 6.8 nM) with low nanomolar affinities featuring a conformation in which the key residues (F19, W23, and β-cyclobutyl-L-alanine (Z)), the staple, as well as an additional interaction of Y22 mediate binding to MDMX. The peptide shows efficient cell penetration and strong efficacy in multiple human cancer cell lines as well as in xenograft models. 100 According to surface plasmon resonance experiments, ATSP-7041 exhibits a slower dissociation rate from MDM2 than the nutlin family members (43 min compared to 6 min). Its effect on p53, MDM2, and p21 protein levels in MCF-7 cell lines persists significantly longer following drug removal when compared with RG7112, a phase I clinical candidate of the nutlin family (48 h compared to 4 h). Upon intravenous administration, ATSP-7041 exhibits low clearance and long plasma half-life in models of mouse, rat, and monkey, and a [3H]-radio-labeled analogue indicated broad organ distribution. Recent reports have questioned the cellular uptake and in vivo activity of stapled peptides targeting the p53−MDM2/ MDMX interaction and indicate a dependency on the serum content of the assay medium suggesting that these stapled peptides compromise membrane integrity at low serum concentrations.43 In earlier studies, diminished activities at increasing serum concentrations had already been observed for SAH-p53−8, ATSP-7041, and sMTide peptides.96,100 However, despite this effect, the optimized lead compound ATSP-7041 retains sufficient cellular potency in the presence of serum and ultimately showed efficacy in different in vivo models.100 Overall, these results underpin the necessity of carefully selected assay conditions for the evaluation of cellular uptake and activity and suggest that a rigorous optimization process can eventually overcome these issues. Ongoing clinical trials with the next-generation peptide ALRN-6924 will further investigate the general applicability of stapled peptides as therapeutic agents.

Figure 3. Inhibitors of the p53−MDM2/MDMX interaction. (a) Peptide sequences and binding affinities toward MDM2 and MDMX for representative members of three series of peptides compared to the small molecule inhibitor nutlin-3A. Interacting key residues (F19, W23, and L26) are highlighted (aref 88, bref 96, cref 100). (b) Superimposed crystal structures of wild type transactivation domain of p53 (dark gray, PDB 1YCR) and of stapled peptide SAH-p53−8 (wheat, PDB 3V3B) with MDM2 in surface representation. The interacting key residues and staple (red) are shown explicitly. (c) Nonnatural amino acids incorporated into the second generation MDM2/ MDMX-binding peptides: J = 6-L-chloro-tryptophan, Z = β-cyclobutylL-alanine.

and b) inspired the development of various peptide-derived PPI inhibitors.5 These efforts resulted in inhibitors that disrupt the p53−MDM2/MDMX interaction and restore tumor suppressive activity of p53. Most successful inhibitors involve stapled peptides and small molecules such as the nutlins,83,84 some of which have reached clinical trials.85,86 On the basis of the α-helical domain of p53, a set of i, i + 7 stapled peptides was designed87 including SAH-p53−8, which shows low nanomolar affinities for both MDM2 and MDMX (Figure 3a). SAH-p53-8 exhibits high α-helical content (85%) and moderate cell penetration and causes dose-dependent inhibition of cell viability and reactivation of the p53 tumor suppressor pathway in cancer cell lines with elevated MDM2 and/or MDMX levels.88 Similarly, SAH-p53-8 showed activity in melanoma cell lines and mouse xenograft models, with elevated MDMX protein levels and synergies upon combined treatment with other antitumor agents.89 In contrast, small molecule inhibitors of the nutlin family are not able to target MDMX and only demonstrate activity in cells with elevated MDM2 expression levels. In agreement with molecular dynamics simulations,90−92 the crystal structure of the SAHp53-8−MDM2 complex93 (Figure 3b) revealed an elongated E


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5. INHIBITION OF INTERACTIONS BETWEEN BCL-2 FAMILY PROTEINS Stapled peptides have been developed as tools to modulate individual steps of programmed cell death.101 A vast number of studies in this field have been conducted on the stabilization of BCL-2 homology 3 (BH3) domains, which interfere with interactions between various BCL-2 family members. These proteins constitute a key control point in apoptosis by regulating the release of apoptogenic factors from mitochondria, and they are involved in other important cellular processes such as energy metabolism, Ca2+ homeostasis, and mitochondrial morphology. Their activity is controlled by a complex network of PPIs in which pro-apoptotic proteins (e.g., BID, BAD, BIM) can activate multidomain BH3 pro-apoptotic members (e.g., BAK, BAX) to promote cell death. On the contrary, antiapoptotic family members (e.g., BCL-2, BCL-XL, MCL-1) sequester these proteins to prevent signaling and promote cell survival (Figure 4a).102 In their pioneering work, Verdine and co-workers generated a set of stabilized peptides derived from the BID BH3 domain with low nanomolar affinity, high helical content, and cell permeability, which show efficacy in mouse xenograft models of human leukemia.28 Thereafter, a variety of different BH3 domains were used as templates for the synthesis of stapled PPI inhibitors and were subjected to biological and computational studies (precursor: BID,28,103,104 BAD,103,105−107 BIM,41,108,109 Mcl-1110,111). Besides the modulation of apoptosis, stapled BH3 binding domains have also been used to study binding modes,108 to identify or validate specific interaction partners of their individual parent protein,103,105−107 and to screen for novel small molecular PPI inhibitors.111 In the search for a specific inhibitor of the antiapoptotic MCL-1 protein, a set of stapled peptides originating from 13 different BCL-2 family proteins (Figure 4b) was generated to obtain peptides with varying selectivity, including MCL-1 specific peptide MCL-1 SAHBA.110 Already, a single point mutation (V220F) was able to abolish MCL-1 selectivity, whereas scanning of the staple position yielded the selective peptide MCL-1 SAHBD, demonstrating high target affinity (Kd = 10 nM) and sensitizing effects on leukemia cancer cells that depend on MCL-1. A crystal structure of MCL-1 SAHBD in complex with MCL-1 revealed hydrophobic contacts of the staple with the protein surface contributing to binding affinity,110 as supported by molecular dynamics simulations.112 Moreover, it was shown that binding specificity which is mediated by post-translational modifications in the parent protein can be transferred to stapled peptides. For instance, the incorporation of phosphorylated serine into a stabilized BAD BH3 helix attenuated the pro-apoptotic activity of the peptide, instead promoting glucose-stimulated insulin secretion in BAD deficient beta cells by activation of glucokinase.105,107

Figure 4. Stapled BH3 domain derived peptides targeting BCL-2 family members. (a) Sequence alignment of BH3 domains from antiand pro-apoptotic BCL-2 family members (sequences obtained from www.uniprot.org). (b) Crystal structure of the complex of BIM wild type peptide (dark gray) with MCL-1 (PDB 2NL9). Key interacting residues are shown explicitly. (c) Superimposed crystal structure of BIM wild type peptide (dark gray) and the stapled peptide MCL-1 SAHBD (wheat, PDB 3MK8).110 Key residues for the interaction with MCL-1 and the interacting peptide staple (red) are shown explicitly.

does not only depend on the binding of its steroid hormone ligand but also on interactions with a coactivator protein.121 A leucine rich pentapeptide motif (LXXLL) has been identified as a core sequence of these coactivator proteins, and cocrystallization with ER reveals an α-helical conformation of the bound motif (Figure 5a).122 On the basis of these observations, a series of stapled peptides (SP1−6) has been developed to bind to the coactivator binding site.113 NMR structures of unbound peptides as well as cocrystal structures with ER allow a detailed investigation of the binding (Figure 5b). These studies confirm that the site of staple incorporation has to be chosen carefully as it can not only affect the conformational restraint of the peptide but also its interaction with the target protein itself. For SP1, the introduction of the staple shifts the interacting residues out of register by a quarter turn, changing the LXXLL binding motif into an IXXS5L motif of lower affinity. A 2-fold increase in affinity can be observed for SP2 where the staple replaces a leucine within the LXXLL motif (LXXS5L), putting

6. RECEPTORS AND SIGNAL TRANSDUCTION Stapled peptides have also been used to inhibit or activate different receptors. Among others, stapled peptides were designed to target the estrogen receptor (ER),113 the ABCA1 transporter,114 different neuronal receptors,115,116 and Gprotein coupled receptors (GPCR) like VPAC 2 117 or EGFR.118,119 Estrogen Receptor. The ER is a member of the nuclear receptor (NR) superfamily and plays an important role in the expression of a large number of target genes.120 ER signaling F


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excellent results after injection are believed to result from their prolonged metabolic stability. Cholesterol and Insulin Receptor. Stapled peptides were also designed to mediate cholesterol efflux114 and insulin secretion.117 Stapling of the last helix of the apolipoprotein A-I (apoA-I) promotes cholesterol efflux via the ABCA1 transporter.114 The unmodified peptide is not able to trigger reverse cholesterol transport and is prone to proteolytic degradation.126 Although, a stapled analogue of the vasoactive intestinal peptide (VIP) displayed enhanced agonist potency on glucose-dependent insulin secretion via activation of the VPAC2 receptor and exceeds α-helical stabilization via lactam bridge formation, this stapled peptide was not able to withstand degradation by pepsin.117 Notably, the stapled VIP derivative involves 31 amino acids, and the incorporation of a single i, i + 4 staple may not be sufficient to protect the peptide from proteolytic degradation. EGFR. Uncontrolled signaling via cell-proliferation and survival pathways is implicated in various forms of cancer and can be caused by mutations in membrane-associated receptors, small GTPases (especially the Ras proto-oncogene), or downstream signaling proteins like phosphatidylinositol 3kinase (PI3K).6,127,128 The epidermal growth factor receptor (EGFR)129−131 tyrosine kinase is a major target in cancer research, and a number of different drugs have already been approved for EGFR inhibition such as the monoclonal antibody cetuximab and several tyrosine kinase inhibitors like gefitinib.132 An alternative approach involves the inhibition of coiled coil formation of the juxtamembrane (JM) segment within the receptor.118,119,133 Dimerization of JM is essential for receptor activation134,135 and can be impaired by a short JM sequence (EGFR residues 645−662), either when fused to a cellpenetrating TAT sequence133 or after introduction of an i, i + 7 staple.118 Both approaches lead to decreased levels of phosphorylated EGFR, Akt, and Erk, indicating the inhibition of downstream signaling. Small GTPases. Targeting of small GTPases, which are key regulators of several pathways, has been pursued extensively with only very limited success.6 The absence of hydrophobic pockets thwarts the design of high affinity small molecules. A peptide stabilized by a hydrogen-bond surrogate was reported to exert effects on the small GTPase Ras in vitro.136 However, the development of i, i + 4 stapled peptide StRIP3, which binds the activated Ras homologue Rab8a with low micromolar affinity (Kd = 22 μM) and inhibits a Rab PPI in vitro,137 fuels new hopes for the development of small GTPase binding peptides. Kinases. Mutations in PI3K can circumvent its activation by Ras and trigger downstream signaling. A mutation in the catalytic p110α subunit of PI3K causes its direct interaction with insulin receptor substrate 1 (IRS1) and activates its kinase activity.138 A stapled peptide derived from the mutated p110α subunit is able to disrupt the interaction with IRS1 thereby reducing downstream Akt phosphorylation. It also proved effective in mouse xenograft models.138 Modulation of kinase activity has also been achieved by interfering with their spatial and temporal regulation. Stapled peptides based on the Akinase anchoring proteins (AKAPs) were designed and turned out to be selective disruptors of the AKAP-protein kinase-A (PKA-RII) interaction in cell culture.139 Furthermore, stapled peptides showed activity in cell-based assays of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL) via impairing the linear ubiquitin chain assembly

Figure 5. Peptides binding to the ER. (a) Crystal structure of the NR coactivator peptide (gray) bound to estrogen receptor α (PDB 2QGT). The three leucine residues within the LXXLL interacting motif are shown as sticks and are highlighted in the sequence. (b) Overlay of the three stapled peptides SP1, SP2, and SP6 (wheat, PDB 2YJD, 2YJA, and 2LDD) with the NR peptide (gray). Interacting residues and sites of stapling are highlighted in the sequence.

the peptide back in register with the parent coactivator peptide. Placing the staple around the two C-terminal leucine residues (LS5XLLS5) as done for SP6 gives an additional 5-fold increase in binding affinity compared to SP2. The NMR structure of the unbound peptide confirms an excellent preorganization of SP6. Superimposition of NMR-derived structures of unbound SP6 and the wild type coactivator peptide reveals the good agreement with the starting structure and shows that the hydrocarbon staple can also substitute for native hydrophobic interactions. Neuronal Receptors. Modulation of neuronal receptors like the N-methyl-D-aspartate (NMDA) receptor family or the galanin (Gal) and neuropeptide Y (NPY) receptors is involved in seizure activity and nociception. The 17 amino acid peptide conantokine-G (con-G) is isolated from the marine snail Conus geographus and blocks the ion flow through NMDA receptors.123,124 In the presence of Ca2+ ions con-G forms a stable α-helix via chelatization of the metal ions with its γcarboxyglutamic acid residues (Gla).125 Peptide stapling of conG enhances helicity, serum stability, and receptor subtype selectivity.116 When tested in a mouse model of pharmacoresistant epilepsy via intracerebroventricular injection, conG[11−15,Si,i+4S(8)] protected 75% of tested mice from seizures, notably without the induction of motor impairments. In analogy to the con-G peptides, stapling of the Gal and NPY sequence yielded peptides with increased α-helicity and metabolic stability.115 Although these stapled peptides show slightly decreased binding affinity toward their receptors, stapled peptide Gal-S1 sufficiently suppressed seizures in mouse models without causing any motor impairment. The G


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Figure 6. Overview of β-catenin binding domains and corresponding stapled peptides. (a) Crystal structure of β-catenin (white) with the β-catenin binding domains of TCF4 (green, helical and extended region) and BCL9 (gray, PDB 2GL7). (b) Detailed view of the BCL9-β-catenin interaction. Selected interacting residues are shown explicitly and are highlighted in the sequence. Red spheres indicate sites of staple incorporation for SAHBCL9B. (c) Top: Superimposition of StAx-35 (PDB 4DJS) with the parent Axin sequence (PDB 1QZ7). Selected interacting residues are shown as sticks (compared to StAx-35, most active peptide StAx-35R bears a Q740R variation). Bottom: Overlaid β-catenin binding domain of Axin (PDB 1QZ7) and helical part of TCF4 (transparent green, PDB 2GL7).

complex (LUBAC) by mimicking a α-helix of the RNF31/ RBCK1 PPI.140

in a destruction complex and marked for proteasomal degradation. Wnt activation inhibits the destruction complex resulting in accumulation of β-catenin and translocation into the nucleus where it binds to transcription factors of the LEF/ TCF family (e.g., TCF4, Figure 6a), thereby recruiting coactivators such as B-cell lymphoma 9 protein (BCL9). Two different families of stapled peptides directly targeting β-catenin have been developed. The SAH-BCL9 family143 is derived from the β-catenin-BCL9 interface, whereas the StAx peptides142 originate from Axin, a member of the destruction complex. SAH-BCL9 inhibits the interaction of β-catenin with the BCL9 coactivator, thereby affecting only a subset of Wnt target genes (Figure 6b). Notably, these genes have been implicated as oncogenic drivers in some types of cancers. SAH-BCL9B suppresses tumor growth, invasion, and angiogenesis in mouse xenografts models. StAx-35 and StAx-35R, the most potent members within the StAx peptide family, are the first examples of selective inhibitors of the interaction between βcatenin and transcription factors of the LEF/TCF family. These peptides were designed based on the β-catenin binding domain of Axin, which shares a binding site with these transcription factors (e.g., TCF4, Figure 6c bottom). Development of StAx peptides into high affinity, cell permeable β-catenin binders required extensive sequence optimization of the initial sequence obtained by i, i + 4 stapling (Figure 6c top). This process involved phage display-based affinity optimization and the introduction of positively charged residues to increase cellular uptake.142 It was shown that target affinity and subcellular localization of β-catenin targeting peptides are crucial for the desired biological activity.147 The eukaryotic translation initiation factor eIF4E is involved in cancer formation by binding to oncogenic mRNAs and pronouncing their translation.150,151 eIF4E is regulated via various PPIs, and it was possible to identify α-helical peptides

7. TRANSCRIPTIONAL AND TRANSLATIONAL MODULATORS Selective modulation of gene activation proved to be particularly challenging, mainly due to the involvement of numerous PPIs in the regulation of these processes. For this reason, stapled peptides were considered for a targeting of transcription factors and transcriptional coactivators. Examples involve inhibitors of the Notch141 or Wnt142,143 signaling pathway, which represent extremely challenging targets for classic small molecules. NOTCH Signaling Pathway. NOTCH signaling occurs through conserved pathways and plays an important role in cell differentiation, proliferation, and death.145 Upon activation of the NOTCH receptor, its intracellular domain (ICN1) translocates to the nucleus where it forms a trimeric complex with a coactivator protein of the mastermind-like (MAML) family and the transcription factor CSL. Hyper-activation of NOTCH signaling is associated with numerous types of cancer.146 On the basis of the trimeric protein complex of CSL-MAML1-ICN1 with DNA, various α-helical stretches of MAML1 were used for the design of hydrocarbon stapled peptides.141 One of the obtained i, i + 4 stapled peptides (SAHM-1) shows increased binding affinity and helicity, represses genome-wide NOTCH1 target gene expression, and proved efficient in a mouse model of NOTCH1-driven T-cell acute lymphoblastic leukemia. Wnt Signaling Pathway. Stapled peptides were also designed to modulate the canonical Wnt signaling cascade.147,148 The Wnt pathway is involved in the regulation of embryonic development as well as adult tissue homeostasis.149 In the absence of Wnt ligand, the protein β-catenin is trapped H


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ACS Chemical Biology capable of inhibiting crucial interacitons.152 This peptide served as starting point for the design of stapled peptides with improved binding properties.153 Surface plasmon resonance measurements followed by molecular dynamics (MD) simulations were used to analyze eIF4E binding, which resulted in an optimization cascade yielding a peptide with 20-fold increased affinity. The study points toward the potential of computational studies to improve the binding properties of stapled peptides. In another study, a peptide which binds to a subunit of replication protein A (RPA70N) was optimized to obtain a high affinity, cell permeable stapled peptide.154 RPA70N binds single stranded DNA and modulates DNA replication and repair.155 The combination of several approaches (alanine scanning, insertion of amino acids from another binding epitope, elimination of negatively charged residues, and introduction of an unnatural amino acid) resulted in cell-permeable stapled peptides with almost 1000-fold increased affinity. However, the optimized peptide was not able to disrupt DNA binding. Besides the direct inhibition of mRNA- and DNA-binding proteins, an addressing of epigenetic modulation opens the possibility to control gene expression. Trimethylation of lysine 27 in histone H3 is performed by the PcG protein complex and associated with a reduction of tumor suppressor activities. Within the PcG complex, the enhancer of zeste homologue 2 (EZH2) resembles N-methyltransferase activity.156 Disruption of the interaction between EZH2 and embryonic ectoderm development (EED) complex using i, i + 4 stapled peptide SAH-EZH2A(42−68) reduces the EZH2 methyltransferase activity and leads to growth arrest in cell-based assays.144 In contrast to GSK126 (a small molecule targeting the active site of EZH2), SAH-EZH2A(42−68) reduces EZH2 protein levels and represents the first inhibitor of a PPI between an epigenetic writer (EZH2) and its cofactor.

penetrating ability of some stapled peptides has recently become the subject of controversy. Reports on stapled peptides targeting the p53-MDM2/MDMX interaction raised the question of whether assay conditions such as a serum-free medium considerably support cellular uptake and activity of certain stapled peptides.43,96 Importantly, this is a trend that has also been observed for other classes of peptides. Moreover, the cellular uptake of stapled peptides has mostly been studied monitoring the fluorescence signal of labeled peptides in the context of fixed cells, which can affect proper readout.157,158 Inspired by classic cell-penetrating peptides that harbor a large number of positively charged amino acids, the optimization of stapled peptides often involves an increase of the overall positive charge. While this has led to some success, it might not be the optimal solution as indicated by hemolytic activity of several stapled peptides.70 Importantly, the preclinical MDM2/ MDMX inhibitor ATSP-7041 features an excellent pharmacokinetic profile while exhibiting a slightly negative overall charge. A thorough investigation of these features is required to enable a more efficient implementation of cell penetration properties. In addition, a general protocol for the evaluation of cell penetration properties is highly desirable to ensure better comparability of reported data. While stapled peptides mostly demonstrate an increased resistance to proteolytic degradation compared to their corresponding parent peptide sequences,159 peptide stapling alone does not necessarily provide the extent of stability, required for in vivo application. In the case of the 31-mer VIP, neither peptide stapling nor lactam cross-links were able to enhance stability against trypsin degration.117 Therefore, the stabilization of lengthy peptide sequences may require the incorporation of an additional staple, a stitched architecture, or unnatural amino acids. For instance, the insertion of two i, i + 4 staples into the 37-mer sequence of the HIV-fusion inhibitor vT649v(626−662) resulted in significantly increased stability against different proteases as well as overall enhanced pharmacokinetic properties. A general comparison of peptide stapling with alternative stabilization approaches or with smallmolecular helix mimetics is complicated. Only a few target proteins, such as MDM2/MDMX or BCL-2 family proteins, were addressed with several of these techniques.12 The inhibition of very challenging targets, such as NOTCH or Wnt transactivational complexes, has only been achieved with stapled peptides so far. However, this may reflect the overproportional use of stapled peptides and does not necessarily exclude the applicability of alternative approaches. Aiming for a most efficient conformational stabilization of αhelical peptides, alternative approaches should also be taken into consideration. In some cases, lactam bridge-containing peptides exceed stapled peptides (i, i + 4) in respect to rigidity and helical content.160 The same results were obtained, in a setup inhibiting the chaperone HSP90, where lactam constrained peptides showed better inhibition than their stapled analogous.161 In particular, a combination of peptide stapling, lactam bridges, hydrogen-bond surrogates, and β-amino acids may provide hybrid peptides with improved target affinity and bioavailability. In some cases, it was observed that the most helical peptide did not necessarily provide the highest target affinity.115−117,137 These observations point toward mechanistic questions that have not been elucidated so far: e.g., “does the binding process require the peptide to partially unfold?” or “do some stapled peptides adopt a conformation upon binding that is not perfectly α-helical?” Therefore, a detailed investigation of

8. CONCLUSION Since their introduction in 2000 by Verdine and co-workers, stapled peptides have evolved into a promising class of bioactive agents, as evidenced by the rapidly increasing number of applications. Among other techniques, they have started to fill the gap between small molecules and biologics, giving fresh impetus in addressing challenging biological targets such as PPIs. Stapled peptides were designed for numerous diseaserelevant targets linked to infections and various forms of cancer with the most advanced examples inhibiting the p53-MDM2/ MDMX interaction. It has been shown that peptide stapling is capable of enhancing drug-like properties of α-helical peptides such as proteolytic resistance, cell permeability, target affinity, and plasma half-lives. Notably, two stapled peptides have reached clinical trials: ALRN-5281, an agonist of the longacting growth-hormone-releasing hormone (GHRH) tested for the treatment of orphan endocrine disorders (phase I safety study completed), and ALRN-6924, a dual specific p53MDM2/MDMX inhibitor (phase I recruiting). The most prominent feature associated with the incorporation of a hydrocarbon staple is the enhanced ability of these peptides to penetrate cellular membranes. Over the past decade, a variety of stapled peptides demonstrated considerable cellular uptake as well as activity in xenograft models of human cancer. Nonetheless, peptide stapling does not always result in cell-permeable peptides,41,117 and in many cases, a poststapling optimization process appears to be required to generate peptides with sufficient cellular activity.45,46 The membrane I


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(7) Kaspar, A. A., and Reichert, J. M. (2013) Future directions for peptide therapeutics development. Drug Discovery Today 18, 807−817. (8) Bock, J. E., Gavenonis, J., and Kritzer, J. A. (2013) Getting in Shape: Controlling Peptide Bioactivity and Bioavailability Using Conformational Constraints. ACS Chem. Biol. 8, 488−499. (9) Garcia, A., and Camarero, J. (2010) Biological Activities of Natural and Engineered Cyclotides, a Novel Molecular Scaffold for Peptide-Based Therapeutics. Curr. Mol. Pharmacol. 3, 153−163. (10) Poth, A. G., Chan, L. Y., and Craik, D. J. (2013) Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers 100, 480−491. (11) Hill, T. A., Shepherd, N. E., Diness, F., and Fairlie, D. P. (2014) Constraining cyclic peptides to mimic protein structure motifs. Angew. Chem., Int. Ed. Engl. 53, 13020−13041. (12) Pelay-Gimeno, M., Glas, A., Koch, O., and Grossmann, T. N. (2015) Structure-based design of inhibitors of protein-protein interactions: Mimicking peptide-binding epitopes. Angew. Chem., Int. Ed., DOI: 10.1002/anie.201412070R1. (13) Klein, M. A. (2014) Stabilized helical peptides: a strategy to target protein-protein interactions. ACS Med. Chem. Lett. 5, 838−839. (14) Milroy, L.-G., and Brunsveld, L. (2013) Pharmaceutical implications of helix length control in helix-mediated protein−protein interactions. Future Med. Chem. 5, 2175−2183. (15) Jochim, A. L., and Arora, P. S. (2009) Assessment of helical interfaces in protein-protein interactions. Mol. Biosyst 5, 924−926. (16) Guarracino, D. A., Bullock, B. N., and Arora, P. S. (2011) Protein-protein interactions in transcription: A fertile ground for helix mimetics. Biopolymers 95, 1−7. (17) Mahon, A. B., and Arora, P. S. (2012) End-Capped α-Helices as Modulators of Protein Function. Drug Discovery Today: Technol. 9, e57−e62. (18) Henchey, L. K., Jochim, A. L., and Arora, P. S. (2008) Contemporary strategies for the stabilization of peptides in the αhelical conformation. Curr. Opin. Chem. Biol. 12, 692−697. (19) Azzarito, V., Long, K., Murphy, N. S., and Wilson, A. J. (2013) Inhibition of α-helix-mediated protein−protein interactions using designed molecules. Nat. Chem. 5, 161−173. (20) Toniolo, C., Crisma, M., Formaggio, F., Valle, G., Cavicchioni, G., Precigoux, G., Aubry, A., and Kamphuis, J. (1993) Structures of peptides from alpha-amino acids methylated at the alpha-carbon. Biopolymers 33, 1061−1072. (21) Doig, A. J., and Baldwin, R. L. (1995) N- and C-capping preferences for all 20 amino acids in alpha-helical peptides. Protein Sci. 4, 1325−1336. (22) Doig, A. J. (2002) Recent advances in helix−coil theory. Biophys. Chem. 101−102, 281−293. (23) Bracken, C., Gulyas, J., Taylor, J. W., and Baum, J. (1994) Synthesis and Nuclear Magnetic Resonance Structure Determination of an.alpha.-Helical, Bicyclic, Lactam-Bridged Hexapeptide. J. Am. Chem. Soc. 116, 6431−6432. (24) Jackson, D. Y., King, D. S., Chmielewski, J., Singh, S., and Schultz, P. G. (1991) General approach to the synthesis of short.alpha.-helical peptides. J. Am. Chem. Soc. 113, 9391−9392. (25) Blackwell, H. E., and Grubbs, R. H. (1998) Highly Efficient Synthesis of Covalently Cross-Linked Peptide Helices by Ring-Closing Metathesis. Angew. Chem., Int. Ed. Engl. 37, 3281−3284. (26) Schafmeister, C. E., Po, J., and Verdine, G. L. (2000) An AllHydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides. J. Am. Chem. Soc. 122, 5891−5892. (27) Kim, Y.-W., Kutchukian, P. S., and Verdine, G. L. (2010) Introduction of All-Hydrocarbon i, i +3 Staples into α-Helices via Ring-Closing Olefin Metathesis. Org. Lett. 12, 3046−3049. (28) Walensky, L. D., Kung, A. L., Escher, I., Malia, T. J., Barbuto, S., Wright, R. D., Wagner, G., Verdine, G. L., and Korsmeyer, S. J. (2004) Activation of Apoptosis in Vivo by a Hydrocarbon-Stapled BH3 Helix. Science 305, 1466−1470. (29) Guerlavais, V., and Sawyer, T. K. (2014) Advancements in Stapled Peptide Drug Discovery & Development, pp 331−345, Elsevier.

the binding process may be required to ensure optimal preorganization. Recent studies indicate that hydrocarbon stapling can also be used to constrain irregular peptide structures which may expand the applicability of the hydrocarbon stapling technology.162 Overall, stapled peptides have exceeded the developmental stage of most α-helix mimetics and have successfully completed phase I clinical trials. Despite their initial success, the approval of a stapled peptide-based drug still remains elusive, and the upcoming years will reveal if stapled peptides can stand up to the high expectations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS P.M.C. is thankful to the Studienstiftung des Deutschen Volkes for a fellowship. J.S. acknowledges financial support by Fonds der Chemischen Industrie. T.N.G. thanks the German Research Foundation (DFG, Emmy Noether program GR3592/2-1) and AstraZeneca, Bayer CropScience, Bayer HealthCare, Boehringer Ingelheim, Merck KGaA, and the Max Planck Society for their support.

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NOMENCLATURE α-helix, protein secondary structure motif formed by a highly ordered hydrogen bond network of the backbone amine and carbonyl groups of the amino acids i and i + 4 of the peptide sequence; cancer, group of diseases which involve abnormal cell growth and invasive behavior; cell penetration, cellular uptake of molecules into a cell which can be mediated by different mechanisms like endocytosis, macropinocytosis, or passive membrane diffusion; PPI-inhibitor, molecular compound that impairs the formation of a protein−protein interaction; protein−protein interaction, noncovalent interaction between two or more proteins; receptors, protein which is able to receive a molecular or environmental signal to generate a defined response; stapled peptides, stabilized α-helical peptides containing an all-hydrocarbon tether to connect two turns of the helix to lock the peptide in a α-helical conformation; transcription, first step of gene expression in which genomic DNA is transcribed into RNA

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REFERENCES

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Hydrocarbon Stapled Peptides as Modulators of Biological Function

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