Medicinal Chemistry

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Medicinal Chemistry

Stapled peptides: providing the best of both worlds in drug development

Peptide-based drug discovery has experienced a remarkable resurgence within the past decade due to the emerging class of inhibitors known as stapled peptides. Stapled peptides are therapeutic protein mimetics that have been locked within a specific conformational structure by hydrocarbon stapling. These peptides are highly important in selectively impairing disease-relevant protein–protein interactions and exhibit significant pharmacokinetic advantages over other forms of therapeutics in terms of affinity, specificity, size, synthetic accessibility and resistance to proteolytic degradation. A series of stapled peptides are currently in development, and the potential successes of these peptides, either as single-agent treatments or as combinational treatments with other therapeutic modalities, could potentially change the landscape of protein therapeutic development. Here, we provide examples of successful discovery efforts to illustrate the research strategies of stapled peptides in drug design and development. First draft submitted: 14 May 2016; Accepted for publication; 4 August 2016; Published online: 21 September 2016 Keywords:  drug development • protein–protein interactions • stapled peptides

Background With the philosophical shift over the past decade toward designing highly specific targeted therapies for drug development, determining the exact cellular processes and molecular interactions that can be therapeutically inhibited within specific diseases have become the critical focus for connecting scientific discoveries to clinical utility. This targeted-therapy approach has led to a new era of therapeutic agent development and exponentially increased the availability of strategies needed for reversing the effects of certain diseases. Most of the recently developed agents can be divided into two classes: smallmolecule and large-molecule targeted inhibitors. Small-molecule inhibitors are chemically synthesized stable compounds comprised of different organic and/or inorganic substrates. Although most of small molecules have the advantage of penetrating cells and target-

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specific intracellular proteins, the large binding surfaces of intracellular protein–protein interactions (PPIs) as well as the inaccessible residues of certain disease-driving proteins often make small-molecule modulators ineffective [1] . By contrast, large molecules known as biologics provide therapeutic benefits not offered by small-molecule inhibitors based on their ability to disrupt the construction and functionality of extracellular PPIs. However, biologics are often unable to effectively permeate cells and are highly susceptible to proteolytic instability. Because of the pharmacological limitations of both classes of inhibitors, approximately 80% of potential drug targets are considered ‘undruggable’ [2] . Nevertheless, different modalities of small molecules or biologics have been developed to overcome the relative disadvantages inherent to these classes of therapeutic agents. Specifically, a novel class of therapeutics called stapled peptides [3] has

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Xiayang Xie1, Lixia Gao2, Austin Y Shull3 & Yong Teng*,2,4,5 1 Department of Pediatrics, Emory Children’s Center, Emory University, Atlanta, GA 30322, USA 2 Department of Oral Biology, Dental College of Georgia, Augusta University, Augusta, GA 30912, USA 3 Department of Biology, Presbyterian College, Clinton, SC 29325, USA 4 Georgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA 5 Department of Biochemistry & Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA *Author for correspondence: Tel.: +1 706 446 5611 Fax: +1 706 721 9415 [email protected]

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ISSN 1756-8919

Review  Xie, Gao, Shull & Teng emerged to seek to marry the best of both modalities by being able to both target intracellular PPIs as well as provide cell permeability and stability to potent, bi­oactive p­eptides (Table 1) [4] . Although the concept of using peptides to modulate intracellular processes has been investigated, they are inherently unstable within the body. Therefore, several chemical modifications have been implemented in order to maintain their proper conformation for therapeutic activity [3] . For example, peptides with an α-helix structure are rich with positively charged residues and can readily pass through the cell membrane [5–7] . Nevertheless, the conformational structures of α-helix-based peptides are relatively unstable, so approaches like disulfide-bridged framework [8] and lactam bridges [9] have been used to stabilize α-helical peptides. However, these methods are not highly effective in stabilizing peptide conformation when the peptides were placed under physiological conditions. It was not until the seminal study from Blackwell and Grubbs that shown the feasibility of ring-closing metathesis in the peptide context. In this study, the group performed an metathesis reaction on a pair of O-allylserine residues in order to form a covalent bond (Figure 1A) [10] . This work was then followed by Verdine and colleagues who designed unnatural amino acids at the α-carbon to create diverse configurations of all-hydrocarbon cross-links (Figure 1B) [11] . This revolutionary work provided great potential for targeting ‘undruggable’ PPIs based on the ability of yielding peptide constructs with marked α-helical stabilization. The study in this field was further advanced by Bernal et al. who reported another ‘peptide stapling’ strategy in which an all-hydrocarbon cross-link is generated in natural peptides by a ruthenium-catalyzed olefin metathesis of inserted α,α-disubstituted non-natural amino acids bearing olefinic side chains [12] . Although ‘stapled peptides’ are nowadays used more widely for macrocyclic helical peptides, this review mainly focuses on ‘h­ydrocarbon stapled peptides’. In design terms, the key components of the system are R-methylated amino acids 1, bearing olefinic side chains of varying length and configured with either R or S stereochemistry, the two non-natural amino acid alkenes, and each of them is α-methylated (Figure 2A)  [10–12] . Multiple types of all-hydrocarbon staples have been optimized through systematic variation of the conglutinate position, cross-linker length and stereochemistry (Figure 2B) . For example, the placement of one unit of R8 cross-linking amino acid at the i position and one unit of S5 at the i+7 position results in a stabilized i, i+7 stapled peptide (Figure 2B). The all-hydrocarbon stapling system is unique not only in its combination of two distinct helix-stabilizing strategies (cross-linking and α-methylation), but also in its avoidance of hetero-

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atoms in the macrocyclic bridge [13] . This revolutionary system is highly adaptable to most peptide sequences, leading to favorable ruthenium-catalyzed ring-closing metathesis kinetics, helix stabilization and promotion of cellular uptake [13] . The ongoing efforts in drug development are to move promising discovery candidates into commercial production. Although the concept of stapled peptides is not new, they have only recently been considered as a viable option for clinical development. There are numerous alternative approaches to stabilize α-helix formation, and these approaches still face challenges pertaining to cost and delivery. However, stapled peptides have the promising potential in becoming the next prominent class of therapeutic agents. Applications of stapled peptides in oncology Selective modulation of transcriptional and translation mechanisms is particularly challenging due to the involvement of specific PPIs that regulate numerous biological processes. Unlike classic small-molecule modulators, peptides can overcome the challenge provided by PPIs by disrupting the larger contact surfaces that are crucial for specific PPI-driven events. Based on their conformational and binding abilities, stapled peptides have been widely used in cancer prevention and treatment. The efficient targeting ability and other unique characteristics of stapled peptides offer unprecedented advantages in disrupting previously ‘undruggable’ mechanisms critical for oncogenesis. Targeting Wnt signaling pathway

The Wnt signaling pathway regulates cell morphology, movement, proliferation and differentiation [14] . It is critically important for embryonic development, tissue regeneration and adult tissue homeostasis. Elevation of canonical Wnt/β-catenin signaling has been linked to neurologic diseases, inflammatory diseases, diabetic and endocrine disorders and bone metabolism in adults [15–17] . Dysregulation of Wnt signaling can completely alter the proper specification of cell fate as well as proper formation of organs. The critical events driven by Wnt signaling suggest that manipulating this pathway with either activators or inhibitors may provide therapeutic potential to diseases driven by deregulated Wnt signaling. β-catenin is a central effector in this pathway, and its stability is regulated by a complex containing a protein known as Axin. Axin will directly interact with β-catenin and enables the GSK3β kinase to phosphorylate β-catenin, causing subsequent ubiquitination and degradation of β-catenin [18,19] . Understanding the consequences of this interaction, Cui’s group designed cell-permeable stapled peptides that were able to activate Wnt signaling by selectively disrupting the Axin–β-catenin interaction [20] . The

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Stapled peptides as promising drugs 

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Table 1. Comparison of different molecular weight drugs.  

Small molecules

Biologics

Stapled peptides

Molecular weight

10 4

103 –5 × 103

Merits

– High stability – High permeability – Low cost

– Low toxicity – High affinity – High selectivity – Ability to disrupt intercellular PPIs

– Low toxicity – High in vivo stability – High permeability – Ability to target intracellular and extracellular PPIs – High affinity and selectivity

Demerits

– Severe side effects – Inability to disrupt intercellular PPIs

– Poor in vivo stability – Inability to traverse cell membrane – High cost

– High cost – Unknown pharmacokinetic properties

PPI: Protein–protein interaction.

stapled peptides targeting this interaction called SAHPAs were developed by incorporating non-natural amino acids at neighboring suitable positions along one face of the α-helix structure. The two stapled α-helical peptides, SAHPA1 and SAHPA2 (Table 2), were generated to target the Axin–β-catenin complex and are able to efficiently disrupt the endogenous Axin–β-catenin complex by competitively binding to β-catenin [20] . In contrast to the Axin–β-catenin complex, another protein known as the BCL9 protein binds to β-catenin and instead enables proper Wnt/β-catenin-mediated transcription in human cell lines [35] . Based on the role BCL9 plays in enabling the effects of Wnt pathway activation, inhibiting its association with β-catenin would mitigate Wnt activation. Based on this interaction, another family of stapled peptides called SAHBCL9s (stabilized α helix of BCL9s) have been successfully utilized to directly target β-catenin–BCL9 interactions  [36] . SAH-BCL9B was generated by replacing native residues on the noninteracting surface of the BCL9 HD2 domain with olefinic non-natural amino acids. This family of stapled-peptides exhibited great ability in disrupting the β-catenin–BCL9 complex, suppressing Wnt-mediated transcriptional response and inhibiting tumorigenic progression [37] . Therefore, targeting affinity and subcellular localization of β-catenin by specifically designed stapled peptides is crucial for determining the desired biological activity elicited. Targeting Notch signaling pathway

Notch proteins (Notch1, 2, 3 and 4) are involved in a conserved signaling pathway that regulates cell differentiation, proliferation and death [38,39] . Notch signaling participates in a variety of complex functions, including hematopoiesis [40] , T-cell development [41] , angiogenesis  [42] and other physiologically relevant processes.

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When the Notch receptor is activated, its intracellular domain (ICN1) translocates to the nucleus, binds to the transcription factor CSL (CBF1, Suppressor of Hairless, Lag-1) and recruits coactivator proteins from the mastermind-like (MAML) family to form a trimeric complex  [43] . Although the duration and strength of Notch signal pathway are usually tightly controlled, mutations found in a variety of diseases including several types of cardiac diseases [44] , neurological diseases [45] and cancers [46] can cause uncontrolled activation or suppression of the pathway. The complexity of the pathway provides several possible targets when developing therapeutic agents against the Notch pathway. Moellering et al.

O

O

O

O

RCM: O-allyl serine residues

CH3

CH3

CH3 CH3 O O

RCM: α, α-disubstitution, olefin tether

Figure 1. Application of ruthenium-catalyzed olefin metathesis to install macrocyclic cross-links into synthetic peptides. (A) The metathesis reaction on a pair of O-allylserine residues. (B) α,α-disubstituted non-natural amino acids bearing all-hydrocarbon tethers. RCM: Ruthenium-catalyzed ring-closing metathesis.

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Review  Xie, Gao, Shull & Teng

Cl Cl

PCy3 Ru PCy3

O

Fmoc-R8

Fmoc-S5

FmocHN

OH

O FmocHN

OH

a 2

R5

S5 i, i + 3

b n

2

n

2

S5

S5 i, i + 4

c Linear peptide

5 R8

2 S5 i, i + 7

Stapled peptide Figure 2. Schematic illustration of peptide stapling and three common types of all-hydrocarbon stapled peptides. (A) Two nonnatural amino acid alkene (R8 and S5) are inserted in the peptide chain, and future cross-linked by ring-closing olefin metathesis at two positions in the peptide chain. (B) Resin-bound peptide is treated with Grubbs I olefin metathesis catalyst to produce a cross-link between the two non-natural amino acids, resulting in a stapled peptide that is braced in an α-helical conformation. i, i+3 stapled peptides: R5 at the i position and S5 at the i+3 position. i, i+4 stapled peptides: two units of S5 at the positions i and i+4. i, i+7 stapled peptides: R8 at the i position and S5 at the i+7 position. Adapted with permission from [4] © The Royal Society of Chemistry.

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Stapled peptides as promising drugs 

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Table 2. The most promising targets and stapled peptides. Target

Stapled peptides

Biological function

Clinical trial?

Axin–β-catenin

SAHPAs

Activate an enhance Wnt/β-catenin signaling in a ligand-dependent manner

No

[20]

MAML1

SAHM1

Inhibit the Notch signaling pathway in human and mice

No

[21]

VPAC2

I17-VIP-GK

Regulate high blood sugar disease

No

[22]

p53–MDM2/MDMX SAH-P53–8

Restore p53 activity through direct inhibition of both No MDM2 and MDMX

[23]

p53–MDM2/MDMX ATSP-7041

Restore p53 activity through direct inhibition of both No MDM2 and MDMX

[24,25]

p53–MDM2/MDMX ALRN-6924

Restore p53 activity through direct inhibition of both Yes (NCT02264613) MDM2 and MDMX

BCL-2

SAHBs

Activate apoptotic pathway in leukemia cells

No

[27]

CYFIP1–WAVE3

WAHMs

Inhibit cancer cell invasion

No

[28]

HIV

NYAD-1

Inhibit HIV activity

No

[29,30]

GHRH

ALRN-5281

Boost the release of growth hormone in people with Yes (NCT01775358) rare endocrine disorders

HCV

SAHH-5

Inhibit activity against HCV infection

No

[31]

RAS

SAH-SOS1A

Block downstream of KRAS and impair KRAS-driven cancer cell viability

No

[32]

Rab8a

StRIP3

Inhibit a Rab8a-effector interaction

No

[33]

conG[11-15, Si, S(8)]

Block NR2B-containing NMDA receptors and provide No protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice

[34]

NMDA

i+4

Ref.

[26]

 

conG: Conantokin G; GHRH: Growth hormone-releasing hormone; HCV: Hepatitis C virus; MDM2: Murine double minute 2; MDMX: Murine double minute X; NMDA: N-methyl- d -aspartate; SAHB: Stabilized α helix of BCL-2; WAHM: WASF helix mimic; VIP: Vasoactive intestinal peptide. 

designed α-helix peptides using a series conformation locking based on the trimeric protein complex structure of CSL-MAML1-ICN1 with DNA [21] . By screening the performance of these stapled peptides, Moellering et al. identified that SAHM1 (Table 2), which utilized an i, i+4 pattern to increase binding affinity and helicity of the stapled peptide, ultimately elicited substantial repression of Notch1 targets genome-wide when implemented [21] . Most importantly, SAHM1 efficiently inhibited the Notch-driven acute T lymphoblastic leukemia cell production in both human cells and mice xenografts. Therefore, SAHM1 demonstrates great the­ rapeutic ability and high potential for clinical treatment. Targeting Wiskott–Aldrich syndrome protein family regulatory complex

Wiskott–Aldrich syndrome protein family (­W­AVE/WASF) members bind to monomeric actin and the ARP2/3 complex to facilitate actin polymerization and cytoskeleton reorganization [47] . CYFIP1 is one of WAVE regulatory complex (WRC) components, and activation of the WRC occurs through interaction with RAC. One study from our group has

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shown that inactivation of CYFIP1 prevents the association of RAC with the WAVE protein, leading to loss of breast cancer cell invasion [28] . Our group successfully designed stapled peptides referred to as WASF helix mimics (WAHMs) (Table 2) based on existing crystallographic data demonstrating the interface between WAVE1 and CYFIP1 proteins [28] . In the current proof-of-principle series of experiments, we demonstrated that WAHMs can destabilize the WRC and suppress RAC binding to WAVE proteins in serumfree-cultured breast cancer cells. As a result, increased numbers and thickness of the actin stress fibers as well as reduced cell motility and invasion were observed in WAHM-treated breast cancer cells (Figure 3) . Therefore, WAHMs may represent a powerful th­erapeutic strategy for preventing tumor invasion and metastasis. Targeting the p53–murine double minute 2/murine double minute X interaction

The tumor suppressor p53 protein is a human transcription factor that plays a central role in guarding the cell in response to cellular stress and oncogenic activation [48] . The association of p53 with its negative regulators

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Review  Xie, Gao, Shull & Teng murine double minute 2 (MDM2) and murine double minute X (MDMX) is one of the more intriguing targets in anticancer strategies. Small molecules like Nutlin 3-a, discovered by chemical library screening, can inhibit the interaction between MDM2 and p53 and allow continued stabilization of p53. Recently, stapled peptides SAH-p53 were developed as effective disruptors of the p53–MDM2/MDMX interaction and enabled restoration of p53 tumor suppressive activity [49] . Compared with traditional small molecules, stapled peptides like SAH-p53–8 (Table 2) significantly improve the membrane permeability and proteolytic resistance for the targeting peptide [23] . Biological investigations using a series of solid tumor cell lines expressing differential levels of MDM2 and MDMX reveal that SAH-p53–8, in contrast to the MDM2-specific antagonist Nutlin-3, was able to activate the p53 tumor suppressor pathway and induce apoptosis regardless of MDMX levels. Although its affinity for MDM2 was 25-fold higher than MDMX, SAH-p53–8 could still effectively block the formation of the p53–MDMX complex. Intravenous administration of the dual-specific MDM2/MDMX antagonist SAHp53–8 to mice induced a p53 response in the tumor cells and suppressed tumor growth [23] . The optimized i, i+7-stapled peptide ATSP-7041 (Table 2) was then later generated by Aileron Therapeutics (MA, USA) and demonstrated improved binding affinity for both MDM2 and MDMX [24] . This p53-activating peptide can elicit p53-independent cytotoxicity, which is inhibited by serum and different from Nutlin-3a induced off-target effects [25] . Compared with the Nutlin family of inhibitors, ATSP-7041 exhibits low clearance and long plasma half-life in mouse models. Moreover, ATSP-7041 (10 μM) reached full p53–MDM2 inhibition much slower (4 h) than Nutlin-3a (20 min) [25] . The next-generation peptide ALRN-6924 (Table 2) binds to both MDM2 and MDMX and interferes with their interaction with the transcriptional activation domain of the tumor suppressor protein p53 [26] . A safety study for oral administration of ALRN6924 is currently ongoing in patients with advanced solid tumors or lymphomas (Phase I, trial IDs: NCT02264613)  [26] , indicating MDM2/MDMX double targeting peptides as having a great deal of potential within a clinical setting. Targeting interactions between BCL-2 family proteins

The BCL-2 family proteins govern mitochondrial outer membrane permeabilization and act as promoters or inhibitors of cell apoptosis [50,51] . An increasing number of studies have been conducted in determining the potential of BCL-2 homology 3 domains in interfering with interactions between proapoptotic members, such

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as BID, BAD and antiapoptotic members (e.g., BCL-2 and BCL-XL) [52] . Verdine et al. developed a branch of stapled peptides derived from the BID BCL-2 homology 3 domain by α-helix fragment and showed apoptotic inducing capability in mouse xenograft models [53] . Another set of stapled peptides known as SAHBs (Table 2), originating from 13 different BCL-2 family members, were generated to improve membrane penetration and enzymatic stability [27] . Walensky’s group designed stable α-helix peptides with varying selectivity, including SAHBD, which specifically targets the MCL-1 protein. These stapled peptides provide a prototype for the development of therapeutics for cancer as well as other diseases in­volving the BCL-2 protein family [54–59] . Targeting small GTPases

Small GTPases are molecular switches using GDP/GTP alternation to control numerous vital cellular processes. Mutationally activated small GTPases are frequent mechanisms utilized in cancer development and progression. Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N- and H-RAS, and are associated with reduced patient survival and chemoresistance [60] . Leshchiner et al. reported a new application of all-hydrocarbon stapling to recapitulate the native primary sequence and secondary structure of the RAS-interacting α-helix of SOS1 [32] . They found that stapled peptide SAHSOS1A (Table 2) can target the SOS1-binding pocket on both wild-type and mutant forms of KRAS with nanomolar affinity [32] . SAH-SOS1A treatment not only blocked the phosphosignaling cascade downstream of KRAS but also impaired KRAS-driven cancer cell viability in a dose-dependent manner [32] . Importantly, the effects of SAH-SOS1A extended to an in vivo context in a Drosophila model containing inducible mutant RAS expression [32] . The same research group also designed a stapled peptide StRIP3 (Table 2) based on crystal structures of Rab proteins bound to their interaction partners. This peptide selectively binds to activated Rab8a and inhibits an Rab8a–effector interaction in vitro [61] . Most interestingly, by replacing the olefin metathesis using an alkyne cross-link, these modified peptides displayed more than a threefold increased potency and a 15-fold increased binding affinity when compared with the parent hydrocarbon stapled peptide StRIP3 [33] . Therefore, a peptide-stapling approach to targeting the regulatory groove on small-GTPases may be a viable strategy for therapeutic development. Applications of stapled peptides in infectious diseases The ability of ‘stapling’ peptide helices to generate high-fidelity, protease-resistant mimics of antigenic

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Stapled peptides as promising drugs 

CYFIP1

Review

CYFIP1

WASF3

WASF3 + WAHMs

Figure 3. The working principle of WASF helix mimic-stapled peptides. WAHMs were designed to target an α-helical interface between WAVE3/WASF3 and CYFIP1 proteins to destabilize the components of WRC. WAHM: WASF helix mimic; WAVE/WASF: Wiskott–Aldrich syndrome protein family; WRC: WAVE regulatory complex.

structures provides substantial promise in vaccine development and infectious disease control. Many of the infectious diseases that can be effectively reversed by stapled peptides include HIV as well as hepatitis C. Targeting HIV type 1

HIV type 1 (HIV-1) attacks the body’s immunity, resulting in loss of a proper antigenic immune response. Because of the compromised immune system, HIV-1 can ultimately lead to a variety of diseases including AIDS and cancer [62,63] . It has been reported that the capsid assembly inhibitor (CAI), a 12-mer peptide, can target in vitro the HIV-1 capsid C-terminal domain and disrupt HIV-1 particle formation [64] . However, this precursor peptide has low cell permeability and protease stability, ultimately limiting bioavailability. Based on high-resolution x-ray crystal structure of the CAI in complex with capsid C-terminal domain [65] , Zhang et al. stabilized the α-helical structure of CAI and converted it into a cellpenetrating peptide [29] . Optimized peptide NYAD-1 (Table 2) had enhanced α-helicity, broad spectrum antiviral activity against HIV-1 and enhanced proteolytic resistance compared with long peptides [29,30] . As well, Bird et al. synthesized carbon–carbon (i, i+4) backbone, conformation-locked enfuvirtide peptides through olefin metathesis and determined the biophysical and biopharmacological properties of the peptide [66] . In this study, they found that hydrocarbon double-stapling peptides have significant oral bioavailability, providing new op­portunities for treatment against AIDS. Targeting hepatitis C virus

Hepatitis C virus (HCV) is a capsule of single-strand RNA virus belonging to the Flavivirus family that drives the infectious disease hepatitis C [67,68] . Human cell surface protein CD81 is an important receptor of HCV, and the binding of CD81 to HCV envelope glycoprotein E2 is necessary for HCV infection [69,70] . Therefore, the ability to block HCV and CD81 interaction is crucial for blocking HCV infection. Cui et al. generated a set of stapled peptides to impair HCV

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membrane fusion [31] . Among them, SAHH-5 (Table 2) demonstrated significantly higher α-helicity and inhibitory activity against HCV infection as well as increased resistance to proteolysis compared with its linear peptide counterpart [31] . These observed advantages have provided motivation in developing novel peptide-based anti-infective agents over existing therapeutic options. Applications of stapled peptides in nervous system Therapeutic interventions using neuropeptides are being developed to modulate neuronal network activity over prolonged periods of time [71,72] . However, the fulllength or truncated analogs of neuropeptides are prone to rapid degradation in serum stability. Green et al. introduced a hydrocarbon stapling to stabilize the helical conformation of bioactive peptides and active fragments of galanin and neuropeptide Y [73] . Green et al. established that these stapled analogs improved physicochemical features and metabolic stability dose dependently while maintaining their anticonvulsant activities in the 6-Hz seizure mouse model [73] . Conantokins, the small helical peptides derived from the venoms of marine cone snails, can antagonize N-methyl- d -aspartate receptors but are not stabilized by disulfide cross-links [74] . Platt et al. added a dicarba bridger at i, i+4 intervals to covalently stabilize helicity of conantokin G (conG) peptide analogs, with varying connectivities in terms of staple length and location along the face of the α-helix  [34] . Unlike native conG, conG [11–15, Si, i+4S(8)] (Table 2) produced no behavioral motor toxicity and improved its pharmacological properties with significant protection in the 6-Hz psychomotor mouse model of pharmacoresistant epilepsy. These findings extend the applications of stapled peptides with extracellular targets and provide a means for the treatment of nervous system disorders. Applications of stapled peptides in other diseases Based on the intrinsic features of stapled peptides, several biological processes that can potentially be miti-

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Review  Xie, Gao, Shull & Teng gated by stapled peptides include cell surface receptor signaling pathway, as well other intracellular cascades responsible for driving several abnormalities. Targeting cell receptors

Stapled peptides have also been used to manipulate the activation of several different cell receptors. These targeted receptors include estrogen receptor [75] , ATP-binding cassette transporter ABCA1 [76] , EGF receptor  [77] and G-protein-coupled receptors [78] . One specific utilization of stapled peptides involves the vasoactive intestinal peptide (VIP), a 28-amino acid neurotransmitter widely distributed in both the central and the peripheral nervous system [79] . There are two known receptors for VIP called VPAC1 and VPAC2, both of which are G-protein coupled receptors [80] . VIP is a member of the secretin/glucagon class of proteins that potently agonizes VPAC2, and agonists of VPAC2 facilitate glucose-dependent insulin secretion [22] . However, VIP has a short serum half-life and poor pharmacokinetics in vivo. Giordanetto et al. thus developed stapled VIP-based peptides as VPAC2 agonists to improve VPAC2 agonism and glucosedependent insulin secretion [22] . An in vivo study showed that one of these peptides I17-VIP-GK (Table 2) significantly enhanced VPAC2 agonist potency and induced glucose-dependent insulin secretion activity in rat pancreatic islets [22] . These observations indicate that VIP–VPAC2 is an attractive candidate for stapled peptide-based Type 2 diabetes and hyperglycemia treatment. Stapled peptide drug ALRN-5281 (Table 2) is a proprietary, long-acting growth hormone-releasing hormone agonist for treating orphan endocrine disorders, including adult growth hormone deficiency and HIV lipodystrophy, as well as broader patient populations that exhibit a wide variety of metabolic/endocrine diseases. The initial Phase I trial evaluated the safety and tolerability of single ascending doses of ALRN-5281 administered by subcutaneous injection in healthy adult subjects (Phase I, trial IDs: NCT01775358) and revealed that ALRN-5281 was safe, tolerable and effective for longer periods co­mpared with traditional peptide hormones. Targeting Cullin3–Bric-a-brac, Tramtrack and Broad complex interface

A key factor of protein ubiquitination and an essential component of the Cullin 3-Ring ubiquitin Ligases system, Cullin3 (Cul3) is able to interact with dozens of different proteins containing Bric-abrac, Tramtrack and Broad Complex domains [81] . Because its alteration is linked to several human diseases, hydrocarbon stapled peptides were designed to target the Cul3–Bric-a-brac, Tramtrack and Broad

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complex interface by using an intriguing approach of stabilizing the α-helical conformation of Cul3based peptides [82] . Evaluating the impact of stapling biochemical/biophysical properties of Cul3-derived peptides obtained by the stapling different Cul349–68 regions suggest that these peptides can p­otentially m­odulate fu­ndamental processes involving Cul3. Conclusion Targeting PPIs with stapled peptides is one of the promising strategies that effectively bridge the techniques of chemical biology with the native molecular processes among diseases. Hydrocarbon stapled peptides represent a promising class of pharmaceuticals that are intermediate in size, affinity, specificity, permeability and cellular stability when compared with traditional organic pharmacophores and protein drugs. By direct chemical modification for stabilization of bioactive peptide confirmations, stapled peptides can mimic proteins ranging from small hormones to large growth factors. Currently, stapled peptide analogs have been shown to be effective in the control or modulation of many biological processes, including regulation of cell receptors, inhibition of apoptotic pathways and suppression of oncogenic cascades. Therefore, stapled peptides hold a great deal of potential in bridging the gap between small molecules and biologics as well as providing new opportunities for disease prevention and treatment. Future perspective Naturally occurring peptides are often not directly suitable for use as convenient treatment because of poor chemical and physical stability, short circulating plasma half-life, poor penetration into target tissue and adverse effects in healthy cells. However, stabilized helical peptides mimicking the original proteins at their binding sites while demonstrating properties similar to small molecules in regard to absorption, distribution and metabolism, have gained increased interest as a safe and efficacious therapeutic strategy. In the global peptide drug market, approximately 140 peptide therapeutics are currently being evaluated in clinical trials [83] , with Aileron Therapeutics having pioneered the first clinical trial of stapled peptides. Nevertheless, more research groups and biotechnology companies are getting involved in this research discipline since the advantageous qualities of these peptides provide motivation for the development of effective therapies. Nevertheless, despite their initial success, cost and largely unknown pharmacokinetic properties make stapled peptides hard to develop compared with other biologics like therapeutic antibodies. There is still much to learn regarding the development of stapled

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Stapled peptides as promising drugs 

peptides as potent and specific inhibitors of PPIs. For example, rational design of specific stapled peptides should start with a known crystal structure of key compounds bound to the target, and this design requires at least one α helix between the binding sites of the targeting proteins. In some cases, serum can antagonize peptide induced membrane damage [25] . It is also true that cell penetration of stapled peptides has to be improved by sequence optimization. Therefore, finding new strategies to enhance oral availability of these peptides is an imminent need. Although approval of stapled peptide-based drug faces several challenges, an increasing number of preclinical advances as well as clinical investigation of stapled peptides (such as ALRN-5281 and ALRN-6924) provide examples of

Review

the efforts being made for determining the full potential of peptide drugs against disease progression. We foresee that these emerging peptides will offer enormous potential as effective therapeutics against poorly treated diseases. Financial & competing interests disclosure This work was supported in part by grant from Department of Defense (W81XWH-14-1-0412). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the m­anuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary • Hydrocarbon stapled peptides are designed to mimic an α-helical structure through a constraint imposed by covalently linking two residues on the same helical face. This ‘stapling’ enhances cellular membrane permeability, therapeutic selectivity and proteolytic stability. • Direct chemical modification of stapled peptides improves bioavailability and their potential as therapeutic agents. • Design of stapled peptides requires protein crystal structure and α-helical interfaces between targeted proteins. • The most prominent feature of stapled peptide-based drugs is combining the desirable properties of the two existing drug classes: their ability to lock-in to target proteins giving them the specificity of biological molecules like biologics; and their small size (around 50 amino acids) allowing them to penetrate cells similar to small-molecule inhibitors. • Stapled peptides can reach previously ‘undruggable’ targets and provide a broad solution to human diseases, spanning cancer, infectious diseases, metabolism and neuroscience.

References

8

Almaaytah A, Tarazi S, Mhaidat N et al. Mauriporin, a novel cationic alpha-helical peptide with selective cytotoxic activity against prostate cancer cell lines from the venom of the scorpion Androctonus mauritanicus. Method Mol. Biol. 19(4), 281–293 (2013).

9

Aihara K, Komiya C, Shigenaga A et al. Liquid-phase synthesis of bridged peptides using olefin metathesis of a protected peptide with a long aliphatic chain anchor. Org. Lett. 17(3), 696–699 (2015).

10

Blackwell HE, Grubbs RH. Highly efficient synthesis of covalently cross-linked peptide helices by ring-closing metathesis. Angew. Chem. Int. Ed. Engl. 37(23), 3281–3284 (1998).

11

Schafmeister CE, Po J, Verdine GL. An all-hydrocarbon cross-linking system for enhancing the helicity and metabolic stability of peptides. J. Am. Chem. Soc. 122(24), 5891–5892 (2000).



Describes the nail carbohydrates enhance the helical stability of peptides by diverse configurations of allhydrocarbon cross-links. 

12

Bernal F, Tyler AF, Korsmeyer SJ et al. Reactivation of the p53 tumor suppressor pathway by a stapled p53 peptide. J. Am. Chem. Soc. 129(16), 5298–5298 (2007).

Papers of special note have been highlighted as: • of interest; •• of considerable interest 1

Nero TL, Morton CJ, Holien JK et al. Oncogenic protein interfaces: small molecules, big challenges. Nat. Rev. Cancer 14(4), 248–262 (2014).

2

Gongora-Benitez M, Tulla-Puche J, Albericio F. Multifaceted roles of disulfide bonds. Peptides as therapeutics. Chem. Rev. 114(2), 901–926 (2014).

3

Walensky LD, Bird GH. Hydrocarbon-stapled peptides: principles, practice, and progress. J. Med. Chem. 57(15), 6275–6288 (2014).

4

Chu Q, Moellering RE, Hilinski GJ et al. Towards understanding cell penetration by stapled peptides. MedChemComm 6(1), 111–119 (2015).

5

6

7

Cronican JJ, Thompson DB, Beier KT et al. Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem. Biol. 5(8), 747–752 (2010). Lawrence MS, Phillips KJ, Liu DR. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 129(33), 10110–10112 (2007). Jenssen H, Aspmo SI. Serum stability of peptides. Method Mol. Biol. 494, 177–186 (2008).

future science group

www.future-science.com 10.4155/fmc-2016-0102

Review  Xie, Gao, Shull & Teng 13

Kim YW, Grossmann TN, Verdine GL. Synthesis of allhydrocarbon stapled α-helical peptides by ring-closing olefin metathesis. Nat. Protoc. 6(6), 761–771 (2011).

14

Alexander MS, Kawahara G, Motohashi N et al. MicroRNA199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation. Cell Death Differ. 20(9), 1194–1208 (2013).

15

Krimpenfort P, Berns A. Wnt down, tumors wind up? Cell 161(7), 1494–1496 (2015).

16

Rossini M, Gatti D, Adami S. Involvement of WNT/betacatenin signaling in the treatment of osteoporosis. Calcif. Tissue Int. 93(2), 121–132 (2013).

17

Cui HK, Zhao B, Li YH et al. Design of stapled alpha-helical peptides to specifically activate Wnt/beta-catenin signaling. Cell Res. 23(4), 581–584 (2013).

18

Gao C, Xiao G, Hu J. Regulation of Wnt/beta-catenin signaling by posttranslational modifications. Cell Biosci. 4(13), 1–20 (2014).

ALRN-6924 in patients with advanced solid tumors or lymphomas.  27

Walensky LD, Kung AL, Escher I et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305(5689), 1466–1470 (2004).

28

Teng Y, Bahassan A, Dong D et al. Targeting the WASF3CYFIP1 complex using stapled peptides suppresses cancer cell invasion. Cancer Res. 76, 965–973 (2016).



Demonstrates that disruption of Wiskott–Aldrich syndrome protein family regulatory complex with WAHM peptides represents a promising therapeutic strategy for preventing breast and prostate cancer invasion and metastasis.

29

Zhang H, Zhao Q, Bhattacharya S et al. A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J. Mol. Biol. 378(3), 565–580 (2008).

30

Zhang H, Curreli F, Zhang X et al. Antiviral activity of alpha-helical stapled peptides designed from the HIV-1 capsid dimerization domain. Retrovirology 8(28), 1–18 (2011).

19

Xu Y, Lee SH, Kim HS et al. Role of CK1 in GSK3betamediated phosphorylation and degradation of snail. Oncogene 29(21), 3124–3133 (2010).

31

20

Cui HK, Zhao B, Li Y et al. Design of stapled alpha-helical peptides to specifically activate Wnt/beta-catenin signaling. Cell Res. 23(4), 581–584 (2013).

Cui HK, Qing J, Guo Y et al. Stapled peptide-based membrane fusion inhibitors of hepatitis C virus. Bioorgan. Med. Chem. 21(12), 3547–3554 (2013).

32



Demonstrates the cell-permeable stapled peptides activate Wnt signaling with a good selectivity to disrupt the Axin–β-catenin interaction. 

Leshchiner ES, Parkhitko A, Bird GH et al. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. Proc. Natl Acad. Sci. USA 112(6), 1761–1766 (2015).

33

21

Moellering RE, Cornejo M, Davis TN et al. Direct inhibition of the NOTCH transcription factor complex. Nature 462(7270), 182–188 (2009).

Cromm PM, Schaubach S, Spiegel J et al. Orthogonal ringclosing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides. Nat. Commun. 7, 11300 (2016).

22

Giordanetto F, Revell JD, Knerr L et al. Stapled vasoactive intestinal peptide (VIP) derivatives improve VPAC2 agonism and glucose-dependent insulin secretion. ACS Med. Chem. Lett. 4(12), 1163–1168 (2013).

34

Platt RJ, Han TS, Green BR et al. Stapling mimics noncovalent interactions of gamma-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-d-aspartic acid receptors. J. Biol. Chem. 287(24), 20727–20736 (2012).

23

Chang YS, Graves B, Guerlavais V et al. Stapled alpha-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc. Natl Acad. Sci. USA 110(36), 3445–3454 (2013).

35

Sustmann C, Flach H, Ebert H et al. Cell-type-specific function of BCL9 involves a transcriptional activation domain that synergizes with beta-catenin. Mol. Cell Biol. 28(10), 3526–3537 (2008).

••

Reports a potent and selective dual inhibitor of murine double minute 2 and murine double minute X, ATSP-7041 stapled peptide, which effectively activates the p53 pathway in tumors in vitro and in vivo.

36

Kawamoto SA, Coleska A, Ran X et al. Design of triazolestapled BCL9 alpha-helical peptides to target the betacatenin/B-cell CLL/lymphoma 9 (BCL9) protein–protein interaction. J. Med. Chem. 55(3), 1137–1146 (2012).

24

Chang Y, Graves B, Guerlavais V et al. ATSP-7041, a dual MDM2 and MDMX targeting stapled a-helical peptide exhibits potent in vitro and in vivo Efficacy in xenograft models of human cancer. Eur. J. Cancer 48(6), 68–69 (2012).

37

Takada K, Zhu D, Bird GH et al. Targeted disruption of the BCL9/beta-catenin complex inhibits oncogenic Wnt signaling. Sci. Transl. Med. 4(148), 148ra117 (2012).

38

Eberhart C. Multiple cilia suppress tumour formation. Nat. Cell Biol. 18(4), 368–369 (2016).

25

Li YC, Rodewald LW, Hoppmann C et al. A versatile platform to analyze low-affinity and transient protein–protein interactions in living cells in real time. Cell Rep. 9(5), 1946–1958 (2014).

39

26

Johnston SJ, Carroll JS. Transcription factors and chromatin proteins as therapeutic targets in cancer. BBA Rev. Cancer 1855(2), 183–192 (2015).

••

Describes the clinical data of murine double minute 2 and murine double minute X double targeting inhibitors

Levite M. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDANR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren’s syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors,

10.4155/fmc-2016-0102 Future Med. Chem. (Epub ahead of print)

future science group

Stapled peptides as promising drugs 

decrease glutamate receptor’s expression, impair glutamateinduced signaling and function, activate blood–brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy. J. Neural. Transm. 121(8), 1029–1075 (2014). 40

54

Gavathiotis E, Reyna DE, Davis ML et al. BH3triggered structural reorganization drives the activation of proapoptotic BAX. Mol. Cell 40(3), 481–492 (2010).

55

Moldoveanu T, Grace CR, Llambi F et al. BID-induced structural changes in BAK promote apoptosis. Nat. Struct. Mol. Biol. 20(5), 589 (2013).

56

Labelle JL, Katz SG, Bird GH et al. A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers. J. Clin. Invest. 122(6), 2018–2031 (2012).

57

Okamoto T, Zobel K, Fedorova A et al. Stabilizing the proapoptotic BimBH3 helix (BimSAHB) does not necessarily enhance affinity or biological activity. ACS Chem. Biol. 8(2), 297–302 (2013).

58

Edwards AL, Gavathiotis E, Labelle JL et al. Multimodal Interaction with BCL-2 family proteins underlies the proapoptotic activity of PUMA BH3. Chem. Biol. 20(7), 888–902 (2013).

59

Dai HM, Pang YP, Ramirez-Alvarado M et al. Evaluation of the BH3-only protein puma as a direct bak activator. J. Biol. Chem. 289(1), 89–99 (2014).

60

Stephen AG, Esposito D, Bagni RK et al. Dragging ras back in the ring. Cancer Cell 25(3), 272–281 (2014).

61

Pekny M, Pekna M, Messing A et al. Astrocytes: a central element in neurological diseases. Acta Neuropathol. 131(3), 323–345 (2016).

Spiegel J, Cromm PM, Itzen A et al. Direct targeting of Rab-GTPase-effector interactions. Angew. Chem. Int. Ed. Engl. 53(9), 2498–2503 (2014).

62

Ntziachristos P, Lim JS, Sage J et al. From fly wings to targeted cancer therapies: a centennial for Notch signaling. Cancer Cell 25(3), 318–334 (2014).

Ouyang L, Shi Z, Zhao S et al. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Proliferat. 45(6), 487–498 (2012).

63

Freed EO. HIV-1 assembly, release and maturation. Nat. Rev. Microbiol. 13(8), 484–496 (2015).

64

Lee SK, Cheng N, Hull-Ryde E et al. A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid. Biochemistry 52(29), 4929–4940 (2013).

65

Sakalian M, Mcmurtrey CP, Deeg FJ et al. 3-O-(3´,3´dimethylsuccinyl) betulinic acid inhibits maturation of the human immunodeficiency virus type 1 Gag precursor assembled in vitro. J. Virol. 80(12), 5716–5722 (2006).

66

Bird GH, Madani N, Perry AF et al. Hydrocarbon doublestapling remedies the proteolytic instability of a lengthy peptide therapeutic. Proc. Natl Acad. Sci. USA 107(32), 14093–14098 (2010).

67

Battaglia AM, Hagmeyer KO. Combination therapy with interferon and ribavirin in the treatment of chronic hepatitis C infection. Ann. Pharmacother. 34(4), 487–494 (2000).

68

Monga HK, Rodriguez-Barradas MC, Breaux K et al. Hepatitis C virus infection – related morbidity and mortality among patients with human immunodeficiency virus infection. Clin. Infect. Dis. 33(2), 240–247 (2001).

69

Meredith LW, Wilson GK, Fletcher NF et al. Hepatitis C virus entry: beyond receptors. Rev. Med. Virol. 22(3), 182–193 (2012).

70

Wong-Staal F, Syder AJ, Mckelvy JF. Targeting HCV entry for development of therapeutics. Viruses 2(8), 1718–1733 (2010).

71

Kovac S, Walker MC. Neuropeptides in epilepsy. Neuropeptides 47(6), 467–475 (2013).

Kushwah R, Guezguez B, Lee JB et al. Pleiotropic roles of Notch signaling in normal, malignant, and developmental hematopoiesis in the human. EMBO Rep. 15(11), 1128–1138 (2014).

41

Ebens CL, Maillard I. Notch signaling in hematopoietic cell transplantation and T cell alloimmunity. Blood Rev. 27(6), 269–277 (2013).

42

Serra H, Chivite I, Angulo-Urarte A et al. PTEN mediates Notch-dependent stalk cell arrest in angiogenesis. Nat. Commun. 6(7935), 1–13 (2015).

43

Arnett KL, Hass M, Mcarthur DG et al. Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat. Struct. Mol. Biol. 17(11), 1312–1317 (2010).

44

Luxan G, D’amato G, Macgrogan D et al. Endocardial Notch signaling in cardiac development and disease. Circ. Res. 118(1), 1–18 (2016).

45

46

47

Sossey-Alaoui K, Su GF, Malaj E et al. WAVE3, an actinpolymerization gene, is truncated and inactivated as a result of a constitutional t(1;13)(q21;q12) chromosome translocation in a patient with ganglioneuroblastoma. Oncogene 21(38), 5967–5974 (2002).

48

Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat. Rev. Cancer 13(2), 83–96 (2013).

49

Bernal F, Wade M, Godes M et al. A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell 18(5), 411–422 (2010).

50

Shamas-Din A, Kale J, Leber B et al. Mechanisms of action of Bcl-2 family proteins. CSH Perspect. Biol. 5(4), a008714 (2013).

51

Volkmann N, Marassi FM, Newmeyer DD et al. The rheostat in the membrane: BCL-2 family proteins and apoptosis. Cell Death Differ. 21(2), 206–215 (2014).

52

Renault TT, Elkholi R, Bharti A et al. B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro-and anti-apoptotic BCL-2 family proteins. J. Biol. Chem. 289(38), 26481–26491 (2014).

53

Verdine GL, Walensky LD. The challenge of drugging undruggable targets in cancer: lessons learned from targeting BCL-2 family members. Clin. Cancer Res. 13(24), 7264–7270 (2007).

future science group

Review

www.future-science.com 10.4155/fmc-2016-0102

Review  Xie, Gao, Shull & Teng 72

Schwarzer C. Neuropeptides in epilepsy: molecular basis to treatment options. J. Neurochem. 134, 72–73 (2015).

73

74

75

78

Green BR, Klein BD, Lee HK et al. Cyclic analogs of galanin and neuropeptide Y by hydrocarbon stapling. Bioorgan. Med. Chem. 21(1), 303–310 (2013).

Bellmann-Sickert K, Beck-Sickinger AG. Peptide drugs to target G protein-coupled receptors. Trends Pharmacol. Sci. 31(9), 434–441 (2010).

79

Huang LX, Balsara RD, Castellino FJ. Synthetic conantokin peptides potently inhibit N-methyl-d-aspartate receptormediated currents of retinal ganglion cells. J. Neurosci. Res. 92(12), 1767–1774 (2014).

Delgado M, Ganea D. Vasoactive intestinal peptide: a neuropeptide with pleiotropic immune functions. Amino Acids 45(1), 25–39 (2013).

80

Speltz TE, Fanning SW, Mayne CG et al. Stapled peptides with gamma-methylated hydrocarbon chains for the estrogen receptor/coactivator interaction. Angew. Chem. Int. Ed. Engl. 55(13), 4252–4255 (2016).

Harmar AJ, Fahrenkrug J, Gozes I et al. Pharmacology and functions of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide: IUPHAR review 1. Br. J. Pharmacol. 166(1), 4–17 (2012).

81

Errington WJ, Khan MQ, Bueler SA et al. Adaptor protein self-assembly drives the control of a cullin-RING ubiquitin ligase. Structure 20(7), 1141–1153 (2012).

82

De Paola I, Pirone L, Palmieri M et al. Cullin3-BTB interface: a novel target for stapled peptides. PLoS ONE 10(4), 1–21 (2015).

83

Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discov. Today 20(1), 122–128 (2015).

76

Sviridov DO, Drake SK, Freeman LA et al. Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter. Biochem. Bioph. Res. Co. 471(4), 560–565 (2016).

77

Sinclair JKL, Schepartz A. Influence of macrocyclization on allosteric, juxtamembrane-derived, stapled peptide inhibitors of the epidermal growth factor receptor (EGFR). Org. Lett. 16(18), 4916–4919 (2014).

10.4155/fmc-2016-0102 Future Med. Chem. (Epub ahead of print)

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