Protein epitope mimetic macrocycles asbiopharmaceuticalsAnatol Luther1, Kerstin Moehle2, Eric Chevalier1, Glenn Dale1

and Daniel Obrecht1

Available online at www.sciencedirect.com

ScienceDirect

Fully synthetic medium-sized macrocyclic peptides mimicking

the key b-hairpin and a-helical protein epitopes relevant in many

protein–protein interactions have emerged as a novel class of

drugs with the potential to fill an important gap between small

molecules and proteins. Conformationally stabilized macrocyclic

scaffolds represent ideal templates for medicinal chemists to

incorporate bioactive peptide and protein pharmacophores in

order to generate novel drugs to treat diseases with high unmet

medical need. This review describes recent approaches to

design and generate large libraries of such macrocycles, for hit

identification, and for their efficient optimization. Finally, this

review describes some of the most advanced protein epitope

mimetic (PEM) macrocycles in clinical development.

Addresses1 Polyphor Ltd, Hegenheimermattweg 125, 4123 Allschwil, Switzerland2Department of Chemistry, University of Zurich, Winterthurerstrasse

190, 8057 Zurich, Switzerland

Corresponding author: Obrecht, Daniel (daniel.obrecht@polyphor.com)

Current Opinion in Chemical Biology 2017, 38:45–51

This review comes from a themed issue on Next generationtherapeutics

Edited by David Craik and Sonia Troeira Henriques

http://dx.doi.org/10.1016/j.cbpa.2017.02.004

1367-5931/ã 2017 Elsevier Ltd. All rights reserved.

IntroductionDespite an enormous global effort in academic and

industrial pharmaceutical research and development, cur-

rent drugs address only roughly 30% of the estimated

3000 disease-relevant targets [1�,2], leaving patients and

doctors without appropriate treatment options for many

fatal diseases.

Many of the untapped targets with potential therapeutic

applications are extracellular and intracellular protein–

protein interactions (PPIs), including multiprotein net-

works [3,4], where binding interfaces are large and often

flat. Although some success stories of small molecule

approaches addressing PPIs have been reported in the

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past 15 years [5,6,7�], these examples are not an indication

of a generalized successful approach.

So far PPIs have mostly been addressed by therapeutic

proteins which since their very successful introduc-

tion have constantly increased their market share [8,9].

Particularly, therapeutic antibodies, Fc-fused therapeutic

proteins, and an increasing number of alternative protein

scaffolds [10,11] have emerged as powerful molecules to

address targets with large surface interactions. Despite

the large diversity of scaffolds, the targets for therapeutic

proteins are so far dominated by soluble proteins (e.g.,

cytokines: TNF-a, IL4/13, IL5, IL6, IL17 etc.; enzymes:

PCSK9) and some cell-surface proteins and receptors

(PD-1, PD-L1, CTLA-4; CD-20 etc.). In addition, thera-

peutic proteins show limited tissue penetration, are usually

not cell-permeable, and limited mainly to extracellular

targets. Furthermore, therapeutic proteins have been less

successful for modulating signaling receptors (GPCRs,

ion channels, transporters), probably due to their specific

pharmacokinetic and kinetic binding properties, and are

usually administered parenterally, being generally not

suitable for oral and inhaled applications (Figure 1).

Extensive efforts are currently made to overcome such

limitations by the development of novel formulation

strategies [12]. Therefore, it is clear that novel drug

classes that are able to address intracellular and extra-

cellular PPIs with complementary drug-like properties

to therapeutic proteins, allowing for example alternative

administration routes such as oral and inhaled adminis-

tration, will fill an important gap and complement the

existing drug arsenal (Figure 1).

Recently medium-sized natural and synthetic macrocycles

in the range of 500–4000 Daltons (Da) have emerged as

privileged scaffolds and a potential alternative to small

molecules and large biopharmaceuticals to address the

untapped target space and overcome some of the present

limitations (Figure 1). Macrocyclic natural products for

example have provided important drugs, especially in

the antiinfective and oncology areas [13–15]. An analysis

of approved natural product-derived drugs between 1976–

2006 shows a high proportion of macrocyclic products

underscoring their importance for drug discovery and

development [16,17].

Properties of medium-sized macrocycles andtarget spaceMedium-sized macrocycles combine two important fea-

tures [18�]. First, their larger size as compared to small

Current Opinion in Chemical Biology 2017, 38:45–51

46 Next generation therapeutics

Figure 1

0.5 1.0 2.0 5.0 10 100

Oral

Inhaled

Injectable

200

mAbsADCsFusion

proteins PEM

Macrocycles

Peptides

SmallProteins

LargeProteins

Macrocyclic PeptidesbRo5

MacrocyclesSmall

Molecules

TNF-αCaspofungin

Avastin

Medium-sized Macrocycles

Imatinib

Aspirin

Molecule Weight in kDa

CytokinesChemokinesInterferonsHormones

POL6014

Cyclosporin A

Rapamycin

Route ofAdministration

bRo5Macrocycles

MacroFinder

4.0

StapledPeptides

CyclotidesBicycles

ALRN-6924

Vancomycin

Balixafortide

Murepavadin

Insulin

Liraglutide

Current Opinion in Chemical Biology

Medium-sized macrocycles complement the chemical space between small molecules and therapeutic proteins:

X-axis: drug molecule classes according to increasing molecule weight (MW).

Y-axis: general trends of applicable administration routes for different drug molecule classes:

- Small molecules (MW�0.5 kDa): oral route is preferred (e.g., aspirin, MW: 180.16 Da), F = 68%; imatinib, MW: 493.62 Da, F = 97%).

- Beyond rule of 5 (bRo5) molecules (MW: 0.5–1.2 kDa): some molecules are also orally bioavailable (e.g., rapamycin, MW: 914.19 Da, F = 14%;

cyclosporine A, MW: 1203.64 Da, F = 30%), however, many molecules are administered parenterally (e.g., caspofungin, MW: 1093.33 Da) [25�].- Macrocyclic peptides, peptides (MW: 1–5 kDa): administration is generally parenterally (e.g., vancomycin; Balixafortide; ALRN-6924). PEM

macrocycles are chemically very stable and can be formulated for inhalation (e.g., POL6014). High lung and low systemic exposure allows to

minimize potential systemic side-effects.

- Small and large therapeutic proteins (5–200 kDa): administration is generally parenterally (e.g., TNF-a, avastin).

molecules allows macrocycles to bind to ‘hot spots’

[19��,20�] with binding sites typically in the range of

800–1200 A2 [21�], and second, their conformational

semi-rigidity provides the necessary flexibility for

induced fit [22]. The latter point is critical as many PPIs

involve dynamic surfaces [23,24]. In addition, the confor-

mational adaptability of macrocycles beyond the rule of 5

(bRo5) translates into favorable physicochemical proper-

ties (e.g., molecular weight (MW): �1000 Da; total polar

surface area (TPSA): �250 A2) as highlighted in several

recent reviews [25�,26,27]. As a result, oral bioavailability is

observed for some natural and synthetic macrocycles that

significantly violate the rule of 5 [28]. The highest restric-

tions seem to be imposed by high lipophilicity and the

number of solvent-exposed hydrogen bond donors [26,27].

Some macrocycles exhibit chameleonic conformational

Current Opinion in Chemical Biology 2017, 38:45–51

behavior favoring intramolecular H-bonds in apolar envi-

ronment facilitating cell-permeation, and conformations

exposing H-bond donors important for binding to a target

in polar surrounding [22]. A classical representative for

such a molecule is cyclosporin A (CsA; MW: 1204 Da)

which is orally bioavailable (Figure 1). Several other

examples of orally bioavailable cyclopeptides containing

N-methylated amino acids showing chameleonic confor-

mational behavior have also been described [29].

A number of approaches have used such structural features

derived from the analysis of natural products to design fully

synthetic macrocycle libraries with good drug-like proper-

ties (Table 1),oneexample is theMacroFinder1 libraryasa

typical representative for bRo5 macrocycles with the

potential of oral bioavailability [30].

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Protein epitope mimetic macrocycles Luther et al. 47

Table 1

Macrocycle-based library approaches

Approach Company/Organization

Cyclopeptides:

MATCH cyclopeptides [52] Tranzyme Pharma (now

Ocera Therapeutics)

Protein epitope mimetics (PEM) [34�] Polyphor

Stapled peptides [45��] Aileron Therapeutics

DNA-encoded cyclopeptides [18�] Ensemble Therapeutics

RNA-encoded cyclopeptides [53�] PeptiDream, Ra

Pharmaceuticals

Bicyclic peptides [38] Bicycle Therapeutics,

Pepscan

Nacellins [54] Encycle Therapeutics

Cell-permeable cyclopeptides [29] Circle Pharma

SICLOPPS [55] University of Southampton

Cyclic cell-penetrating peptides [56] The Ohio State University

Phylomer peptides [60] Phylogica

Non peptide-based macrocycles:

DOS macrocycles [57] Broad Institute

MacroFinder [30] Polyphor

Nanocyclix [58] Oncodesign

CMRT Cyclenium Pharma

Among the various types of macrocycles, cyclopeptides

are particularly well-suited to modulate PPIs [31], as

peptides in general display better packed protein–protein

interfaces making significantly more intermolecular

hydrogen bonds, mainly those involving the peptide

backbone [32�]. Systematic analysis of protein interfaces

reveal a large proportion of recurring secondary structure

motifs such as b-strands [33], b-hairpins [34�], a-helices[35], and various turn-like loop structures [36]. Cyclopep-

tides which mimic such privileged structural motifs

should therefore be highly preferred to target PPIs

[37�,38]. In recent years several novel cyclopeptide-based

drug discovery screening and hit identification technolo-

gies have been developed (Table 1).

Protein epitope mimetics (PEM)The b-hairpin and a-helical secondary structure motifs

have been reported to allow the most densely packed

interfaces with proteins [39] and are key locations on

the surface of proteins for modulating protein–protein

interaction. Many peptidomimetic approaches are based

on locking important bioactive peptide pharmacophores,

such as exposed loops and b-turns [36], b-hairpins[34�], and a-helices [35] into macrocyclic scaffolds

[37�] (Figure 2). Furthermore, macrocyclic cysteine-rich

natural peptides such as cyclotides (Figure 1) [40] and

conotoxins and their synthetic analogues combine several

privileged epitopes in one stable scaffold and are success-

fully developed as a potential new class of therapeutics

(PTG-100, Table 2).

One of the first and most versatile peptidomimetic

approaches is the PEM approach [34�]. Here, a b-hairpinloop structure is mimicked in a chemically and

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conformationally stable type-II’ b-turn template (e.g.,DPro-LPro)-induced cyclopeptide scaffold [34�] (Figure 2).

Integrating 4–20 amino acid building blocks into the

PEM scaffold allowed to synthesize hairpin mimetics

of different size, essentially mimicking most of the

naturally occurring hairpin loops. It is interesting to note

that the PEM scaffold can also be successfully used to

mimic a-helical epitopes as successfully exemplified

with the design and synthesis of potent inhibitors of

the p53-HDM2 interaction [41] (Figure 2).

Alternatively, the stapled peptide approach mimics

a-helical conformations by means of a side-chain hydro-

carbon linkage [42] (Figure 2). Although linear peptides

with polar side-chains usually show very limited cell-

permeability, peptide-stapling coupled with chemical

optimization can yield helix mimetics with appreciable

cell-permeability [43,44]. ALRN-5281 and ALRN-6924

are the first stapled peptides that entered clinical devel-

opment (Table 2), the latter targeting the p53-HDM1/2

interaction (Figure 1) [45��].

PEM drug discoveryPEM drug discovery takes advantage of several proprie-

tary elements that were jointly developed by the Univer-

sity of Zurich and Polyphor Ltd. The PEMfinderJ library

was designed and synthesized by integrating bioactive

peptide and protein sequences into various PEM scaf-

folds. To this end, sequences derived from protein

loops, GPCR ligands, blockers and activators of ion

channels and transporters, and from host defense pep-

tides (Figure 2) were successfully integrated into the

PEM universe. The synthetic PEMfinderJ library is

supported by the proprietary PEMphageJ approach,

where libraries of 107–108 hairpin-like peptides are dis-

played on the surface of filamentous phage. Screening of

PEMfinderJ and PEMphageJ combined with structure-

based design has provided many excellent starting

points for drug discovery projects [34�]. It is interesting

to note that all three clinical-stage PEM compounds

Murepavadin (POL7080), Balixafortide (POL6326) and

POL6014 originated from pharmacophores derived from

host-defense peptides [34�].

PEM macrocycles can be efficiently synthesized and

purified by a unique 576-parallel process allowing rapid

iterative optimization of biological activity, selectivity,

and ADMET properties required for progressing com-

pounds through the different drug discovery stages.

PEM drugs in clinical developmentTo date several PEM macrocycles and other macrocyclic

peptide mimetics are in clinical development (summary

Table 2).

Murepavadin (POL7080) represents the first member

of a novel class of outer membrane protein targeting

Current Opinion in Chemical Biology 2017, 38:45–51

48 Next generation therapeutics

Figure 2

(c)

(b)

(a)

Current Opinion in Chemical Biology

The protein epitope mimetic (PEM) approach.

Colours: a-helix backbone (red); b-hairpin backbone (cyan); pharmacophore side-chains (orange).

(a) Naturally occuring cyclic peptide inhibitor from sunflower seeds in complex with trypsin (PDB: 1SFI) and a representative solution NMR

structure of a derived peptidomimetic inhibitor [34�].(b) Surface exposed protein loops (e.g., antibody CDR loops, PDB: 1GIG) transplanted to a template to stabilize the bioactive conformation in a

macrocyclic peptidomimetic (NMR structure) [34�].(c) Crystal structure of human MDM2 bound to the p53 tumor processor transactivation domain (PDB: 1YCR) and two potent peptidomimetic

scaffolds mimicking the a-helical pharmacophore. The crystal structure of a macrocyclic b-hairpin peptide in complex with HDM2 (PDB: 2AXI) is

shown at the top right [41], and the crystal structure of a stapled peptide in complex with HDM2 (PDB: 3V3B) is shown at the bottom right [45��].Note that p53 is turned 180� around the vertical axis compared to the top view in order to visualize the helix staple.

antibiotics (OMPTA). POL7080 was inspired by host

defense antimicrobial peptides, such as protegrin I, show-

ing broad-spectrum antimicrobial activity and maintain-

ing good activity against multi-drug resistant Gram-neg-

ative pathogens, which constitute a considerable and

growing threat to human health [46]. However, antimi-

crobial peptides generally exhibit unfavorable drug prop-

erties. By transferring antimicrobial peptide pharmaco-

phores onto a 14-residue PEM scaffold, PEM libraries

were designed and synthesized [47]. Among those librar-

ies PEM macrocycles with remarkable Pseudomonas-spe-cific antimicrobial activity were discovered. Further opti-

mization led to the discovery of the clinical candidate

POL7080 [48��]. Mechanism of action studies showed

Current Opinion in Chemical Biology 2017, 38:45–51

that POL7080 interacts with the essential outer-mem-

brane b-barrel transporter LptD and blocks LPS assem-

bly in the outer membrane [48��]. In vitro, POL7080

displays potent antimicrobial activity against Pseudomonasaeruginosa (PA) with an MIC90 at 0.12 mg/L when tested

against >1200 PA clinical isolates from various geographic

locations, including MDR strains. In vivo, POL7080 dis-

plays linear pharmacokinetics, is dose proportional with a

good penetration into the epithelial lung fluid which

underscores its potent in vivo activity in lung infection

models including XDR isolates. The favorable in vitroand in vivo properties of POL7080 combined with an

appropriate safety pharmacology and toxicology profile

led to the clinical development of POL7080 (Table 2) for

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Protein epitope mimetic macrocycles Luther et al. 49

Table 2

Selected macrocyclic peptides in clinical developmen

Company Compound Clinical phase Indication/disease area

Aileron Therapeutics ALRN-6924 Phase 2 Cancer

ALRN-5281 Phase 1 Metabolic diseases

Apeptico Solnatide Phase 2 Pulmonary edema, acutlung injury,

(AP-301) lung transplant rejection

Bristol-Myers Squibb PD-L1 inhibitor Phase 1 Immuno-oncology

Eli Lilly LY-2510924 Phase 2 Small-cell lung cancer, renal cell carcinoma

Polyphor Murepavadin Phase 2 Pseudomonas infections

(POL7080)

Balixafortide Phase 1 Advanced metastatic breast cancer, other oncology indications

(POL6326)

POL6014 Phase 1 Cystic fibrosis, non-cystic fibrosis bronchiectasis,

alpha-1 antitrypsin deficiency

Protagonist Therapeutics PTG-100 Phase 1 Ulcerative colitis

Information from Thomson Reuters CortellisTM (https://cortellis.thomsonreuterslifesciences.com) and company homepages.

the treatment of serious infections caused by PA. Cur-

rently POL7080 is planned to enter Phase III in the first

half of 2017.

Excessive extracellular human neutrophil elastase

(HNE) plays an important role in airway mucus hyperse-

cretion, sustained lung tissue inflammation, and destruc-

tion leading to emphysema or chronic infection [49].

Pharmacological inhibition of lung HNE by inhalation

holds great promise to provide novel and highly effective

treatment options for patients with airway neutrophilic

diseases like cystic fibrosis (CF), non-cystic fibrosis bron-

chiectasis (NCFB), alpha-antitrypsin deficiency (AATD)

or chronic obstructive pulmonary disease (COPD).

Although the target elastase and its role in lung function

deterioration has been known for many years, no suitable

oral small molecule compound has been identified or

brought to the market yet, possibly because of limited

lung exposure or safety.

POL6014 is a novel potent and fully reversible HNE

inhibitor and originated from PEMfinderJ library hits

inspired by the natural sunflower trypsin inhibitor (SFTI)

[34�] (Figure 2). The PEM approach was applied to

optimize potency, selectivity, ADME and physicochemi-

cal properties in rapid iterative cycles to finally identify

POL6014 as a suitable clinical candidate [34�]. POL6014

potently inhibits HNE in vitro (IC50 = 1.4 nM) [34�] and

also ex-vivo in the bronchoalveolar lavage from CF,

AATD, and NCFB patient sputa with comparable activ-

ity to the endogenous alpha-1 antitrypsin. POL6014 also

shows significant efficacy in different rodent models

induced with exogenous NE (e.g., acute lung injury

model) or endogenous NE (lipopolysaccharide and

tobacco smoke) induced models by local administration

with ED50 �0.3 mg/kg [34�]. POL6014 shows favorable

solubility and stability compatible with deep lung

(alveoli) respirability after aerosolization. A favorable

pharmacokinetic profile was observed for POL6014 after

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direct lung application in different species and is charac-

terized by sustained lung retention and a >100-fold ratio

of airway-to-plasma concentrations, thus maximizing its

local effect and minimizing potential side effects associ-

ated with a high systemic exposure [59]. The favorable

physicochemical properties combined with an excellent

safety pharmacology and toxicology profile led to the

clinical development of POL6014 using the Pari eFlow1

aerosol inhaler. POL6014 is currently in Phase Ib clinical

studies in CF patients and underscores the potential of

PEM macrocycles for the treatment of serious lung

diseases.

The chemokine receptor CXCR4 and its ligand CXCL12

(SDF-1a) are key regulators of hematopoetic stem cell

and cancer cell growth and migration. Inhibition of

CXCR4 is being clinically used to harvest hematopoetic

stem/progenitor cells from the bone marrow. The

CXCR4/CXCL12 axis has also an important role in tumor

growth and metastasis, is critically involved in cancer cell-

tumour microenvironment interaction, and angiogenesis.

Inhibitors of CXCR4 have shown promise as novel cancer

therapeutics in combination with standard chemothera-

peutics against various cancer types [50] such as breast

cancer [51]. Balixafortide (POL6326) is a potent and

selective PEM CXCR4 antagonist that was inspired by

the hairpin-shaped host defense peptide Polyphemusin II

isolated from the American horseshoe crab [34�].POL6326 is well tolerated and in Phase Ib for combina-

tion therapy in advanced metastatic breast cancer

(Table 2).

Summary and outlookIn recent years fully synthetic medium-sized macrocycles

have emerged as a potential novel class of biopharma-

ceuticals able to address complex biological targets

including PPIs. By virtue of addressing a complementary

target space and exhibiting complementary physico-

chemical properties compared to either small molecules

Current Opinion in Chemical Biology 2017, 38:45–51

50 Next generation therapeutics

and therapeutic proteins, macrocycles may well fill an

important gap in the current drug arsenal to treat diseases

with high unmet medical need. We highlight here

PEM macrocycles mimicking key peptide pharmaco-

phores as the currently most advanced class in clinical

development. The approval of one of these front-runner

molecules will be a crucial milestone and proof of

concept for this interesting class of compounds as novel

biopharmaceuticals.

Conflict of interestAnatol Luther, Eric Chevalier, Glenn Dale and Daniel

Obrecht are employees of Polyphor Ltd, Switzerland.

Kerstin Moehle is an employee of the University of

Zurich and a part-time employee of Polyphor Ltd.

AcknowledgementsWe thank Dr. Christian Bisang for his contributions, Prof. John A. Robinson(University of Zurich) and Prof. Brian Cox (University of Sussex) for proof-reading and their valuable support. We would like to thank Dr. MichaelAltorfer for his many inspiring contributions to the development ofPolyphor Ltd, PEM technology and products.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

1.�

Santos R, Ursu O, Bento AP, Donadi RS, Bologa CG, Karlsson A,Al-Lazikani B, Hersey A, Oprea TI, Overington JP: Acomprehensive map of molecular drug targets. Nat Rev DrugDiscov 2017, 16:19-34 http://dx.doi.org/10.1038/nrd.2016.230Advance online publication.

A recent comprehensive analysis and overview on disease related drugtargets.

2. Overington JP, Al-Lazikani B, Hopkins AL: How many drugtargets are there? Nat Rev Drug Discov 2006, 5:993-996.

3. Ruffner A, Bauer A, Bouwmeester T: Human protein-proteininteraction networks and the value for drug discovery. DrugDiscov Today 2007, 12:709-716.

4. Thompson AD, Dugan A, Gestwicki JE, Mapp AK: Fine-tuningmultiprotein complexes using small molecules. ACS Chem Biol2012, 7:1311-1320.

5. Sperandio O, Reynes CH, Camproux AC, Villoutreix BO:Rationalizing the chemical space of protein-proteininteraction inhibitors. Drug Discov Today 2009, 15:220-229.

6. Smith MC, Gestwicki JE: Features of protein-proteininteractions that translate into potent inhibitors: topology,surface areas and affinity. Exp Rev Mol Med 2012, 14:e16.

7.�

Arkin MR, Tang Y, Wells JA: Small-molecule inhibitors ofprotein-protein interactions: progressing toward the reality.Chem Biol 2014, 21:1102-1114.

A comprehensive overview describing successful approaches and casestudies of small molecule inhibitors of PPIs.

8. Dimitrov DS: Therapeutic Proteins: methods and protocols. InMethods in Molecular Biology. Edited by Voynov V, Caravella JA.Human Press; 2012:1-26.

9. Reichert JM: Antibodies to watch in 2014. mAbs 2014, 6:5-14.

10. Binz HK, Amstutz P, Pluckthun A: Engineering novel bindingproteins from nonimmunoglobulin domains. Nat Biotechnol2005, 23:1257-1268.

11. Boersma YL, Pluckthun A: DARPins and other repeat proteins.Curr Opin Biotechnol 2011, 22:849-857.

Current Opinion in Chemical Biology 2017, 38:45–51

12. Mitragorti S, Burke PA, Langer R: Overcoming the challenges inadministering biopharmaceuticals: formulation and deliverystrategies. Nat Rev Drug Discov 2014, 13:655-672.

13. von Nussbaum F, Brands M, Hinzen B, Weigand S, Habich D:Antibacterial natural products in medicinal chemistry-exodusor revival? Angew Chem Int Ed 2006, 45:5072-5129.

14. Obrecht D, Robinson JA, Bernardini F, Bisang C, DeMarco SJ,Moehle K, Gombert F: Recent progress in the discovery ofmacrocyclic compounds as potential anti-infectivetherapeutics. Curr Med Chem 2009, 16:42-65.

15. Newman DJ, Cragg GM: Natural products as sources of newdrugs from 1981-2014. J Nat Prod 2016, 79:629-661.

16. Wessjohann LA, Ruijter E, Garcia-Rivera D, Brandt W: What can achemist learn from nature’s macrocycles?-A brief, conceptualview. Mol Divers 2005, 9:171-189.

17. Ganesan A: The impact of natural products upon modern drugdiscovery. Curr Opin Chem Biol 2008, 12:306-317.

18.�

Driggers EM, Hale SP, Lee J, Terrett NK: The exploration ofmacrocycles for drug discovery-an underexploited class.Nat Rev Drug Discov 2008, 7:608-624.

Important overview on the relevance of macrocycles in drug discovery.

19.��

Clackson T, Wells JA: A hot spot of binding energy in ahormone-receptor interface. Science 1995, 267:383-386.

Landmark paper on the relevance of hot spots in protein interfaces.

20.�

Bogan AA, Thorn KS: Anatomy of hot spots in proteininterfaces. J Mol Biol 1998, 280:1-9.

Comprehensive analysis of the dominant forces and anatomy of hot spotsin protein interfaces.

21.�

Villar EA, Beglov B, Chennamadhavuni S, Porco JA Jr, Kozakov D,Vajda S, Whitty A: How proteins bind macrocycles. Nat ChemBiol 2014, 10:723-731.

Comprehensive analysis of X-ray structures of macrocycles bound totheir targets.

22. Whitty A, Zhong M, Viarengo L, Beglov D, Hall DR, Vajda S:Quantifying the chameleonic properties of macrocycles andother high-molecular-weight drugs. Drug Discov Today 2016,21:712-717.

23. Meszaros B, Tompa P, Simon I, Dosztanyi Z: Molecular principlesof the interactions of disordered proteins. J Mol Biol 2007,372:549-561.

24. Meszaros B, Simon I, Dosztanyi Z: Prediction of protein bindingregions in disordered proteins. PLoS Comput Biol 2009,5:1000376.

25.�

Giordanetto F, Kihlberg J: Macrocyclic drugs and clinicalcandidates: what can medicinal chemists learn from theirproperties? J Med Chem 2014, 57:278-295.

Comprehensive analysis of drug-like properties of macrocyclic drugs anddrug candidates.

26. Doak BC, Over B, Giordanetto F, Kihlberg J: Oral druggablespace beyond the rule of 5: insights from drugs and clinicalcandidates. Chem Biol 2014, 21:1115-1141.

27. Over B, Matsson P, Tyrchan C, Artursson P, Doak BC, Foley MA,Hilgendorf C, Johnston SE, Lee MD IV, Lewis RJ et al.: Structuraland conformational determinants of macrocycle cellpermeability. Nat Chem Biol 2016, 12:1067-1074.

28. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ: Experimentaland computational approaches to estimate solubility andpermeability in drug discovery and development settings.Adv Drug Deliv Rev 1997, 23:3-25.

29. Bockus AT, McEwen CM, Lockey RS: Form and function incyclic peptide natural products: a pharmacokineticperspective. Curr Top Med Chem 2013, 13:821-836.

30. Ermert P, Moehle K, Obrecht D: Macrocyclic inhibitors ofGPCR’s, integrins and protein-protein interactions. InMacrocycles in Drug Discovery. RCS Drug Discovery Series No.40. Edited by Levin J. The Royal Society of Chemistry; 2015:283-338. [Chapter 8].

www.sciencedirect.com

Protein epitope mimetic macrocycles Luther et al. 51

31. Wahyudi H, McAlpine SR: Predicting the unpredictable: recentstructure-activity studies on peptide-based macrocycles.Bioorg Chem 2015, 60:74-97.

32.�

London N, Movshovits-Attias D, Schueler-Fuhrman O: Thestructural basis of peptide-protein binding strategies.Structure 2010, 18:188-199.

An analysis rationalizing how peptides bind to proteins.

33. Watkins AM, Arora PS: The anatomy of b-strands at proteininterfaces. ACS Chem Biol 2014, 9:1747-1754.

34.�

Obrecht D, Chevalier E, Moehle K, Robinson JA: b-Hairpinprotein mimetic technology in drug discovery. Drug DiscovToday 2012, 9:e63-e69.

A review on the concept and applications of PEM-drug discovery.

35. Watkins AM, Wuo MG, Arora PS: Protein-protein interactionsmediated by helical tertiary structure motifs. J Am Chem Soc2015, 137:11622-11630.

36. Gavenonis J, Sheneman BA, Siegert TR, Eshelman MR, Kritzer JA:Comprehensive analysis of loops at protein-protein interfacesfor macrocycle design. Nat Chem Biol 2014, 10:716-722.

37.�

Tsomaia N: Peptide therapeutics: targeting the undruggablespace. Eur J Med Chem 2015, 94:459-470.

A comprehensive review on the role of linear and macrocyclic peptidetherapeutics to address the undruggable target space.

38. Heinis C: Tools and rules for macrocycles. Nat Chem Biol 2014,10:696-698.

39. London N, Raveh B, Schueler-Fuhrman O: Druggable protein-protein interactions-from hot spots to hot segments.Curr Opin Chem Biol 2013, 23:894-902.

40. Poth AG, Chan LY, Craik DJ: Cyclotides as grafting frameworksfor protein engineering and drug design applications.Biopolymers (Peptide Science) 2013, 100:480-491.

41. Fasan R, Dias RLA, Moehle K, Zerbe O, Vrijbloed JW, Obrecht D,Robinson JA: Using a b-hairpin to mimic an a-helix: cyclicpeptidomimetic inhibitors of the p53-HDM2 interaction.Angew Chem Int Ed 2004, 43:2109-2112.

42. Schafmeister CE, Po J, Verdine GL: An all-hydrocarboncross-linking system for enhancing the helicity and metabolicstability of peptides. J Am Chem Soc 2000, 122:5891-5892.

43. Verdine GL, Hilinski GJ: All hydrocarbon-stapled peptides assynthetic cell-accessible mini-proteins. Drug Discov Today2012, 9:e41-e46.

44. Moellering RE, Cornejo M, Davies TN, Del Bianco C, Aster JC,Blacklow SC, Kung AL, Gilliland DG, Verdine GL, Bradner JE:Direct inhibition of the Notch transcription factor complex.Nature 2009, 462:182-188.

45.��

Chang YS, Graves B, Guerlevais V, Tovar C, Packman K, To KH,Olson KA, Gangurde P, Mukherjee A, Baker T et al.: Stapleda-helical drug development: a potent dual inhibitor of MDM2and MDMX for p53-dependent cancer therapy. Proc Natl AcadSci USA 2013, 110:E3445.

Describes the discovery and development of the stapled-peptide clinicaldrug candidate ATSP-7041.

46. Obrecht D, Bernardini F, Dale G, Dembowsky K: Emergingnew therapeutics against key Gram-negative pathogens.

www.sciencedirect.com

In Ann Reports Med Chem, 46. Edited by Macor J. Burlington:Academic Press; 2011:245-262.

47. Robinson JA, Shankaramma SC, Jetter P, Kienzl U,Schwendener RA, Vrijbloed JW, Obrecht D: Properties andstructure-activity studies of cyclic b-hairpin mimetics basedon the cationic antimicrobial peptide protegrin I. Bioorg MedChem 2005, 13:2055-2064.

48.��

Srinivas N, Jetter P, Ueberbacher BJ, Werneburg M, Zerbe K,Steinmann J, Van der Meijden B, Bernardini F, Lederer A, Dias RLAet al.: Peptidomimetic antibiotics target outer-membranebiogenesis in Pseudomonas aeroginosa. Science 2010,327:1010-1013.

Describes discovery and development of Murepavadin as the first anti-biotic targeting an outer-membrane protein.

49. Davies PB, Drumm M, Konstan MW: Cystic fibrosis. Am J RespirCrit Care Med 1996, 154:1229-1256.

50. Domanska UM, Kruizinga RC, Nagengast WB, Timmer-Bosscha H, Huls G, de Vries EGE, Walenkamp AME: A review onCXCR4/CXCL12axis in oncology: no place to hide. Eur J Cancer2013, 49:219-230.

51. Zhang Z, Chao N, Wuzhen C, Wu P, Wang Z, Yin J, Huang J, Qiu F:Expression of CXCR4 and breast cancer prognosis: asystematic review and meta-analysis. BMC Cancer 2014, 14:49.

52. Marsault E, Petersen ML: Macrocycles are great cycles:applications, opportunities, and challenges of syntheticmacrocycles in drug discovery. J Med Chem 2011, 54:1961-2004.

53.�

Bashiruddin NK, Suga H: Construction and screening of vastlibraries of natural-product-like macrocyclic peptides using invitro display technologies. Curr Opin Chem Biol 2015, 24:131-138.

Comprehensive overview on in vitro display technologies to discovermacrocyclic peptide hits.

54. Yudin AK: Macrocycles: lessons from the distant past, recentdevelopments, and future directions. Chem Sci 2015, 6:30-49.

55. Foster AD, Ingram JD, Leitch EK, Lennard KR, Osher EL,Tavassoli A: Methods for the creation of cyclic peptide librariesfor use in lead discovery. J Biomol Screen 2015, 20:563-576.

56. Trinh TB, Upadhyaya P, Qian Z, Pei D: Discovery of a direct Rasinhibitor by screening a combinatorial library of cell-permeable bicyclic peptides. ACS Comb Sci 2016, 8:75-85.

57. Dandapani S, Marcaurelle LA: Current strategies for diversity-oriented synthesis. Curr Opin Chem Biol 2010, 14:362-370.

58. Tigno-Aranjuez TJ, Benderitter P, Rombouts F, Deroose F, Bai XD,Mattioli B, Cominelli F, Pizarro TT, Hoflack J, Abbott DW: In vivoinhibition of RIPK2 kinase alleviates inflammatory disease.J Biol Chem 2014 http://dx.doi.org/10.1074/jbc.M114.591388.

59. von Nussbaum F, Li VMJ: Neutrophil elastase inhibitorsfor thetreatment of (cardio)pulmonary diseases: into clinical testingwith pre-adaptive pharmacophores. Bioorg Med Chem Lett2015, 25:4370-4381.

60. Milech N, Longville BAC, Cunningham PT, Scobie MN,Bogdawa HM, Winslow S, Anastasas M, Connor T, Ong F,Stone SR et al.: GFP-complementation assay to detect CPPand protein delivery into living cells. Sci Rep 2015, 5:18329.

Current Opinion in Chemical Biology 2017, 38:45–51