The Current State of Peptide Drug Discovery: Back ... - ACS Publications

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The Current State of Peptide Drug Discovery: Back to the Future? Antoine Henninot,† James C. Collins,† and John M. Nuss* Ferring Research Institute, 4245 Sorrento Valley Boulevard, San Diego, California 92121, United States ABSTRACT: Over the past decade, peptide drug discovery has experienced a revival of interest and scientific momentum, as the pharmaceutical industry has come to appreciate the role that peptide therapeutics can play in addressing unmet medical needs and how this class of compounds can be an excellent complement or even preferable alternative to small molecule and biological therapeutics. In this Perspective, we give a concise description of the recent progress in peptide drug discovery in a holistic manner, highlighting enabling technological advances affecting nearly every aspect of this field: from lead discovery, to synthesis and optimization, to peptide drug delivery. An emphasis is placed on describing research efforts to overcome the inherent weaknesses of peptide drugs, in particular their poor pharmacokinetic properties, and how these efforts have been critical to the discovery, design, and subsequent development of novel therapeutics.

1. INTRODUCTION Peptides have profoundly impacted the development of the modern pharmaceutical industry and have contributed significantly to the advancement of biological and chemical science.1 Fundamental studies in the first half of the 20th century aimed at understanding the structures and physiological role of peptide hormones such as insulin, oxytocin, gonadotropin-releasing hormone, and vasopressin have catalyzed many major advances in pharmacology, biology, and chemistry as well as other enabling technologies essential for what we now know as modern drug discovery. The investigators involved in these efforts reads like a list of the giants of 20th century science: de Vigneaud, Banting and Macleod, Schally and Guillemin, Sanger, and Merrifield, Nobel laureates all. There is no better illustration of our opening statement than the discovery and development of the 51 amino acid (aa) hormone insulin (Figure 1), which stands as one of the monumental scientific achievements of the era.2 Following its initial isolation in 1921, the pace of development was remarkable, reaching patients just a year later and then becoming the first commercially available peptide drug in 1923. Considered a “miracle drug”, insulin was key to the growing public appreciation of the power and societal value of scientific research. This pioneering success preceded by decades the understanding of insulin’s sequence, structure and molecular pharmacology, and indeed the structure of peptides and proteins in general. In another pivotal moment for the field, human insulin was introduced in 1982 as the first recombinant drug, ultimately leading to the recent discontinuation of the original animal-derived product after a mere 86 years on the market.3 Insulin’s more recent history features a litany of impressive scientific advances that have allowed diabetic patients to achieve tight glycemic control,4 yet further © XXXX American Chemical Society

discussion largely falls outside the scope of this review of the discovery, design, and development of synthetic peptide drugs. However, elegant new approaches as outlined in section 3.4 have brought insulin analogues within reach of chemical synthesis and its powerful toolkit for molecular design. Despite this pedigree, peptides as a class of drugs were considered by the end of the twentieth century as, frankly, a niche area. Even now, it has been our experience when discussing drug discovery with experienced “drug hunters”, most would never think to use a peptide to begin a drug discovery program and would always opt for small molecules or recombinant biological therapeutics (such as antibodies and fusion proteins) as their preferred therapeutic modality for addressing novel biological targets. The reasons for this loss in prominence are quite complex, yet may largely stem from the perception that the disadvantages of peptide drugs greatly outweigh the strengths (see section 2) and that most of the “low hanging fruit” in this area, i.e., work on known and wellunderstood hormones, had been harvested. However, the last two decades have seen a significant renaissance in peptide drug discovery; since 2000 there have been 28 new, noninsulin peptide drugs approved worldwide (Table 1), with several achieving significant market success.5 The synchronous and spectacular success of recombinant biologics has also driven reexamination of the peptide field for new opportunities due to their shared biological characteristics (vide infra) and scientific advances relevant to both areas. These achievements, viewed in the harsh light of the multidecadal exponential death spiral of research productivity termed “Eroom’s Law”,6 have encouraged pharmaceutical Received: February 27, 2017 Published: July 24, 2017 A

DOI: 10.1021/acs.jmedchem.7b00318 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Two- and three-dimensional structures of insulin.

Table 1. Non-insulin Peptides Approved in the Years 2000−2016, Including Region of Launch generic name

year of approval

therapeutic area

country of approval

generic name

year of approval

therapeutic area

country of approval

atosiban taltirelin aviptadil carbetocin nesiritide teriparatide enfuvirtide abarelix ziconotide pramlintide exenatide icatibant romiplostim degarelix

2000 2000 2000 2001 2001 2002 2003 2003 2004 2005 2005 2008 2008 2008

obstetrics CNS urology obstetrics cardiovascular osteoporosis antiinfective oncology pain metabolic disease metabolic disease hematology hematology oncology

EU JP EU EU US US US US US US US EU US US

mifamurtide liraglutide tesamorelin lucinactant peginesatide pasireotide carfilzomib linaclotide teduglutide lixisenatide albiglutide oritavancin dulaglutide afamelanotide

2009 2009 2010 2012 2012 2012 2012 2012 2012 2013 2014 2014 2014 2014

oncology metabolic disease antiinfective pulmonary hematology endocinology oncology gastroenterology gastroenterology metabolic disease metabolic disease antiinfective metabolic disease dermatology

EU EU US US US EU US US EU EU EU US US EU

companies to go “back to the future” and significantly increase their investments in peptide drug discovery. Analysis of the peptide drug market and development climate as of 2016 shows that there are >50 peptide drugs marketed worldwide (Table 1; this table does not include the various insulin derivatives on the market), with another ca. 170 in various stages of clinical development (Table 2) and >200 others at preclinical stages.5 When these figures are broken out by therapeutic area (TA), as in Figure 2, it is apparent that two TA’s predominate, namely metabolic disease and oncology.

Inclusion of insulin analogues further increases the share of the metabolic disease TA. World-wide sales of pharmaceuticals exceeded $1 trillion for the first time in 20147 and are expected to continue to rise at ca. 4% per year.6,7 Estimates place the annual global sales of noninsulin peptide drugs in 2013 at ca. $19 billion; when combined with global sales of insulin derivatives of ca. $28 billion and assuming steady growth in recent years, the total sales of peptide drugs in 2015 can be estimated to be ca. $50 billion or about 5% of 2015 total global pharmaceutical sales.8 While peptides make up just a fraction of global pharma sales, it is likely that as the incidence of metabolic disease and cancer in the western world continues to increase, and as advances in technology that address the inherent liabilities of peptides continue to positively impact their design and development, the demand for peptide drugs will continue to grow. With a projected annual growth rate of 9−10%, peptide drug sales are likely to exceed $70 billion in 2019 (insulins included); this growth rate is slightly higher than the rate predicted for worldwide pharmaceuticals.8

Table 2. Peptides Approved and in Active Development by Clinical Phase as of 2015 (2016 Figures) phase 1 phase II phase IIIa approved total a

55 94 29 56 234

Including preregistration. B



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Figure 2. Peptides approved and in active development by therapeutic area.

Figure 3. Top-selling non-insulin peptides in 2015.

Further examination of these data reveals an interesting aspect of the peptide market. Of the >50 commercial, noninsulin peptide drugs, a small fraction are responsible for the vast majority of the worldwide sales, as seen in Figure 3. For example, Copaxone (glutramer), a 6.4 kDa synthetic copolymer of repeating alanine, lysine, glutamic acid, and tyrosine units used to treat multiple sclerosis, had estimated sales of $4 billion in 2015. The six glucagon-like peptide 1 (GLP-1) analogues currently on the market had combined sales of just over $4 billion in 2014, with Victoza (liraglutide) the leader at $2.4 billion. In the area of oncology, the gonadotrophin releasing hormone (GnRH) agonists Lupron (leuprolide) and Zoladex (gosarelin) together had sales of nearly $2 bn, with the somatostatin analogues octreotide and lanreotide at a comparable level. Overall, the top 6−7 selling peptide drugs are responsible for a significant fraction of worldwide sales; conversely, many of the approved agents have marginal sales (< $100 million), demonstrating once again that approval of a drug does not guarantee meaningful sales, a harsh lesson that

many companies, both large pharma and small biotech, have learned. Having given the reader this historical perspective of the peptide drug field, as well as an outlook for current and future commercial prospects for the peptide market, with this review we will attempt describe the current state of the art in peptide drug discovery. In our discussion, we will focus on important recent technological advances and new approaches to the discovery and development of amino acid based therapeutics that have led to the field’s resurgence as well as the implications of these developments on the future of peptide drug discovery.

2. ADVANTAGES AND DISADVANTAGES OF PEPTIDE DRUGS Peptides, historically defined as polypeptides having 2−50 aa, play critical roles in human physiology, acting as hormones, neurotransmitters, growth factors, and antibacterial agents, inter alia, and their inherent properties define their strengths and weaknesses as drug molecules.9 C



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mediated oxidation) is generally not significant, and therefore drug−drug interactions and nonmechanistic-based toxicology are rarely observed. These are certainly major issues with small molecule drugs. Furthermore, predicting human doses of peptides through allometric scaling is generally more straightforward than for small molecules; this can greatly facilitate early dose-range finding studies in the clinic.10 Biologics, and in particular antibodies, traditionally offer considerable pharmacokinetic advantages over peptides,11,12 yet peptides also have advantages here such as ease of production and lower immunogenicity. It has also been claimed by some that peptide drug discovery suffers from a lower attrition rate than small molecule discovery efforts;13 while this may be true historically, current attrition rates may turn out to be no different than that seen with small molecules or biologics as the targets and indications addressed by modern peptide drug discovery programs are riskier than earlier efforts, which were mainly looking at older, more validated targets. Finally, peptide discovery optimization has a significant resource advantage over small molecules; the relatively simplicity and increasing automation of the required synthesis allows successful prosecution by much smaller teams of medicinal chemists, many-fold smaller than that necessary for a comparable small molecule effort. One strategic aspect of peptide drug discovery, in the experience of the authors and alluded to above, is that development of efficacious peptide agonists is often more straightforward than that of peptide antagonists. This can be explained by consideration of basic pharmacokinetic/pharmacodynamic (PK/PD) principles.14 Agonists are, in general, more potent at binding to and activating their cognate receptor than are the corresponding antagonists and require lower plasma concentrations to achieve a pharmacological response. Competitive antagonists, while competing with the natural agonist for binding sites on the receptor, require high or nearly complete receptor occupancy, higher trough levels, and longer exposure to demonstrate the desired effect. Peptide agonists therefore generally require lower doses (and hence lower production volumes), lower injection volumes, and can have a more favorable dosing schedule than a peptide antagonist. Small molecules and antibodies may therefore be the preferred modalities when attempting to design and develop antagonists.11 Small molecules can be dosed orally, multiple times per day, to achieve the high exposures needed for effective pharmacological antagonism. Therapeutic antibodies, though requiring parenteral injection, have pharmacokinetic advantages over peptides that allow them to be administered biweekly or monthly to achieve the requisite therapeutic exposures.12 section 7 and section 9 describe current approaches that are proving successful in closing this traditional gap in achievable drug exposures between peptides and antibodies. A final consideration is the cost of peptide production and the necessary HPLC purification, which, although decreasing as advances continue to be made, is still high in comparison to small molecules. Current chemical production is limited to peptides of ca. 50 aa; beyond this, the efficiency and costs of synthesis become problematic and recombinant methods become competitive.15 Even now, while the majority of peptide drugs are produced synthetically using solid-phase peptide synthesis (SPPS) methods, some peptide drugs, including all of the various insulin derivatives on the market, are made using recombinant techniques. Recombinant production is especially relevant for “protein scaffolds”, peptides of between 50 and 100

1. Peptides Are the Natural Biological Messengers for Most Endocrine Signaling Pathways. Peptides are the natural ligands for many cell surface receptors such as Gprotein coupled receptors (GPCRs), ion channels, and growth factor receptors, initiating signal transduction processes inside the cell. It is therefore often the case that peptides are agonists of signal transduction pathways, and most successful peptide drugs are agonists (e.g., insulin, GLP-1, GnRH, etc.). As described in section 3, natural peptides therefore often represent viable and validated leads for drug discovery, ultimately producing extremely potent and selective drugs. 2. Peptides Are Membrane Impermeable. This is a critical property that has historically defined and circumscribed the field. The therapeutic application of peptides is restricted to extracellular and transmembrane targets, and the inability to permeate the intestinal mucosa necessitates parenteral administration via subcutaneous or intravenous injection, with the corresponding detriment to patient convenience and compliance. Peptides are also generally unable to cross the blood−brain barrier, which from a toxicology perspective may be a net positive but effectively precludes CNS targets. Addressing these limitations is currently a vibrant and rapidly progressing field; general and reproducible oral delivery is on the horizon (see section 9), and intracellular delivery has seen promising advances (see section 7). 3. Peptides are Biologically Unstable. As the principal role of peptide hormones is to bind to and activate their cognate receptors, it is no surprise that they have evolved to very effectively serve this purpose via elaborate and highly regulated systems of checks and balances including preprohormone processing and feedback loops. Most peptides therefore necessarily have short plasma half-lives; peptides are produced in response to a biological signal, processed, and released, perform their function, and are then rapidly metabolized, i.e., the signal is turned off. Peptides are cleared primarily by proteolytic degradation and by renal filtration, generally leading to suboptimal pharmacokinetic properties which can necessitate considerable research efforts to discover analogues with sufficient plasma exposure. Section 6 outlines a protocol describing methods by which medicinal chemistry optimization is able to reduce or prevent proteolytic cleavage, and section 7 describes conjugation approaches designed to reduce or eliminate renal filtration. For further assessment, it is instructive to compare these stereotypical properties of peptides with their molecular competition in the drug discovery landscape. Peptides, being neither small molecule nor protein, exist at the nexus of these two classes of therapeutics but are generally classified with protein drugs because of their size, composition, distribution, and routes of administration. Small molecules have continued to dominate the worldwide drug market, with advantages including lower cost and price, oral dosing, and ease of synthesis, while their (often) inherent membrane-penetrating ability allows a much broader range of biological targets to be addressed. The proteolytic instability and rapid clearance of peptides, while an issue when considering pharmacokinetic optimization, does mean that they do not accumulate in tissues; hepatic metabolism of peptides (e.g., cytochrome P450 D



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them the largest family of cell surface receptors. Their prominence is reflected in the vast number of human diseases linked to abnormal GPCR signaling, including metabolic disorders, cardiovascular diseases, mental disorders, neurodegenerative diseases, and cancer. GPCRs represent ca. 40% of the currently marketed drugs,19 and a large majority of peptide drugs address this superfamily of receptors, either as agonists or antagonists. However, even though much is known about the pharmacology and biology of this family, it is still thought that as much as 25% of the GPCRs encoded in the human genome are so-called “orphan” receptors with unknown cognate ligands;20 it is likely some of these receptors are peptidergic in nature and will provide new targets for peptide discovery, if a lead can be found and optimized. De novo peptide ligand discovery has traditionally been done by screening large libraries of peptides, produced either synthetically or biologically. Biological library methods include phage, ribosomal, and mRNA display and have undoubtedly become the standard for peptide discovery, with phage display playing the most prominent role to date. While there still is a role in lead discovery for high-throughput screening of synthetic peptide libraries, this subject has been reviewed extensively21 and will not be covered in this perspective. Genomic or peptidomic/proteomic approaches are also being employed to find heretofore undiscovered peptides of interest in species ranging from microorganisms to humans; these studies have also found new biological activity for known peptides.22 Peptide-derived natural product research has been experiencing a revival, as evidenced by the particularly impressive advances in the discovery of nonribosomally synthesized peptide natural products.23 For the purposes of this review, we will discuss some of the more exciting examples of these technologies reported during the last 5−7 years that have enabled scientists involved in peptide drug discovery to address an ever-widening group of biological targets with peptide therapeutics. 3.1. Naturally Occurring Peptides as Starting Points for Lead Discovery. The traditional approach to peptide drug discovery has used naturally occurring hormones and other known signaling molecules as starting points for lead discovery. Medicinal chemistry strategies to enhance the potency and pharmacokinetic profiles have been very productive at finding analogues of these signaling molecules with more drug-like properties. A particularly successful example is the discovery of analogues of the natural intestinal incretin hormone glucagonlike peptide 1 (GLP-1).24 This 37 aa peptide is involved in insulin secretion and regulation yet exhibits a very short circulating half-life that makes it an unlikely drug candidate. Considerable effort has been made to enhance the stability of this hormone while maintaining its potency and pharmacological effect. Many strategies have been employed, including stabilizing the molecule by reducing susceptibility to proteolytic cleavage by dipeptidyl peptidase-4 (DPP-IV), the protease involved in regulating the deactivation of GLP-1, and conjugating the peptide to lipophilic moieties that bind to serum albumin in order to attenuate the renal clearance of this hormone (see section 7). These efforts have resulted in multiple marketed products which are in the process of revolutionizing the treatment of type 2 diabetes and perhaps ultimately obesity itself.25 Many other hormone-derived drugs been discovered and developed using a similar strategy:

aa residues with stable folds that are thought to have potential for increased stability in plasma (and the GI tract) and the ability to address a wider variety of biological targets (e.g., growth factor and cytokine receptors) vis-a-vis smaller peptides. Chemical synthesis permits access to a much more diverse range of peptides because elements such as unnatural amino acids, peptidomimetic bonds, and novel cyclization methodologies can be incorporated to enhance the properties of the target peptides, for which recombinant methods are not currently viable. Looking ahead, recent technologies have focused on expanding the genetic code to produce noncanonical aa containing proteins (including the use of amber stop codon to incorporate novel aa into proteins,16 new ribosomal-based in vitro technologies17 (vide infra), or by engineering semisynthetic organisms with expanded genetic alphabets18), and the ability to utilize these methods for the large scale recombinant production of peptides and proteins having multiple non-natural aa may be imminent. This would certainly relieve some of the constraints described above and likely further spur peptide drug discovery forward. Overall, we feel there will be a convergence in recombinant and synthetic production of peptides; advanced recombinant technologies will be able to incorporate unnatural aa and macrocyclic structures, and advanced synthetic ligation methods (see section 3.4) will enable the economically feasible production of customized, bespoke peptides or small proteins of upward of 100 aa. The traditional advantages and disadvantages of peptide drugs described here represent both opportunities and challenges for drug discovery teams. The prospects for the field engender an optimistic outlook, and the remainder of this perspective will describe diverse and parallel scientific advances that may soon combine to significantly mitigate these deficiencies in order to exploit the potent pharmacological activity of peptides in the clinic and beyond.

3. ENABLING TECHNOLOGIES IN PEPTIDE LEAD DISCOVERY The discovery or selection of attractive lead compounds is a critical first step in delivering a compound with a profile that will maximize success in the clinic. Historically, the selection of peptide leads for discovery programs has employed one of two general strategies, depending on the biological target of interest and what is known about its associated ligands and molecular pharmacology. The first strategy, the most common and historically productive in peptide discovery, is used when targeting a known signaling peptide, i.e., a hormone or peptide with a known receptor/target and some degree of understanding around its pharmacology. In this situation, the focus of the discovery program is on the manipulation of the parent peptide’s structure and properties (almost always via synthetic medicinal chemistry efforts) to optimize the compound’s halflife, potency, selectivity, or other properties in order to attain the desired compound profile, as described in later sections. Alternative strategies are needed when it is not possible to optimize a cognate ligand for a biological target of interest, either because the ligand is inherently unsuitable for chemical optimization (perhaps because of instability, promiscuous biological activity, or other factors) or the endogenous ligand for the target may be unknown. This situation is not uncommon. For example, genomic studies have shown there are ca. 800 members of the G protein coupled receptor (GPCR) superfamily encoded in the human genome, making E



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Figure 4. Structures of somatostatin and octreotide.

Figure 5. Structures of GnRH, the agonist analogue leuprolide, and the antagonist analogue degarelix.

Figure 6. Structures of arginine vasopressin and the analogue desmopressin.

• Octreotide (Sandostatin) is a synthetic somatostatin agonist that mimics the biological action of the natural hormone, optimized to improve potency, receptor selectivity, and pharmacokinetic profile (Figure 4).26 Octreotide is used to treat growth hormone producing tumors in acromegaly patients and pituitary tumors secreting thyroid stimulating hormone as well as other indications. • Gonadotropin-releasing hormone (GnRH, aka LHRH), a 10 aa peptide produced in the GnRH neurons in the hypothalamus, has been a target for peptide chemists almost since its discovery (Figure 5).27 Leuprolide is a synthetic nonapeptide GnRH receptor agonist, optimized for increased potency and half-life,28 that is used for diverse clinical applications including the treatment of hormone responsive prostate cancer, endometriosis, uterine fibroids, and precocious puberty and additionally for in vitro fertilization.28 Further medicinal chemistry efforts uncovered the key modifications that resulted in

potent GnRH antagonists such as degarelix,29 which has a remarkable sustained-release subcutaneous pharmacokinetic profile (see section 9) and is used in the treatment of hormone responsive prostate cancer and some benign gynecological conditions. • Desmopressin (1-desamino-8-D-arginine vasopressin) was discovered during an extensive campaign aimed at improving the pharmacokinetic properties of the natural antidiuretic hormone arginine vasopressin while retaining its impressive potency (Figure 6). Desmopressin is used to treat diabetes insipidus, bedwetting in children, and nocturia in adults.30 • Oxytocin, the first peptide hormone to be made synthetically,31 is a nonapeptide produced by the hypothalamus and is used to induce labor in case of nonprogression of parturition and for treatment of amenorrhea. The synthetic analogue carbetocin has improved pharmaceutical properties and can be prepared in a temperature-stable formulation.32 This should prove F



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Figure 7. Structures of oxytocin, the agonist analogue carbetocin, and the antagonist analogue atosiban.

Figure 8. Structures of vancomycin and cyclosporin.

Figure 9. Structures of arylomycin B and the synthetic analogue arylomycin M131.

products produced by bacteria and fungi, such as vancomycin and cyclosporin (Figure 8), are not synthesized at the ribosome; the amino acid constituents of these nonribosomally synthesized peptides (NRSPs) are nonstandard, and their diverse structures offer an array of structurally interesting features including an assortment of cyclization motifs, incorporation of D- and N-methylated aa, and other unique features contributing to their unusual structures and biological properties.23,33 The production of these peptides is controlled by clusters of genes termed nonribosomal peptide synthetases; each of these gene clusters, which operate independently of mRNA, is able to synthesize a single NRSP. Astounding progress in bacterial

to be a huge improvement for treating mothers in parts of the developing world such as sub-Saharan Africa, where refrigeration is rare and storage of peptide drugs is problematic. Antagonists of oxytocin such as atosiban, discovered during medicinal chemistry efforts aimed at finding compounds with tocolytic activity, are used to suppress premature labor (Figure 7).30 3.2. Natural Product Derived Peptides as Starting Points for Drug Discovery. a. Non-Ribosomally Synthesized Peptides (NRSPs). The peptides discussed thus far in this review are all biosynthesized using the well-defined machinery of the ribosome and are thus comprised of proteinogenic amino acids. It has been known for decades that some peptide natural G



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exenatide, a GLP-1 agonist, originated from Gila monster venom,25 and ziconotide, an N-type calcium channel blocker used to treat chronic neuropathic pain, derives from a venom peptide from the predatory cone snail Conus magus.40 The venoms traditionally used for drug discovery research have predominantly derived from snakes and scorpions, primarily due to the ease of collection and identification of the toxins. Recent technological advances such as nextgeneration sequencing (NGS) and proteomics have allowed the venoms from many heretofore neglected organisms to be accessed and studied, and this continues to be a fruitful area for new developments in peptide lead discovery. Representing the toxins is linaclotide, a cysteine-rich 14 aa bicyclic peptide, recently approved for the treatment of chronic idiopathic constipation and irritable bowel syndrome, which was derived from the Escherichia coli toxins STh and STp (Figure 11).41 Linaclotide is an agonist of the guanyl cyclase receptor (GCCR) which regulates intestinal water and electrolyte transport and acts to increase chloride secretion and decrease intestinal fluid absorption.42 c. Cyclotides and Lantipeptides. Cyclotides are a family of structurally novel, plant-derived cyclic peptides with interesting biological activity and are found in a variety of plant sources that are used in traditional folk medicine.43 For example, cyclotides are prominent components of an indigenous medicinal tea used in the Democratic Republic of Congo to accelerate childbirth. That particular observation has drawn considerable interest, as it can be inferred that not only do cyclotides have pharmacological activity, but they are thermally stable and orally active. The raison d’être for these structurally intricate peptides is likely to serve as a primitive innate immune system for the host, defending the plant against a variety of challenges; to support this notion, these compounds have been found to have insecticidal activity. Cyclotides have a head-to-tail cyclized backbone and three conserved disulfide bonds, creating a cyclic cysteine knot motif as shown in Figure 12 for the prototypical cyclotide kalata B1. Cyclotides are synthesized at the ribosome and are enzymatically N−C cyclized in a post-translational process. This aesthetically pleasing topology has been shown to bestow enhanced stability in plasma and gastric fluid, oral bioavailability, and a diverse range of biological activities. Additionally, the structures of cyclotides exhibit considerable topological plasticity, as changing the number and type of amino acids in the loops does little to distort the overall shape, making them particularly amenable to molecular grafting strategies. Tam and co-workers have reported a particularly interesting example of this approach, grafting a bradykinin antagonist peptide onto a cyclotide scaffold to afford orally active peptides for the treatment of inflammatory pain (Figure 13).44 Lanthipeptides (previously known as lantibiotics due to the use of these peptides as antimicrobials) are a class of cyclic peptides bearing a macrocyclic thioether linkage, formed by a mechanism involving dehydration of selected serine and threonine residues and subsequent intramolecular addition of the thiol of proximal cysteine residues to the newly formed Michael acceptor (Figure 14).45 Linear peptide precursors are ribosomally synthesized by Gram-positive microbes; these are then post-translationally modified enzymatically to afford the novel macrocyclic peptides. While the potent antibacterial properties of lanthipeptides such as nisin have been appreciated and exploited for decades, the current need for new antibiotics

genetics, genomics, protein biochemistry, and mass spectrometry have delineated the principles of assembly line enzymology for the production of these NRSPs, and this field promises to become one of the most exciting areas of peptide chemistry in the years to come. The unique structures and nonstandard aa present in these NRSPs enable the molecules to overcome some of the inherent weaknesses seen with the majority of peptides. For example, the cyclic structure and N-methylated aa in cyclosporin combine to generate small molecule-like biological properties including enhanced membrane permeability (and an intracellular biological target), high gut stability, and significant oral bioavailability. In a series of seminal papers, Romesberg has reexamined the potential of the arylomycin family of antibiotics, a long neglected family of NRSPs that exhibited a potent but narrow spectrum of activity against Gram positive bacteria (Figure 9).34 By identifying that the molecular target, signal peptidase, is both unique to arylomycins and essential for viability, and via an impressive structure-based medicinal chemistry program, they have been able to transform this family of NSRPs into a broad spectrum class of antibiotics with potent activity against both Gram negative and Gram positive organisms.34,35 A consortium of groups has recently reported the discovery of the new NRSP antibiotic teixobactin (Figure 10), a

Figure 10. Structure of teixobactin.

depsipeptide with significant activity against Gram positive bacteria.36 Considerable excitement accompanied this report, as the putative target of this peptide, the pyrophosphate of lipid II, plays an essential role in cell membrane biosynthesis; it is hoped that this mechanism of action will result in a significantly delayed onset of resistance.37 b. Venoms and Toxins. Peptides derived from venomous organisms have provided productive starting points for many successful drug discovery programs.38 Venom peptides typically target ion channels and other membrane bound receptors. While the variety of these compounds found in nature is enormous, they generally are disulfide-rich cyclic peptides ranging in size from 12−30 residues (as seen in cone snails) to 40−80 residues (in snakes and other organisms). They can be accessed synthetically or made using recombinant methods, and the stability afforded by the disulfide bridges relative to linear peptides make these venoms extremely attractive starting points for drug discovery programs; many prominent blockbuster drugs have resulted from optimization of venom derived peptides. For example, the ACE inhibitor captopril was discovered during the optimization of a venom peptide from the Brazilian viper.33,39 Similarly, the antidiabetic agent H



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Figure 11. Structures of E. coli STh toxin and the analogue linaclotide.

Figure 12. Two- and three-dimensional structures of cyclotide kalata B1.

has catalyzed a renewal of interest and a flurry of recent discoveries in this class of peptides. The basic structural feature of lantipeptides, the thioether macrocycle, has been employed by Moll and co-workers in an analogue of angiotensin.46 Ang(1−7) is a heptapeptide that plays a pivotal role in the renin−angiotensin system and possesses important pharmacological activities and is the likely endogenous ligand for the Mas receptor. As with most peptides of this type, the very short half-life of Ang(1−7) limits its therapeutic potential. In an attempt to address these deficiencies, Moll used a Lactococcus lactis variant, equipped with the appropriate lantipeptide modification machinery, to enzymatically produce a thioether-bridged Ang(1−7) from the corresponding linear peptide (Figure 15). The resulting cyclized peptide was found to be fully resistant against purified angiotensin-converting enzyme, had significantly increased

stability in plasma and tissue, and exhibited a ca. 30-fold longer plasma half-life than Ang(1−7) in rat studies. Surprisingly, the cyclized Ang(1−7) analogue A is also 2-fold more potent than Ang(1−7) in inducing the relaxation of precontracted rat aorta rings ex vivo. This study demonstrates the potential of lanthipeptides outside the antibiotic area and that their thioether motifs may represent a transferable tool for peptide medicinal chemistry optimization. 3.3. Use of Peptidomics for the Identification of New Peptide Leads. The field of peptidomics has emerged during the past decade as an important technique for the discovery of new peptides having novel biological function.22 Peptidomics combines peptide sequence identification with the profiling of peptides in various tissues and fluids and aims to systematically catalogue genetically encoded polypeptides; this is facilitated by spectacular advances in mass spectrometry and bioinformatics I



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Figure 13. Grafting of active peptide sequences onto cyclotide loops.

Figure 14. Mechanism of lanthionine synthesis and the structure of the lanthipeptide nisin.

Figure 15. Enzymatic synthesis of lanthipeptide A.

that permit the identification (and sequencing) of peptides present in the tissue of interest with exquisite sensitivity. Saghatelian and co-workers have developed elegant methods to describe endogenous polypeptides encoded by short or small

open reading frames (smORFs) that they refer to as SEPS; SEPs were originally found in bacteria, yeast, and worms, but functional SEPs have also now been found in the human genome that may prove to be biologically significant.47 It has J



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Figure 16. Chemical modification of phage by disulfide reduction and cysteine alkylation.

Figure 17. Principle of mirror image phage display.

facilitated the rapid identification of active peptide sequences because the phenotype (usually affinity) is directly associated with the genotype (sequence). Fueled by the critical advances made in the antibody field, where this has become the sine qua non for antibody discovery,53,54 phage display (and the related technologies mRNA and ribosomal display) have become powerful methods for the generation and optimization of peptide ligands and have been used for the discovery of peptide leads against a vast array of biological targets.52 Technological advances continue to make these techniques ever more efficient and applicable to an increasing range of targets. For example, the traditional screening, or “panning”, protocols generally used to find peptide binders to the target of interest are being impacted by the revolution in high throughput sequencing. Using “next generation sequencing” protocols, more efficient screening paradigms are being developed which allow access to enormous amounts of sequence data after even a single round of panning.55 This sequence data can greatly facilitate the strategy for further screening and library synthesis during the affinity maturation stages, leading rapidly to both more potent binders and a significantly larger structure−activity data set directly from the early panning rounds. A further advantage of reducing the number of iterations of the phage panning cycle is the prevention of a selection bias toward phage clones that more readily replicate; these “parasitic” sequences can quickly

been estimated that >200 SEPs are encoded in the human genome, varying in length from 15 to 138 aa. As an example, a screen of a human cDNA library for the prevention of amyloid precursor protein-induced cell death led to the discovery of humanin, a 24 aa peptide encoded by a smORF on the mitochondrial genome; this represents the first peptide in a new class of mitochondrial-derived peptides (MDP).48 Humanin has also been shown to be synthesized in the cytoplasm as a 21 aa variant and has a range of interesting biological activity, including inhibiting the pro-apoptotic protein BAX and interacting with IGF-1 binding protein.49 Immediate applications of peptidomics will likely be to the discovery of relevant peptide biomarkers that facilitate translational medicine, clinical studies, and even patient stratification, but this developing technology is also proving to be an engine for the discovery of new peptides and peptide drug targets. 3.4. Display Technologies in Peptide Lead Discovery. Simultaneously with seminal discoveries in the area of peptide library synthesis and HTS methods, the first report by Smith50 in 1985 of the ability to engineer and display peptides on the surface of bacteriophage revolutionized the discovery of bioactive peptides and proteins, and important technological developments in this area have continued unabated.51,52 The ability to construct and screen vast libraries of peptide, protein, and antibody motifs using recombinant technologies has K



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extremely large libraries (>1013) of novel, macrocyclic peptides that contain a majority of unnatural amino acids.62 Using a PURE/mRNA-display system that generates the tRNAs charged with various unnatural amino acids in situ, 12 of the 20 natural amino acids were reassigned to introduce a diverse set of unnatural side chain and backbone modifications. They were able to also incorporate aa derivatives that permitted macrocyclization to introduce further conformational restriction and enhanced stability. The technology was exemplified by the discovery of nanomolar-affinity thrombin inhibitors comprising mostly unnatural amino acids that were shown to be essential for binding.63 Similarly, Suga has reported extensively on the use of a ribosomal display method they term the “random non-standard peptides integrated discovery” (RaPID) system, which can be used to produce large libraries of bioactive macrocyclic peptides containing noncanonical aa.17,64 This highly promising technology has been demonstrated to provide hits against an increasingly wide variety of biological targets and features a critical additional element that provides an advantage over the PURE technology described above; the aminoacyl tRNAs do not have to be premade. The use of “flexible” tRNA acylation ribozymes, known as “flexizymes”, facilitate the preparation of a wide array of nonproteinogenic aminoacyl tRNAs and allows ribosomal synthesis of macrocyclic peptides using an almost unlimited number of amino acids, including D-aa.65 In addition to proteolytic stability, the authors claim that these unnatural peptides exhibit enhanced permeability and could potentially have sufficient bioavailability for oral dosing. With these and many other impressive advances in the application of display technologies to peptide and protein drug discovery over the past decade, it is now generally true that binders to nearly any biological target of interest can be found using phage, ribosomal, or mRNA-display methodologies, providing that the protein/receptor/enzyme target can be assayed in a relevant state. This latter caveat has traditionally excluded membrane bound targets such as GPCRs, ion channels, and singlespanning receptor tyrosine kinase (RTK) receptors despite their aforementioned prominence in the spectrum of peptide pharmacology. A number of different approaches have recently been developed to facilitate the study of transmembrane proteins and allow display screening efforts. Modified calixarenes were reported as a next-generation detergent formulation to better mimic the structure and properties of phospholipids, allowing extraction of functional membrane proteins.66 Alternatively, Sligar and co-workers developed a sophisticated synthetic model of a portion of a phospholipid bilayer, termed a nanodisc, using a “double-belt” of membrane scaffold proteins (MSP) to surround and stabilize the exposed hydrophobic surface.67 These nanodiscs have proven highly effective at hosting stable, soluble, and active transmembrane proteins. Finally, the transmembrane protein can be modified by precisely selected mutations for improved thermostability without impairing its pharmacology;68 this is the basis of Heptares’ StaR technology. These techniques allow fundamental structural investigations of GPCRs,69 as well as enabling pharmaceutical screening efforts.70 Another major limitation of screening technologies that has to date excluded a critical part of the peptide field is the difficulty in parsing these huge libraries for receptor agonists despite the powerful pharmacology this might uncover. An inefficient two-step approach is possible, screening for

accumulate to become a majority, seriously hindering the identification of peptides with real affinity for the target.56 By definition, the peptides produced in phage display libraries are comprised of the proteinogenic amino acids, and thus the peptide leads obtained using this technique must usually subsequently be optimized via medicinal chemistry to find compounds with the requisite pharmaceutical properties (potency, PK, solubility, etc.) required of a drug lead. Several ingenious techniques have emerged, however, that could lead to much more “drug-like” peptide leads arising directly from phage libraries. For example, “on-phage” chemical modification of libraries is an area of intense current investigation and promises to significantly expand the capabilities of peptide phage display.57 The compounds produced often have novel, conformationally restricted molecular architectures that represent unusual epitopes; in theory, this leads to improved affinity and increased stability to enzymatic degradation (though without obviating the inherent renal clearance issues). An early example of this from Heinis used a polyvalent electrophile to alkylate multiple reduced cysteine thiols in the same phagedisplayed peptide, leading to the replacement of the cyclic disulfide peptide with a novel, stabilized cyclic bis-thioether peptide (Figure 16).57 An alternative approach uses chemical modification to mimic common post-translational modifications such as glycosylation, effectively allowing the screening of a defined and characterized library of glycopeptides.58 Other approaches to find more pharmaceutically attractive peptides from display technology continue to be reported. Kim demonstrated in an elegant series of papers the concept of “mirror image phage display”. This technique uses an unnatural D-peptide/protein target to select peptides from a standard Lpeptide phage library; the D- mirror images of these active phage-displayed peptides, by symmetry, will then interact with the target protein having natural L- chirality (Figure 17).59 These D-peptides are effectively guaranteed to exhibit complete plasma stability and are very attractive starting points for drug discovery programs. In the first examples of this approach, Kim found D-cyclic peptides that interact with c-SRC and HIVprotease.59,60 The obvious and significant barrier to implementing this technique is the requirement for the synthesis of the Denantiomer of the target protein, an impressive scientific achievement in its own right.61 The continual improvements in synthetic methods for the production of large peptides and small proteins, outlined in section 3.4, may eventually allow routine access to D-proteins, at which point, this screening approach may naturally supersede others to become a dominant peptide discovery tool. The conceptually ideal display technology envisions a system where the chemist has an essentially unlimited toolbox of amino acid building blocks to construct display libraries, comparable to the construction of synthetic peptides, while still exploiting the power of the phenotype/genotype linkage to generate very large libraries and rapidly identify active peptides. If one could use, for example, N-methylated or D-amino acids, or amino acids that can promote the formation of novel cyclization motifs, then display libraries could directly produce stable peptides that would immediately prove valuable for drug discovery programs, obviating the need for significant time and effort in early medicinal chemistry optimization. Several recent technological developments have shown that this goal may be close to realization. Szostak and co-workers have shown that mRNA display can be applied to the evolution, selection, and discovery of L



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Figure 18. Principle of Lerner’s autocrine screening technology.

Figure 19. Comparison of the sequences of GLP-1, exenatide, and the biased agonist P5.

use of a yeast cell line, which has the additional structural element of a cell wall encircling the membrane-bound screening protagonists; the peptides in this example are anchored to the inner face of the cell wall. In theory, this provides a simple solution to the potential for confounding intercell signaling, where a peptide bound to one membrane activates a receptor on an adjacent cell. This technology may have applicability to a wide variety of membrane bound receptors, including GPCRs. These examples highlight the both the utility and versatility of display technologies for the selection of peptide binders against a broad range of biological targets. In addition, we have described some of the key technological advances in this area that have attempted to make the peptide leads discovered in display efforts much more amenable to optimization. As in vitro display technologies continue to advance, their utility in peptide drug discovery programs will no doubt increase further.

competitive binders, then assaying synthesized peptide hits for agonism. The ideal technique would allow direct panning of display libraries against intact cells with a functional signaling readout, yet this approach has faced complex practical problems and challenging interpretation of the screening results. Recently, however, several groups have reported promising advances in this area that may uncover pharmacologically interesting peptide hits against whole classes of previously intractable receptor targets. Lerner and co-workers, in a series of ground-breaking papers, have described a general autocrine-based signaling system that utilizes an engineered cell line containing a membrane-tethered library of potential peptide ligands, introduced via lentivirus infection, coexpressed with the receptor of interest (Figure 18).71 Activity is reported using an artificial signal transduction system, engineered to be observed even in the presence of the endogenous signaling of the receptor−ligand system. Cells having the desired phenotype are identified, sorted, and the peptides of interest identified using standard techniques. Originally used to find agonist antibodies, this system has been extended to the use of libraries of scaffold proteins, peptides, and venoms. An exciting recent report by this group described the discovery of a potent analogue of GLP-1, P5, that uniquely has a differentiated molecular pharmacology profile; it does not recruit β-arrestin and is a weak insulin secretogogue.72 In vivo studies in mice showed this biased agonist (see section 5) more effectively controls glucose levels and reduces HbA1c compared to marketed GLP-1 agonist Exendin-4 (Figure 19). Yoshimoto, Kurota, and co-workers have used a similar approach for the discovery of new peptide ligands for a single membrane spanning receptor tyrosine kinase receptor, endothelial growth factor receptor (EGFR).73 The principle is functionally identical to Lerner’s method, expression of a library of anchored peptides that can demonstrate agonism of the receptor via a fluorescence readout. The key difference is the

4. NEW ENABLING SYNTHETIC METHODS That the synthesis of moderately long peptides (30−50 residues) is now considered relatively routine is a testament to decades of continual optimization, both methodological and technological. The modern infrastructure of solid-phase peptide synthesis (SPPS) would still be recognizable to chemists of earlier generations, but the accumulated knowledge and extensive chemical toolbox has dramatically expanded the scope of potential targets, both for academic research and in the higher-throughput environment of industrial drug discovery programs. The current slate of peptide synthesizers exhibit a range of capabilities; sequential or multiparallel synthesis of up to 192 peptides, at scales from 1 μmol to 5 mmol, IR or microwave heating, flexible programming and preactivation chambers, and UV monitoring of the deprotection step to identify troublesome sequences. Downstream, automated HPLC purification systems are also increasing throughput at this traditional M



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Figure 20. Structure of isoacyl dipeptides and pseudoprolines.

Figure 21. Mechanism of native chemical ligation.

bottleneck, commonly with the addition of MS-directed sample collection. There has been a recent resurgence in peptide reverse phase (RP) flash chromatography, which may provide a cheap and high-throughput alternative, particularly for shorter peptides with good crude purity. Ideally, large multiparallel library synthesis would avoid individual chromatographic purification altogether, and interesting progress is being made in the use of reversible N-terminal capping strategies that allow the physical separation of the desired peptide;74 refinement and incorporation of this technology into a future generation of synthesizers should prove extremely valuable. In Fmoc-SPPS, it is generally understood that the critical problem for the synthesis of long peptides is physical occlusion of the reactive N-terminus by aggregation (effectively desolvation) or the formation of secondary-structural elements on the resin. A range of approaches are now widely used for problematic sequences, in many cases routinely and preemptively. Low-substitution PEG resins such as ChemMatrix allow increased swelling and greater solvation,75 and microwave heating offers the dual advantages of reduced aggregation and short reaction times of 5 min or less.76 Solubilization can be improved using chaotropic agents and optimized solvent mixtures.77 Additionally, there are three common synthetic approaches that are used to purposefully disorder the secondary structure of the growing chain:77,78 • Pseudoprolines are cyclized Ser, Thr, and recently Cys dipeptides that have demonstrated great efficacy in destabilizing β-sheet formation by both masking a backbone NH and altering the overall conformation, consequently improving yield and purity (Figure 20).79 Available, cheap, and readily deprotected by the standard TFA cleavage, pseudoprolines are now often proactively incorporated every 8−10 residues where the sequence allows. • Isoacyl dipeptides, esters of Ser and Thr residues, exert a similar effect on the synthesis (Figure 20). However, the lack of any consistent advantage over pseudoprolines limits their utility, as this approach carries risks of diketopiperazine formation and β-elimination side reactions and requires an additional rearrangement step following cleavage.80 • Removable N-α-alkylation is an effective alternative strategy, most commonly as acid-labile Hmb- or DmbGly derivatives that retain sufficient reactivity for the

subsequent coupling; some dipeptides are also commercially available.81 It is worth noting that in Boc-SPPS the TFA deprotection step effectively destroys aggregates and has proven useful for challenging sequences,78 yet the safety-driven shift toward Fmoc-SPPS has resulted in many peptide synthesis laboratories no longer being equipped for the final HF cleavage step. The second major problem in Fmoc-SPPS of longer peptides is the accumulative formation of aspartimides by base-catalyzed cyclization of aspartic acid esters and their subsequent distribution across up to nine different side products.82 Three strategies have each proven generally effective: bulky ester protecting groups such as 3-ethyl-3-pentyl (Epe),83 buffering of the basicity of the deprotection solution using HOBt,84 Oxyma,85 or formic acid,84 and the use of an N-α-alkyl dipeptide for the especially susceptible Asp-Gly sequence.81 Despite these impressive advances, it is rarely practical to synthesize very large peptides and small proteins as a single chain in SPPS, and perhaps the most vibrant field of peptide synthesis research now involves the development of methods for the controlled ligation of synthesized fragments, which will comprise the majority of this section. As a logical extension from Fmoc-SPPS, the ligation of protected fragments in both solution and solid phase is possible when C-terminal racemization is controlled by the selection of Gly or Pro as the ligation site,86 the use of a C-terminal pseudoproline residue,87 or the careful choice of coupling conditions.88 Although the selectivity of the coupling is guaranteed, the significant limitation to this method is the difficulty in handling and purifying the fully protected peptides prior to the ligation. Native chemical ligation (NCL) is the most widely used and successful fragment coupling approach and has been essential in the expansion of synthetically accessible biological space from large peptides to proteins, culminating in the recent chemical syntheses of single polypeptide chains of over 300 residues.89 NCL takes advantage of the rapid and reversible exchange of thioesters at the C-terminus to induce a highly selective proximity-directed amidation with an adjacent amine (Figure 21).90 Originally developed for ligation at cysteine, which is relatively uncommon in proteins,91 subsequent advances have expanded the possible ligation sites through the use of both removable thiol auxiliaries92 (N-alkyl glycines) and mild desulfurization conditions that convert cysteine to alanine.93 N



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Figure 22. NCL via C-terminal hydrazides.

Figure 23. NCL via C-terminal N-acyl ureas.

Figure 24. NCL via C-terminal bis(2-sulfanylethyl)amides.

Figure 25. Intein-mediated protein ligation (IPL).

The potential of this latter approach has been further extended by the synthesis of the β-thiol analogues of more-common natural amino acids,94 where the desulfurization would again result in a traceless ligation and future commercialization of these building blocks should result in widespread adoption. Turning to the thioester ligation partner, the reactivity has been demonstrated to vary with both the steric hindrance of the C-terminal residue95 and the nature of the chosen thiol; 4mercaptophenylacetic acid (MPAA) is widely used.90 A number of alternative approaches have been developed to access the deprotected thioester fragment via Fmoc synthesis, four of which are highlighted here: • C-Terminal hydrazides are readily synthesized and highly stable yet can be selectively and mildly nitrosated to form reactive acyl azides, which in turn are rapidly converted to thioesters with the appropriate thiol (Figure 22).96 This widely used approach has the additional advantage of simple application in an iterative fragment condensation strategy, where each new ligation product has a C-terminal hydrazide ready for subsequent activation and coupling. • Dawson’s second-generation N-acyl urea approach synthesizes the peptide fragment on a bis-aniline linker (Figure 23).97 Conversion to the urea immediately prior

to cleavage again produces a reactive acyl leaving group for displacement by a thiol. • An elegant strategy takes advantage of the inherently reversible acyl transfer of β-thio-amines. Melnyk’s bis(2sulfanylethyl)amides (SEA), again readily prepared by SPPS using a special linker, are activated by reduction to set up this equilibrium, which can be directed at low pH toward the thioester by protonation of the amine.98 Interception with an added thiol results in a stable thioester ligation partner (Figure 24). • Finally, ligations at the C-terminus of recombinant proteins have been made possible by fusion to an intein sequence. Following purification using an appropriate tag, the intein can be cleaved by addition of a thiol reagent to give the C-terminal thioester (Figure 25).99 Another approach for recombinant expression is the genetic incorporation of α-hydroxy acids, creating an ester that can be displaced by hydrazine to give a ligationcompatible C-terminal hydrazide.100 These methods of initially activating the C-terminus are often also applicable to the synthesis of salicylaldehyde esters, which enable a useful serine/threonine ligation by the selective, reversible formation of oxazolidine intermediates.101 These O



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Figure 26. Mechanism of the Ser/Thr ligation.

Figure 27. Mechanism of the KAHA ligation.

Figure 28. Schematic of butelase ligation, illustrating sequence recognition and the irreversible thioester variant.

Figure 29. Synthesis of CAM esters as substrates for peptiligase ligation.

proteins due to their required five-residue “LPXTG” recognition footprint and a relatively slow, highly reversible reaction that requires a substantial excess of one component.104 Tam’s butelase improves in many aspects, requiring only an asparagine footprint in the final product and demonstrating high rates of ligation and particularly macrocyclization, with excellence tolerance for D-amino acids (Figure 28).105 Tam has recently proposed an irreversible alternative that uses a thioester motif at the ligation site, analogous to NCL approaches but accepting a broad range of nonthiol N-terminal partners.106 The most impressive enzymatic approach to date is the peptiligase family, demonstrated to have a near-perfect set of attributes for peptide and protein ligation applications: traceless combination with no recognition motif, applicability to ligation of almost every combination of primary amino acids, high efficiency in water or with organic cosolvents, no hydrolysis of internal amide bonds, and a surprisingly high synthesis/hydrolysis ratio that promotes the desired ligation.107 The acyl donors are carboxamidomethyl (CAM) esters, which are easily synthesized via conventional SPPS (Figure 29). The enzyme itself has proven amenable to industrial production and protein engineering, with two classes of product envisioned: a high-volume omniligase with maximally broad specificity and

undergo an acyl shift and subsequent hydrolysis to effect the ligation (Figure 26). One alternative recent method of chemical ligation has expanded the possibilities of fragment condensation approaches by uncovering a truly orthogonal and completely selective coupling reaction. Bode’s ketoacid-hydroxylamine (KAHA) ligation uses these eponymous functionalities at the C- and Nterminus, respectively, which rapidly combine to a depsipeptide that rearranges to give the desired product (Figure 27).102 The major limitation is currently the preferred use of oxaproline as the hydroxylamine partner, which incorporates homoserine residues into the final product; the oxazetidine analogue for serine site ligations has recently been demonstrated.103 Serial fragment condensation is possible by the use of protected precursors, and overall further refinement and commercial availability of the building blocks should make this a powerful parallel method to the NCL methods described above, particularly due to the complementary reactivity profiles of Cterminal ligation sites for these two approaches.102 Biochemical ligation approaches to peptide and protein synthesis represent an exciting frontier that has seen rapid advances in the past few years. The use of sortase enzymes has been mostly exploited for functional tagging of recombinant P



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Figure 30. Examples of peptides exhibiting nonclassical GPCR pharmacology.

the basis of the field, a foundation for the exciting advances in peptide pharmacology and their pharmaceutical applications discussed in subsequent sections.

utility and the bespoke development of tight-specificity peptiligases that would allow exquisitely selective ligations of a chosen N-terminus for multifragment approaches, branched products, or even bioorthogonal applications. It is of course not just peptide length that can favor recombinant production but also structural complexity. All insulin on the market is currently produced using recombinant technologies, as the disulfide-bridged heterodimeric structure has proven challenging to synthesize efficiently. Taking inspiration from the biosynthesis, a variety of recent approaches to this class of peptide have demonstrated synthetic accessibility using oxime tethering,108 hydrolyzable depsipeptides,109 and a chemically cleavable bis-linker tether.110 All avoid the conventional synthetic approach that requires a difficult selective dimerization of poorly soluble single strands using orthogonally protected cysteines. The commercialization of peptide drugs represents the culmination of the discovery process and the ultimate challenge for large-scale, cost-efficient production methods. A range of techniques established in the literature have been highly optimized for the synthesis of peptide active pharmaceutical ingredients, including solution-phase and ligation approaches alongside the more common solid-phase and recombinant strategies, and the challenges involved have been described in a recent review.111 The new synthetic methods briefly described here enable peptide chemists to rapidly and efficiently drive the optimization of biologically active peptides and explore new regions of biological space. This ever-growing toolbox forms

5. NEW PARADIGMS IN PEPTIDE PHARMACOLOGY As the natural ligands for many GPCRs, peptides have been at the forefront of the considerable effort to characterize and delineate the intricately woven signaling processes mediated by these receptors. Consequently, modern GPCR pharmacology has developed far beyond simple on/off-switch notions of agonism and antagonism and the attendant assumptions of orthosteric binding, linear efficacy, and uniform receptor activation.112,113 One of the preeminent voices in this field, Terry Kenakin, has described seven-transmembrane (7TM) receptors as shapeshifting conformational ensembles, whose seemingly diverse signaling pathways can be holistically expressed in terms of multidirectional allosteric effects.114 To briefly summarize, all the interacting partners of a GPCR can affect its conformation and thus its binding to any other partner. These partners include traditional extracellular orthosteric ligand peptides but also other membrane receptors and a variety of intracellular signal transduction proteins. If the binding mode of a ligand to the GPCR alters the expected recruitment or activation pattern of these intracellular proteins, the resulting differential signaling is termed functional selectivity or bias. This complexity presents both challenges and opportunities in drug discovery, and new approaches and assays are continually being adopted by drug discovery scientists to not Q



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functionally selective PAR1 inhibition.120 Importantly, by not classically antagonizing the binding of thrombin to the PAR receptors, 2 has been demonstrated to prevent platelet activation and arterial thrombosis without interfering with bleeding or coagulation.120 2 is in clinical development for patients with acute coronary syndrome (ACS) and those undergoing percutaneous coronary interventions (PCI) and has recently completed a phase I trial.121 The concept of functional selectivity is gaining widespread interest in modern drug discovery programs. The biological efficacy of a GPCR ligand hit, lead, or candidate must now be seen as a multidimensional parameter, able to be both measured and optimized in a variety of functional in vitro assays. In the near future, the pioneering drug discovery efforts listed above will provide vital clinical data to begin to validate the current pharmacological signaling hypotheses and models. However, this is a nascent field; even as attempts are made to both standardize and quantify aspects of ligand bias, many questions remain. Is there a temporal difference in pathway activation? Are different signaling pathways favored in different cell types? A further decade or two of research may be required to fully understand the fundamental biology, allow confident translation to meaningful functional selectivity in vivo, and realize the clinical promise of precise control of GPCR signaling.122

only better understand the targeted biological pathway but also to access potentially unexplored pharmacology of established GPCRs. A critical question in the peptide field is whether the pleiotropic effects of many long-established hormones, specifically where a single extracellular receptor binding event causes a multifaceted intracellular signaling response, can be divorced and isolated by careful optimization. As the receptor pharmacology is unraveled, exciting therapeutic opportunities may present themselves; can the desired effect be boosted and/ or the unwanted side effects eliminated? Selected examples are presented below of peptides that were specifically developed to effect therapeutically relevant GPCR signaling outside the realm of classical orthosteric agonists and antagonists: • Trevena’s octapeptide 1 (TRV120027,115 Figure 30a) was developed as a functionally selective ligand of the angiotensin II type 1 receptor (AT1R).113,115 Classical marketed AT1R antagonists are used to block the adverse effects of the natural ligand angiotensin II (predominantly hypertension) but are thought to also prevent ionotropic effects that boost cardiac function. During acute heart failure (AHF), these become opposing therapeutic outcomes, and as such AT1R antagonists have little positive effect on AHF patients. Elucidation of the AT1R signaling pathways found that the vasoconstrictive, hypertensive pharmacology results from activation of G-proteins, whereas the ionotropic effects are mediated by β-arrestin signaling.115 To achieve a beneficial profile, 1 therefore both inhibits G-protein coupling and stimulates β-arrestin recruitment at AT1R. Despite the promise of this functional selectivity, 1 recently missed both primary and secondary end points of a phase IIb trial in AHF. • The parathyroid hormone (PTH) analogue PTH-βarr (Figure 30b), developed at Duke University Medical Center, is a functionally selective agonist for the βarrestin signaling pathway and has been demonstrated in animal models to stimulate bone formation without the accompanying increase in bone resorption that has limited the clinical use of PTH in osteoporosis.116 • Rapastinel (Figure 30c), originally developed by Naurex, binds to an allosteric regulatory site on the N-methyl-Daspartate (NMDA) receptor and functionally acts as a weak partial agonist of the glycine site.117 Modulation of this receptor produces a variety of profound CNS effects, and antagonists such as ketamine and PCP can have powerful antidepressant activity, albeit with a narrow therapeutic window and a range of dangerous psychotomimetic side effects. Rapastinel’s weak partial agonism has been shown to produce only the desired antidepressant (and cognitive enhancing) response, and this drug has recently completed phase II clinical development for treatment-resistant major depressive disorder. In 2016, rapastinel, now owned by Allergan, received Breakthrough Therapy designation from the FDA.118 • Pepducins, developed at Tufts Medical Center, are lipidated peptides that target the intracellular GPCR/ G-protein interface, and can either activate or block the associated signaling pathway.119 Pepducin 2 (PZ-128,120 Figure 30d) was designed to directly mimic the PAR1 receptor loop that binds to the Gα-protein, yielding

6. MEDICINAL CHEMISTRY OPTIMIZATION OF PEPTIDES Successful peptide medicinal chemistry optimization requires a comprehensive multiparametric approach that ideally assesses the impact of each iteration on the key candidate properties of potency, selectivity, stability, solubility, and toxicity.123 There are a number of key differences between the optimization of peptides and small molecules: • Peptide natural products or affinity-matured screening hits often present with high potency, which must then be sufficiently retained during optimization of the other critical properties. • The polymeric nature of peptides readily allows each residue to be individually optimized, with the modifications then often combining to give predictably additive results. • The ADMET properties are inherently restricted: minimal absorption, limited distribution from the plasma, an in vivo peptide metabolism that is essentially limited to proteolytic cleavage of backbone amide bonds, largely renal excretion, and consequently limited toxicological liabilities. The section describes the logical sequence of steps taken in the medicinal chemistry optimization of a hit or lead peptide alongside selected examples of successful applications. One highly context-dependent factor that is also considered throughout this process, yet will not be discussed further, is ensuring intellectual property rights for the final candidate(s). 6.1. Identification of the Minimum Active Sequence. The lead peptide is truncated by iterative removal of amino acids from the C- and N-termini to identify the core sequence critical for the desired biological properties. 6.2. Positional Scanning to Determine Critical Residues. Classically this is done as an L-Ala-scan, replacing each side chain with the smallest alternative that should still R



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• β-aa incorporation may cause a significant conformational change in addition to the positional shift of the side chain yet has proven an effective approach when rationally introduced into α-helices, as exemplified by periodic incorporation in a recent parathyroid hormone inverse agonist.134 • Peptoids (poly-N-alkyl glycines) are a logical extension of these concepts131,135 yet as a global strategy suffer from a problematic decrease in overall conformational rigidity and a corresponding entropic penalty for receptor binding. Retention of the α-stereocenter (e.g., alanine peptoids) has been demonstrated as a viable solution.136 • Similarly, aza-peptides can be effective in isolation in reducing proteolytic degradation but also introduce additional conformational flexibility.130 A range of peptidomimetic approaches have been developed that partially or totally replace peptide backbones with nonnatural oligomeric units,131 but this is rarely attempted as part of a peptide medicinal chemistry optimization program. It is worth noting that maximizing the plasma half-life of peptide compounds, while a very common optimization goal, can of course be unnecessary in indications where the treatment is expected to be particularly acute, and a short half-life may be optimal where rapid clearance allows safer titration of the drug levels to within a narrow therapeutic window. This strategy was successfully pursued by Ferring in the development of selepressin (Figure 31), a vasopressin analogue designed to be both selective and short-acting,137 which is currently in clinical development for the treatment of vasodilatory hypotension in septic shock.138

retain a similar conformational profile and thus determining its importance for the biological activity. The increasing throughput of novel synthetic and purification approaches now also allows positional scanning using a defined set of amino acids that possess a wide spectrum of physical attributes (i.e., hydrophobic, basic, acidic).124 6.3. Protection from Degradation at the Termini. If acceptable to the binding interactions, modification of the Cand N-termini is usually carried out to proactively prevent the degradative activity of carboxy- and aminopeptidases, respectively. The simplest analogues, C-terminal primary amide and N-terminal acetylation, often work well. If these are not tolerated, specific optimization of these residues toward unnatural analogues may prove necessary.10,125 6.4. Identification of Sites of Proteolysis. This initial exploration of the SAR, in conjunction with PK experiments, stability assays, and metabolite detection, should allow identification of the proteolytically labile amide bonds within the sequence.126 6.5. Proteolytic Stabilization by Backbone Modification. As already discussed, proteolytic stabilization with retention of activity is one of the major challenges for the optimization of peptide natural products and screening hits. There are many common strategies available for modification of the labile amide bond(s), yet retaining the desired conformation and binding affinity can be extremely difficult: • D-aa are often the first resort and can be incorporated into the positional scanning libraries. A study comparing backbone modifications has determined that D-aa are the most effective way to prevent chymotrypsin degradation and can confer protection remotely to nearby residues. This effect, also seen for the α-Me-aa strategy discussed next, could be especially useful if the labile amide bond cannot be modified without significant loss of activity.127 The retro-inverso strategy uses a reverse sequence of all D-aa, which in theory should place the side chains in a similar orientation to the original L-peptide;128 this is a conceptually pleasing approach that unfortunately has rarely proven effective, likely due to the major conformational changes produced by the effective transposition of each backbone amide bond. • α-Me-aa occlude protease binding in a similar manner to D-aa, while in theory retaining the side chain in its original spatial location.141,127,129 Quaternary aa also have restricted conformations which can prove beneficial in specific contexts, particularly for helical peptides.130 αMe-aa are now widely commercially available yet can pose synthetic challenges through increased steric hindrance; microwave heating and double-couplings may be required.131 • N-Me-aa generally offer good protease protection127 but may also disrupt intra- or intermolecular hydrogen bonding interactions. This can modulate biological function and with judicious application also potentially decrease unwanted aggregation and improve solubility.132 An intriguing application inspired by the immunosuppressant natural product cyclosporine is the use of selective N-methylation to generate membranepermeable cyclic peptides, which often also have significant oral bioavailability.133 As above, N-Me-aa often require more forceful coupling conditions during the synthesis.131

Figure 31. Structure of selepressin.

6.6. Optimization by Side Chain Modification. The results of the positional scanning usually provide sufficient data for rational modifications of the key binding residues toward improving the affinity and selectivity of the peptide. Each natural amino acid effectively has a set of commonly used close analogues that can often be substituted in at this stage, and non-natural side chains often induce protease resistance. For example, variants of arginine include lysine, ornithine, homoarginine, citrulline, and N-isopropylornithine.137 Aromatic residues have a particularly broad set of available analogues including unnatural heterocycles139 and can also benefit from the introduction of β-methyl groups that rigidify the conformation.140 The residues that were found to not be critically involved in the binding interaction can be rationally modified to alter the physical properties of the peptide, improving solubility or installing an unnatural amino acid that might confer increased proteolytic stability on adjacent positions.127,141 Alternatively, noncritical residues provide sites for conjugation (see section 7) or in some cases cyclization. S



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• An active peptide conjugated in order to modify its pharmacokinetic properties • A peptide used as a targeting agent for an active molecule • A peptide used as a trans-membrane delivery agent for an active molecule In this section, the critical aspects of each of these major categories will be briefly summarized in the context of selected examples of interest. In diseases that have validated targets from multiple biological pathways, studies often find a synergistic therapeutic effect with combined administration.152 Direct conjugation of compatible active agents has advantages for clinical development; for a peptide, this compatibility generally requires the other agent to act extracellularly and have broadly similar pharmacological potency. Peptides are attractive molecular candidates for conjugation, usually providing easy synthetic access to an appropriate functional group spatially distinct from the binding pharmacophore and allowing an array of chemical conjugation strategies. Ipsen’s follow-up program to their somatostatin analogue lanreotide, used to suppress growth hormone (GH) release in acromegaly, has explored the use of small-molecule−peptide conjugates. Medical guidelines for these patients support both somatostatin and dopamine agonists as first-line treatments,54 and the chimeric dual-agonist 3 (BIM-23A760,153 Figure 32)

An impressive recent demonstration of sequence optimization is the rational design of a triagonist by identification and combination of the active partial sequences for three individual hormones.142 The resulting single helical peptide simultaneously and equally activates the GLP-1, GIP, and glucagon receptors, resulting in superior reduction of body weight and diabetic complications in rodent models of obesity. 6.7. Cyclization. It is a central tenet of medicinal chemistry that restricting the conformational flexibility of a molecule to favor the orientation required to bind to the target reduces the entropic penalty of binding and can dramatically improve the affinity and selectivity.10 For peptides, cyclization also often prevents protease access to the backbone amides; these enzymes bind their substrates in a linear, extended conformation. Many natural peptides and screening hits contain one or more disulfide bridges, and this linkage can be further optimized for stability; typically, one of the sulfur atoms is replaced with a carbon atom to generate a cystathionine.46,143 Disulfide positional scanning can also identify the optimal position for cyclization and conformational restraint. For helical peptides, side-chain-to-side-chain cyclization has proven highly effective at promoting and stabilizing the desired conformation. A common approach is a lactam scan,144 coupling together the side chains of lysine and glutamic acid residues on the same face of the helix.145 Ring-closing metathesis of alkenyl side chains produces a hydrocarbon “staple”, and this technique has been used extensively by Aileron in the design of cell-permeable α-helices.146 In recent years a toolbox of chemical approaches has emerged for the one-step orthogonal stapling of cysteine residues, which offer increased throughput, diverse conformations, unnatural functionality for additional interactions, and the potential for direct modification of displayed peptides during the phage screening process.147 This latter approach has been pioneered in an industrial setting by Bicycle Therapeutics, bridging three cysteine residues to create large screening libraries of peptides with unusual fixed conformations.148 Finally, β-turn dipeptide mimetics have proven useful for restricting the conformation of the peptide backbone and precisely mimicking the geometry of this secondary structural motif,149 as demonstrated for a CGRP antagonist using the thioindolizine “BTD”.150 6.8. Solubility. The latter stages of peptide optimization must involve determination of solubility and gelling properties toward achieving the desired pharmaceutical profile. Improvement of the physical properties is a largely empirical process, with some generally applicable trends: inclusion of hydrophilic and charged residues, replacement of unnecessary hydrophobic regions, and modulation of the isoelectric point to ensure sufficient charge at the desired formulation pH. A recent example demonstrated enhanced solubility of a glucagon analogue by the replacement of natural, hydrophobic, aromatic residues with 3- and 4-pyridylalanine as effective bioisosteres.151

Figure 32. Structure of 3.

was developed as a single polypharmacological agent.153 Structurally, 3 is a cyclic peptide containing a branching Nterminal lysine that has both amines capped with a thioetherlinked ergoline-based small-molecule dopamine agonist (Figure 32). Conjugates in this series retained high affinity to somatostatin receptors-2 and -5 and dopamine receptor D2 (0.03, 42, and 15 nM, respectively, for 3) and demonstrated robust GH suppression superior to an equimolar combination of three monospecific agonists in human ex vivo preclinical studies. However, Ipsen’s preliminary phase IIb data for 3 found only weak somatostatinergic activity in acromegaly patients, and the program was discontinued.154 CovX’s development of peptide−antibody fusion protein 4 (CVX-241155) exemplifies both the first and second conjugation strategies listed above; dual pharmacology through the use of a pair of antagonist peptides targeting VEGF and Ang-2. respectively, and conjugation of both to a scaffold antibody (or CovX-Body) in order to confer increased half-life.155 In this example, the pharmacologically silent antibody acts to reduce plasma clearance by both preventing glomerular filtration (due

7. PEPTIDE CONJUGATES Many of the inherent properties of peptides make them attractive candidates for use as drug conjugates, including facile synthesis, high specificity and affinity, narrow pharmacokinetic distribution, and short half-life. The approaches that have received the most interest in drug discovery can readily be classified by strategic intent: • A pharmacologically active peptide conjugated to another active molecule T



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Figure 33. Structures of (a) zoptarelin doxorubicin and (b) 90Y-edotreotide.

liabilities resulting from cellular accumulation and immunogenicity.164 Consequently, there is growing interest in biopolymer analogues such as XTEN165 (including a GLP-1 agonist now known as NB-1001166) or PASylation167 (proline−alanine− serine polymer) that have the additional advantages of recombinant production and monodispersity. Overall, conjugation approaches for half-life extension have proven highly effective in delivering drugs with both compliance-friendly dosing frequencies and continuous target coverage for enhancing efficacy. There are, however, significant optimization challenges, chiefly in retaining the pharmacological activity of the peptide while bound in vivo (covalently or not) to the macromolecule. To take maximal advantage of the multiweek plasma retention offered by these conjugation approaches, the active peptide must also be exceptionally stable to enzymatic degradation. The third distinct conjugation strategy defined above uses the availability of specific high-affinity peptides to target a molecule of interest to a specific receptor or protein in vivo. This encompasses the peptide analogues of antibody−drug conjugates, widely used in the oncology field to confine the powerful cytotoxicity of many small-molecule chemotherapeutics to ideally just the tumor cells, thus substantially mitigating the severe side effects that conventionally afflict these patients. Zoptarelin doxorubicin (AEterna Zentaris) uses a peptide luteinizing hormone-releasing hormone (LHRH) receptor agonist to bind, internalize, and deliver a conjugated anthracycline DNA-intercalating cytotoxic agent (Figure 33a).168 This receptor is aberrantly present on the cell surface of a various tumor types, and the conjugate drug is currently being evaluated in a phase III trial for endometrial cancer.169 In an adjacent approach, 90Y-edotreotide (Molecular Insight) is a targeted radiotherapeutic, comprising a chelate-conjugated somatostatin analogue complexed to a high-energy betaemitting yttrium-90 atom (Figure 33b).170 A phase I trial is underway in a variety of somatostatin-receptor-positive solid tumors.171 Peptide-targeting can also be a useful diagnostic tool, exemplified by Blaze Bioscience’s innovative “Tumor Paint” conjugate BLZ-100,172 which uses a scorpion chlorotoxin peptide with high affinity for tumor-specific ion channels to label these cells with a near-infrared dye.172 This conjugate is currently in phase I clinical trials to assist in the complete and precise surgical excision of various tumor types through realtime visualization.173 The specific targeting of integrins by “RGD” peptides, a therapeutic strategy that with Merck KGaA’s cilengitide has recently stalled in the clinic,174 is widely applied

to increased size) and preventing lysosomal degradation following nonspecific vascular endocytosis (due to the FcRnbinding recycling mechanism). A likely additional beneficial effect is reduction of enzymatic degradation of the peptides by steric occlusion of the plasma proteases. The dual targets of 4 are both established pro-angiogenic pathways in tumors, and evidence suggests simultaneous inhibition should produce an additive or synergistic effect. CovX-Bodies overcome the difficulties of site-specific conjugation to antibodies by the engineering of a single highly nucleophilic lysine in each of the Fab arms, which reacts with azetidinone-containing linkers in order to rapidly generate small-molecule and peptide conjugates in various possible combinations. Unfortunately, as with the Ipsen case above, the dual pharmacology concept did not translate into the clinic; a phase 1 trial in patients with advanced solid tumors was discontinued due to poor efficacy and an unexpectedly short half-life.156 While conceptually attractive, these polypharmacological conjugate approaches represent challenging discovery programs that must simultaneously deliver two or three diverse biological effects to achieve success. Many alternative conjugation approaches have been explored for peptide half-life extension, and the vibrant slate of GLP-1 agonists in clinical development for type 2 diabetes serves as an effective microcosm of the broader peptide field. The antibody conjugation approach has been successfully exploited here with Lilly’s dulaglutide,157 but albumin binding is a more common and conceptually analogous strategy, similarly able to prevent glomerular filtration and take advantage of FcRn recycling.158 Albumin has a plasma half-life in humans of ca. 20 days, potentially allowing weekly-to-monthly dosing of the conjugate. GLP-1 analogues have been covalently attached to albumin through either recombinant fusion (GSK’s albiglutide)159 or through the lone reactive cysteine-34 (Conjuchem’s CJC1131160). Alternatively, conjugation of GLP-1 analogues to an albumin-binding motif can achieve a similar pharmacokinetic effect, as the high albumin plasma concentration (600 μM) results in very low levels of free conjugate even in examples with only a moderate binding affinity. These motifs can range from simple fatty acids (as in Novo Nordisk’s semaglutide)161 to recombinant antibody domains (GSK’s exendin-AlbudAb GSK2374697162). Finally, conjugation of the active peptide to a large, inert, polymeric ballast is again effective in minimizing renal clearance. PEG is the most widely used polymer across the peptide field and in marketed drugs,163 yet its application currently divides opinions due to concerns over potential U



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Figure 34. Structure and sequence of NA-1.

efficacy and alter the PK profile.190 As peptide drug discovery increasingly moves toward small proteins and thus into canonically immunogenic molecular space, this becomes an important criterion for the preclinical derisking of candidates. The complexity and variability of the immunization process is beyond the scope of this review, and attempts to delineate the critical structural factors have failed to provide generally applicable guidelines for the design of nonimmunogenic peptides and proteins; indeed, many axioms have subsequently been disproven or disputed:191,192 • First, shorter peptides are not always poor immunogens. Roche and Ipsen’s projected GLP-1 blockbuster taspoglutide failed in phase III with an antibody response in almost half of patients193 and exemplifies the apparent arbitrariness of immunogenicity by sharing almost the entirety of its structure with successful competitor GLP-1 analogues that avoided a similar outcome. Conversely, a mild antibody response did not prevent the GLP-1 species homologue exenatide from achieving market success.192 • Second, although self-derived therapeutics are not necessarily excluded due to central immune tolerance, this theory has also shaped the GLP-1 peptide field, with Novo Nordisk’s successful development of liraglutide and semaglutide centering on the design criterion to remain as similar to native GLP-1 as possible to avoid immunogenicity.161 The critical hazard facing this strategy is the potential for antidrug antibodies that cross react with the natural ligand, potentially exacerbating the target disease or causing serious side effects.194 • From the opposing angle, there is also debate over whether unnatural peptides can be proteolytically processed and presented as antigens in the first steps of the immune response.195 Examples of all-D peptides have been separately reported to be immunogenically silent and active.196 There has however been significant innovation and success in recent years in developing and validating a hierarchy of empirical immunogenicity assays and models that have varying levels of complexity and predictive power.197 Binding of peptide to the major histocompatibility complex (MHC) is the very first step in the process and can be determined either in vitro or increasingly in silico, providing a relatively cheap and high throughput screening tool.192 These methods inevitably result in a high false positive rate, which the most useful current assays minimize by moving downstream to measure the subsequent CD4+ T-cell activation ex vivo in donor peripheral blood mononuclear cells, albeit with added cost and lower throughput.191,192 A leading example of this technology is Antitope’s EpiScreen, which has demonstrated a good

for diagnostic purposes, including PET imaging of tumor angiogenesis using conjugates containing 18F or 68Ga.175 Future discovery of new targeting peptides may be significantly enhanced by in vivo phage display approaches, where entire phage libraries are administered to animals or humans, and biopsies of the target tissue reveal accumulation of specific sequences of interest.176 The fourth and final conjugation strategy considered here is the use of cell-penetrating peptides (CPPs) to deliver covalently bound active molecules, including large proteins and nucleic acids, to their intracellular targets.177 The majority of research in this large and active field uses amphipathic or polycationic peptides such as TAT, where the membrane translocation mechanism is poorly understood and likely occurs through multiple pathways.178 In general, these CPPs are nonspecific, require high extracellular concentrations (>5 μM), and lead to the cytoplasmic delivery of only a small percentage of their cargo. Nonetheless, a growing set of CPPs have demonstrated clinical efficacy;179 NoNO Inc.’s NA-1180 (TAT conjugated to a peptidic inhibitor of NMDAR-pDP95 (Figure 34)) was recently found to be neuroprotective in a phase II trial of subarachnoid hemorrhage.180 Diverse CPP structures and strategies are currently under preclinical investigation, and evidence of targeted and high-yielding cytoplasmic delivery using submicromolar extracellular concentrations will be of broad interest to the drug discovery community.181 The synthetic challenges in pursuing a conjugation strategy have undoubtedly been eased by the growing arsenal of bioorthogonal chemical techniques that allow controlled and efficient conjugation of unprotected peptides to their chimeric partners. Where both components are chemically synthesized, the alkene−azide Huisgen cycloaddition remains the most reliable and valuable approach.182 Where one or both partners are recombinantly produced, there is now a remarkable toolbox of powerful methods,183 including next-generation reagents for cysteine alkylation,184 oxidation/oxime ligation,185 sequenceencoded reactivity,186 sortase187 and ligase tags (see section 3.4), inteins,188 and genetic encoding of unnatural functionalized amino acids.16,189 These advances should accelerate the discovery, optimization, and preclinical evaluation of peptide conjugates and ultimately increase the number of bifunctional therapeutics entering clinical development.

8. PREDICTING AND PREVENTING IMMUNOGENICITY The potential for peptides and proteins to elicit an immunogenic response has historically existed as a capricious specter haunting drug development scientists, intangible to their optimization process and often materializing only in latestage clinical trials. Production of antidrug antibodies is the most common manifestation and can significantly reduce drug V



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and cargo peptide and usually demonstrate an initial “burst phase” corresponding to the release of surface-absorbed or solvent-accessible peptide; in some contexts, this has been associated with adverse events.205 Many subsequent approaches have targeted tighter control of peptide release, including the incorporation of vitamin-E into the hydrophilic polymers to create dense hydrophobic domains that more effectively trap the peptides.206 Direct drug−polymer conjugation allows the strictest control of peptide pharmacokinetics, particularly in combination with a highly predictable slow-release mechanism;207 ProLynx’s technology uses a tunable β-elimination motif to effect consistent drug release dependent only on the pH of the injection site, reaching theoretical half-lives of over a year.208 An alternative approach to depot formation is self-assembly of the peptide itself. Ferring’s GnRH agonist degarelix demonstrates a remarkable subcutaneous half-life that was found to derive from the rapid formation and slow dissolution of fibril structures.209 Ipsen made a similar serendipitous discovery for somatostatin-analogue lanreotide; under specific conditions, the peptide forms helicoidal nanotubes that proved highly effective as a subcutaneous depot termed Autogel.210 The sustained release allowed monthly dosing, which had not proved possible with a PLGA formulation. A potentially general approach from PhaseBio has demonstrated that self-assembly can be a transferable property, conjugating an optimized elastin domain to therapeutic peptides and retaining precise control over the gelling properties. By selecting a gel-phase transition at slightly below physiological temperatures, these conjugates can be injected in solution only to immediately form gel depots in the subcutaneous space.211 There is also growing interest in technological solutions such as implantable osmotic pumps, which offer controllable zeroorder release kinetics for up to a year. These devices use an osmotic gradient to draw in water into one chamber, incrementally driving a piston to expel the drug from an adjacent reservoir of limited capacity.212 Similarly, a range of wearable or implanted electronic pumps are also at various stages of development. The consistency and guaranteed compliance is of course significantly offset by the inconvenience of the surgical procedures required for both the initial installation and subsequent reservoir replenishment or replacement.213 These technologies have therefore seen the greatest interest from the diabetes field, where long-term, precise, and reliable delivery of small doses of potent hormones is a necessity. The most advanced electronic devices in this field act as an artificial pancreas, incorporating continuous glucose monitoring of the patient to automatically direct a pair of infusion pumps to deliver the appropriate dose of either insulin or glucagon as required, thereby preventing both hyper- and hypoglycemia.214 It is of course oral delivery of peptides that has long been sought after as a truly transformative development for the industry, allowing effective competition with small molecules and access to indications where the expected patient compliance with repeated injections is low. The combined proteolytic and absorptive physiological barriers constitute a powerful defense designed to prevent foreign proteins from entering the body, and both must be evaded to achieve systemic bioavailability.215 The remainder of this section explores the critical elements required through selected examples of marketed oral peptides, those in clinical development, and exciting new technological approaches.

correlation between T-cell activation and clinically observed immunogenicity198 and represents the current industry standard for preclinical assessment. Alternative versions of these assays use epitope mapping of larger peptides to identify the sequences that either contribute to MHC affinity or contact the T-cell receptors.199 This information greatly assists the process of deimmunization, where the candidate can ideally be optimized to retain activity with minimal immunogenicity by the judicious alteration of a few key residues. It has also been demonstrated that shielding the critical region by glycosylation200 or PEG201 conjugation can prove effective at reducing immunogenicity. Looking forward, the development of lowcost, predictive in vivo models is the focus of much research, and recent advances point toward a general model using mice that have been transplanted with a fully functional human adaptive immune system.202 The rapid progress in this area is demystifying and disarming the immunogenic response as a source of late-stage risk and should lead to increased clinical success for peptides and proteins alike.

9. PEPTIDE FORMULATION AND DELIVERY This final section will discuss recent advances in peptide formulation and delivery and by necessity only briefly touch on a number of large, vibrant fields of research, each of which has deservedly warranted many comprehensive reviews.203 Parenteral administration is the canonical delivery method for therapeutic peptides, physically piercing the membrane barriers through which peptides are very poorly absorbed. However, further challenges remain, pre- and postinjection, in achieving both the desired pharmacokinetic profile and high patient compliance. One critical decision is the choice of dosage form: liquid formulation or solid powder. Both share requirements of aqueous solubility and low solution viscosity to allow the use of minimal volumes and narrow-bore “pain-free” needles. Liquid formulations are preferred for subcutaneously self-administered drugs, averting the need for patients to perform a reconstitution step immediately prior to injection yet have the additional obligation of multiyear solution stability. Classical formulation optimization of pH, buffer composition, and additives can in many cases achieve this goal but may be rendered unnecessary by recent technological advances in dual-chamber “mixing pens” that combine patient-friendly reconstitution with highly stable solid powder products.204 The majority of peptide formulation and delivery platforms have been founded with the goal of extending the circulating half-life of the therapeutic peptide, and section 7 discussed albumin and other discrete conjugation approaches that essentially result in a long-acting plasma depot. It is important to note the fundamental difference between a true formulation approach and those examples above and below that involve conjugation to the therapeutic peptide and the concomitant creation of a new chemical entity that must face the full gauntlet of regulatory barriers. Clinical and market success has been achieved using slow-release subcutaneous polymeric depot technologies, in particular suspensions of PLGA (poly(lactic-coglycolic acid)) microspheres,205 with Abbvie’s leuprolide formulation Lupron Depot as the prototypical example approved nearly 30 years ago. PLGA polymers offer proven safety and biocompatibility, predictable degradation, and sufficient encapsulative capacity for 3−6 month dosing frequencies. The release kinetics of PLGA depots depend heavily on the precise conditions of manufacture, composition, W



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built chemical reaction to propel an array of peptide-coated sugar microneedles through the intestinal wall and has achieved bioavailability values comparable to the analogous subcutaneous injection.226 Also successfully used for the delivery of insulin,227 oral microneedle approaches have been demonstrated to cause minimal tissue damage,227 and clinical success of this approach could open an interesting new frontier in peptide delivery. A final route of interest is nasal/pulmonary delivery, which exhibits the twin advantages of a comparatively thin, porous endothelial barrier and high vascularization; the rapid absorption results in an early Tmax comparable to subcutaneous delivery.228 Desmopressin’s bioavailability increases 20−50-fold over oral administration when delivered as a nasal spray to 3− 5%.229,230 The pulmonary route has proven an area of significant interest and controversy in the insulin field, where a variety of companies and technologies are vying to offer what has been a much-anticipated noninvasive alternative product.231 However, despite achieving bioavailability values of over 10%, the market failure of Exubera (Pfizer),232 and market troubles of Afrezza (Sanofi/Mannkind)233 for a complex blend of reasons demonstrate the difficulties in predicting success and compliance, even where the route of administration is theoretically more attractive.234 As with oral dosing, both patient variation in bioavailability and the impact of chronic delivery to sensitive mucosa are significant concerns.230

Ferring’s desmopressin, an analogue of the antidiuretic hormone vasopressin, is administered orally despite a bioavailability of just 0.1%. In this example, the low intrinsic absorption is sufficient to achieve therapeutic doses due to the very high picomolar potency.216 In evading the proteolytic barrier, desmopressin exemplifies some of the basic strategies also discussed in section 6 for improving plasma stability: cyclization, modification of the termini, and conversion of a labile residue to the D-enantiomer. As referenced in that section and extensively covered in a recent review,217 there is renewed interest in general approaches to the discovery and design of membrane-permeable (and thus orally bioavailable) cyclic peptides, using N-methylation and lipophilic side chains to effectively raise the log P to the required level. It remains to be seen whether the resulting permeable peptides will successfully acquire the desirable molecular properties of small molecule drugs without gaining the pharmacological and solubility liabilities that correlate with increased lipophilicity;218 a more elegant approach temporarily shields peptidic hydrophilicity via intramolecular hydrogen-bonding, yet it may be difficult to apply this as a general strategy when hydrophilic side chains or multiple backbone interactions are often critical for biological activity.219 Of course, minimal oral bioavailability can be a desired feature for drug targets in the intestinal lumen. As previously mentioned, linaclotide is an agonist analogue of guanylin that targets GC-C in the colon, stimulating the secretion of water and thus alleviating constipation, with undetectable systemic exposure. Linaclotide’s compact structure ensures improve protease stability, rigidified by three disulfide bonds within the 14-mer sequence.220 Looking to the current slate of peptides in clinical development,221 the oral formulation of Novo Nordisk’s GLP-1 analogue semaglutide exemplifies the current favored technological approach and has the potential to make a considerable impact on the peptide field. This peptide was developed as a long-acting follow-up to Novo’s current blockbuster liraglutide, addressing a known proteolytic liability and optimizing a conjugated albumin-binding fatty acid in order to achieve a plasma half-life sufficient for weekly subcutaneous administration.161 Novo then surprised the field by immediately by following up these successful diabetes trials with an oral formulation of this linear 31-mer peptide, encapsulated with Emisphere’s proprietary technology.222 A range of specialist companies have developed oral peptide formulations using similar combinations of enteric coatings, nonspecific protease inhibitors and, most crucially, permeation enhancers,223 in this case the sodium caprylate analogue SNAC. Despite concerns over epithelial damage and the effect of any long-term increase in intestinal permeability, SNAC has a very high no-observedeffect-level in rodents and primates and is already marketed for delivery of vitamin B12.224 Phase III trials for oral semaglutide are ongoing, and Novo have reported comparable efficacy to the weekly 1 mg subcutaneous dosing with a daily 20−40 mg oral dose and no new safety signals.225 Successful realization of oral semaglutide’s immense market potential over the next decade would demonstrate both the biological and economic feasibility of oral administration of long synthetic peptides and likely spur significant interest and investment in related approaches. The future of oral peptide delivery may ultimately involve cross-disciplinary technological solutions that can invoke themes of medical science fiction yet represent relatively simple engineering challenges. Rani’s “robotic pill” uses an in-

10. SUMMARY The field of peptide drug discovery has never been more active nor had a more promising future, as evidenced by the vast array of new technologies that are being employed to facilitate the discovery of new peptide-based drugs, addressing a wide variety of novel biological targets in many therapeutic areas. We hope that this review has provided a perspective for the reader to appreciate the resurgence that this field has undergone during the past decade. The ultimate measure of success in this field remains, however, the number and impact of peptide drugs approved. Drug development is a long, complicated process, with a cumulative clinical success rate of ca. 10%,235 yet with the dramatic increase in new peptide therapeutics entering the clinic in the past decade, we can confidently predict that the number of approved peptide drugs will increase accordingly, providing patients with innovative new treatments across a wide range of therapeutic areas.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (858)-337-0162. E-mail: [email protected]. ORCID

John M. Nuss: 0000-0003-1213-3233 Author Contributions †

A.H. and J.C.C. contributed equally.

Notes

The authors declare no competing financial interest. Biographies Antoine Henninot obtained his M.Sc. at the Université de Lille 1 in 2011 and his Pharm.D. at the Université de Lille 2 in 2012. He stayed on to complete his Ph.D. (2015) in medicinal chemistry and chemical biology, under the supervision of Prof. Benoit Deprez and in collaboration with LFB Biotechnologie, working on identifying modulators of antibody post-translational modification. He joined X



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Research: Albany, NY, 2012; http://www. transparencymarketresearch.com/peptide-therapeutics-market.html. (9) (a) Fosgerau, K.; Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discovery Today 2015, 20, 122−128. (b) Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D. The future of peptidebased drugs. Chem. Biol. Drug Des. 2013, 81, 136−147. (c) Dunn, B. M. Peptide Chemistry and Drug Design; John Wiley & Sons: Hoboken, NJ, 2015. (10) Di, L. Strategic approaches to optimizing peptide ADME properties. AAPS J. 2015, 17, 134−143. (11) Smith, A. J. New horizons in therapeutic antibody discovery: opportunities and challenges versus small-molecule therapeutics. J. Biomol. Screening 2015, 20, 437−453. (12) Salemi, S.; Markovic, M.; Martini, G.; D’Amelio, R. The expanding role of therapeutic antibodies. Int. Rev. Immunol. 2015, 34, 202−264. (13) Kaspar, A. A.; Reichert, J. M. Future directions for peptide therapeutics development. Drug Discovery Today 2013, 18, 807−817. (14) Diao, L.; Meibohm, B. Pharmacokinetics and pharmacokineticpharmacodynamic correlations of therapeutic peptides. Clin. Pharmacokinet. 2013, 52, 855−868. (15) Lax, R. The future of peptide development in the pharmaceutical industry. PharManufacturing: Int. Pept. Rev. 2010, 10−15. (16) Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 2014, 83, 379−408. (17) Maini, R.; Umemoto, S.; Suga, H. Ribosome-mediated synthesis of natural product-like peptides via cell-free translation. Curr. Opin. Chem. Biol. 2016, 34, 44−52. (18) (a) Malyshev, D. A.; Romesberg, F. E. The expanded genetic alphabet. Angew. Chem., Int. Ed. 2015, 54, 11930−11944. (b) Chen, T.; Hongdilokkul, N.; Liu, Z.; Thirunavukarasu, D.; Romesberg, F. E. The expanding world of DNA and RNA. Curr. Opin. Chem. Biol. 2016, 34, 80−87. (19) (a) Fang, Y.; Kenakin, T.; Liu, C. Editorial: Orphan GPCRs as emerging drug targets. Front. Pharmacol. 2015, 6, 295. (b) Fillmore, D. It's a GPCR world. Mod. Drug Discovery 2004, 7, 24−28. (20) (a) Stockert, J. A.; Devi, L. A. Advancements in therapeutically targeting orphan GPCRs. Front. Pharmacol. 2015, 6, 100. (b) Ngo, T.; Kufareva, I.; Coleman, J. L.; Graham, R. M.; Abagyan, R.; Smith, N. J. Identifying ligands at orphan GPCRs: current status using structurebased approaches. Br. J. Pharmacol. 2016, 173, 2934−2951. (c) Tang, X.-l.; Wang, Y.; Li, D.-l.; Luo, J.; Liu, M.-y. Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets. Acta Pharmacol. Sin. 2012, 33, 363−371. (21) (a) Diller, D. J. The synergy between combinatorial chemistry and high-throughput screening. Curr. Opin. Drug Discovery Dev. 2008, 11, 346−355. (b) Kennedy, J. P.; Williams, L.; Bridges, T. M.; Daniels, R. N.; Weaver, D.; Lindsley, C. W. Application of combinatorial chemistry science on modern drug discovery. J. Comb. Chem. 2008, 10, 345−354. (c) Bononi, F. C.; Luyt, L. G. Synthesis and cell-based screening of one-bead-one-compound peptide libraries. In Peptide Libraries: Methods and Protocols; Springer: New York, 2015; pp 223− 237. (22) Romanova, E. V.; Sweedler, J. V. Peptidomics for the discovery and characterization of neuropeptides and hormones. Trends Pharmacol. Sci. 2015, 36, 579−586. (23) (a) Calcott, M. J.; Ackerley, D. F. Genetic manipulation of nonribosomal peptide synthetases to generate novel bioactive peptide products. Biotechnol. Lett. 2014, 36, 2407−2416. (b) Walsh, C. T. Insights into the chemical logic and enzymatic machinery of NRPS assembly lines. Nat. Prod. Rep. 2016, 33, 127−135. (24) Jones, L. H.; Price, D. A. Medicinal chemistry of glucagon-like peptide receptor agonists. Prog. Med. Chem. 2013, 52, 45−96. (25) (a) Miller, L. J.; Sexton, P. M.; Dong, M.; Harikumar, K. G. The class B G-protein-coupled GLP-1 receptor: an important target for the treatment of type-2 diabetes mellitus. Int. J. Obes. 2014, 4, S9−S13. (b) Christensen, M.; Miossec, P.; Larsen, B. D.; Werner, U.; Knop, F. K. The design and discovery of lixisenatide for the treatment of type 2

the Chemistry department at Ferring Research Institute in San Diego as a postdoctoral researcher in 2013 before transitioning to Scientist. James C. Collins obtained his M.Sc. (2006) and Ph.D. (2011) in organic and medicinal chemistry at Imperial College London under the supervision of Prof. Alan Armstrong, focusing on phosphatase inhibitors. He carried out postdoctoral research at The Scripps Research Institute in La Jolla, on macrocyclizations with Prof. Keith James, and on glycosylated vancomycin analogues with Prof. Dale Boger, before taking up his current position in the Chemistry department at Ferring Research Institute in San Diego in 2013. John M. Nuss obtained his B.S. in chemistry from the University of Kansas and his Ph.D. in organic chemistry at the University of Wisconsin, working in the laboratory of Prof. Howard Zimmerman. Following an NIH postdoctoral fellowship at Stanford University with Prof. Paul Wender, he joined the faculty of UC Riverside. After moving to the pharmaceutical industry in 1994, he has led chemistry and discovery groups at Chiron, Exelixis, and Ferring Research Institute that have been responsible for the discovery of two marketed drugs, Cabozantinib (Cometriq/Cabometyx) and Cobimetinib (Cotellic) as well as more than 25 other clinical candidates. He is currently CSO of Oppilan Pharma, Encinitas, CA.

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ACKNOWLEDGMENTS We are grateful to Dr. Jolene Lau at Ferring Research Institute for her contributions to this article. ABBREVIATIONS USED aa, amino acid; Ang, angiotensin; AT1R, angiotensin II type 1 receptor; CPP, cell-penetrating peptides; GLP-1, glucagon-like peptide 1; GnRH, gondotrophin releasing hormone; GPCR, Gprotein coupled receptor; Epe, 3-ethyl-3-pentyl; NGS, nextgeneration sequencing; NRSP, nonribosomally synthesized peptides; MHC, major histocompatibility complex; PASylation, proline-alanine-serine polymer; PTH, parathyroid hormone; RP, reverse phase; SEPs, smORF-encoded polypeptides; smORF, small open reading frames; SPPS, solid-phase peptide synthesis; TCEP, tris(2-carboxyethyl)phosphine

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