The Future of Peptide-based Drugs - Chem Biol Drug Des 2013; 81: 136-147
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Chem Biol Drug Des 2013; 81: 136–147
Special Issue-Review
Rational Design of Biologics and Peptides
The Future of Peptide-based Drugs
David J. Craik1,*, David P. Fairlie1, Spiros Liras2 of peptides in drug design, it is useful to briefly reflect on
and David Price2 the past, where traditionally peptides were not considered
as drug candidates. Table 1 summarizes some historical
1
Division of Chemistry & Structural Biology, Institute for trends in drug development technologies, targets and
Molecular Bioscience, The University of Queensland, product classes, as well as providing a broad overview of
Brisbane, Qld 4072, Australia the changing regulatory environment. We structure the dis-
2
CVMED Medicinal Chemistry, Pfizer Inc., Worldwide cussion in this section around the major eras of drug dis-
Medicinal Chemistry, 620 Memorial Drive, Cambridge, MA covery (chemistry, biologics, and genomics) to illustrate
02139, USA how they might provide insights into future trends.
*Corresponding author: David J. Craik,
d.craik@imb.uq.edu.au Most drug development in the 20th century can be classi-
fied as being in the ’chemistry era’, where leads were gen-
The suite of currently used drugs can be divided into erated from small molecule natural products (1), either
two categories – traditional ’small molecule’ drugs with based on screening, or rational design processes that
typical molecular weights of 5000 Da that are not orally bioavailable and need to the treatment of a wide range of diseases, although only
be delivered via injection. Due to their small size, con- approximately 20 new chemical entities (NCEs) have been
ventional small molecule drugs may suffer from
registered each year since 1980 (Annual Reports in Medic-
reduced target selectivity that often ultimately mani-
inal Chemistry 1980–2011) and this number has remained
fests in human side-effects, whereas protein therapeu-
tics tend to be exquisitely specific for their targets due fairly constant despite an increase in the number of drug
to many more interactions with them, but this comes targets discovered. Prospective analyses of the types of
at a cost of low bioavailability, poor membrane perme- compounds that were successful in the clinic led to broad
ability, and metabolic instability. The time has now generalizations that have subsequently been useful in
come to reinvestigate new drug leads that fit between guiding design of small molecular weight compounds. The
these two molecular weight extremes, with the goal of most widely known of these analyses, that is, the ’rule-of-
combining advantages of small molecules (cost, con- five’ (2) noted, among other criteria, a preference for a
formational restriction, membrane permeability, meta-
molecular weight of
Peptides in Drug Development
Table 1: Changing trends in drug design
Year Technological era Molecular classes and ⁄ or approaches Regulatory environment
1960 Chemistry Natural products, screening, rational design Activity paramount
?
?
?
?
?
1980 Molecular biology Biologics (insulin, growth factors, EPO) ?
?
2000 Genome & proteomics New target identification and validation y
2020 Peptide drugs? Personalized medicine Safety paramount
Plant factories? Increased specificity
Cheaper manufacture
drugs are generally referred to as ’biologics’ and include secreted due to regulation at the protein level. However,
molecules such as insulin, growth factors, and engineered the potential is clearly there to realize many new drug tar-
antibodies. These proteinaceous molecules disobey every gets over the next decade or two. Because this review is
one of the rule-of-five parameters and, not surprisingly, are future looking, we speculate that many of the new targets
generally not suitable for oral delivery. They typically from sequencing efforts are likely to involve large protein–
require injection (via subcutaneous, intramuscular or intra- protein interactions, featuring shallow interaction sites that
venous routes) or intranasal delivery. Nevertheless, biolo- span large surface areas. Traditionally, this target class
gics have been an extremely successful class of has not been very tractable for small molecule drugs, but
therapeutics, both economically and in treating certain dis- is potentially more suitable for peptides due to their larger
eases. Surprisingly, antibodies have been found to persist surfaces areas and suitability for array technologies (3).
in vivo often for weeks following a single administration Furthermore, along with genomics technologies, transcri-
and several antibodies and proteins are now in the ’block- ptomics and proteomics technologies are developing rap-
buster’ category, including adalimumab ⁄ Humira Pen and idly, becoming more heavily used and are likely to
etanercept ⁄ Enbrel, both for rheumatoid arthritis ($8b sales contribute to more widespread delineation of protein–pro-
in 2011), infliximab ⁄ Remicade for arthritis and ritux- tein interactions as targets in drug design. Thus, one pre-
imab ⁄ Rituxan for non-Hodgkin’s B-cell lymphoma ($7b dicted outcome of these new technologies is a greater
sales in 2011), bevacizumab ⁄ Avastin for colorectal cancer emphasis on proteins and peptides as prospective drugs.
and trastuzumab ⁄ Herceptin for breast cancer ($5–$6b
sales in 2011). There are other soluble proteins such as Another predicted outcome of ’omics’ studies in humans
insulin glargine ⁄ Lantus ($4.8b, insulin receptor for diabetes is a greater emphasis in the future on personalized medi-
type I and II), Neulasta ⁄ pegfilgrastim ($3.5b, G-CSF recep- cines based on identification of an individual’s genetic
tor for myelosuppression), Epogen ($2.5b, erythropoietin make-up. Interpatient variations in responses to drugs
receptor for renal anemia), and Avonex ($2.5b, interferon have long been observed, but only recently have the
beta receptor for multiple sclerosis). Among top selling genetic tools for understanding the mechanistic basis of
injectable peptides are the 10 amino acid immunomodula- these variations become available, allowing therapy to
tor Copaxone ⁄ glatiramer acetate ($3b) for multiple sclero- potentially be tailored accordingly. Again, peptides have
sis, the 9 aa gonadotropin receptor agonists great potential here, as they are components of the very
Lupron ⁄ leuprorelin ($1.5b) and Zoladex ⁄ goserelin ($1.1b) proteins that are differentially expressed between patients
for breast and prostate cancer, endometriosis and fibroids, and result in the different interpatient responses to drugs.
the 8 aa inhibitor of secretion of growth hormone and Peptides are also very amenable to site-specific modifica-
other hormones Sandostatin ⁄ octreotide ($1.3b) for acro- tion that might be used to tailor therapeutics to individual
megaly and cancer, and the two amino acid proteasome patients. Nevertheless, truly personalized medicine may be
inhibitor Velcade ⁄ bortezomib ($1.5b) for multiple myeloma. a lot further away than currently mooted for many practical
reasons.
Genome sequencing efforts that became possible at the
turn of the 21st century were originally touted as having One of the drivers of personalized medicine will be the
the potential to lead to a boom in drug development via need for a more targeted approach to therapy, both from
the identification of a vast range of new targets. Although efficacy and safety perspectives. This trend toward
genome technologies have indeed led to massive amounts demands for higher safety in therapeutic products is noted
of gene expression data, this has not yet translated into in Table 1 and has been responsible for the much longer
large numbers of new validated targets, largely because time frames now required from drug discovery to drug reg-
most contemporary drug targets are proteins and the field istration, due to increasingly more stringent safety stan-
of proteomics is still in relative infancy. High expression of dards imposed by regulatory authorities. This has altered
specific genes also often does not correlate with levels of the drug development landscape, bringing into favor more
corresponding proteins, which may not be expressed or biologics like antibodies that feature a common scaffold
Chem Biol Drug Des 2013; 81: 136–147 137Craik et al.
and that can realize higher earnings per unit volume. This
change in emphasis has been further catalyzed by some Peptides as Drugs
well-published failures among registered small molecule
drugs, including the withdrawal from the market of Vio- Some of the potential advantages and disadvantages of
xx for well-documented cardiovascular side-effects (4) peptides as drugs, compared to small molecule drugs, are
within a few years after its introduction for the treatment summarized in Table 2. A primary reason for interest in
of inflammatory conditions. An important reason for the peptides and proteins is that they bind with exquisite
increasing market share of biologics has been their specificity to their in vivo targets, resulting in exceptionally
much higher target specificity, which is largely driven by high potencies of action and relatively few off-target side-
their larger size than small molecule drugs. This enables effects. This high degree of selectivity in their interactions
many more, and much stronger, interactions and thus is the product of millions of years of evolutionary selection
less promiscuity in drug targeting. Balanced against this for complementary shapes and sizes from among a huge
major advantage are their disadvantages, such as their array of structural and functional diversity. Thus, peptides
lack of membrane permeability, poor oral bioavailability, have been fine-tuned to interact specifically with biological
and generally lower metabolic stability than small mole- targets, evolving into potent endogenous hormones,
cule drugs. growth factors, neurotransmitters and signaling molecules,
as well as immunologic and defense agents. In principle,
Following this introduction, we now turn to identifying gaps peptides and proteins could have many valuable applica-
in current therapeutic approaches that need to be filled in tions in medicine, but so far applications of chemically syn-
the future. The two main classes of successful drugs, that thesized peptides have been severely limited by their low
is, small molecules (5000 Da) systemic stability, high clearance, poor membrane perme-
clearly are separated by a significant gap in molecular ability, negligible activity when administered orally, and
weight that has not, as yet, been serviced by the pharma- their high costs of manufacture.
ceutical industry, as illustrated in Figure 1. Peptides offer
the potential of filling this gap and represent a class of Despite these generic disadvantages of peptides (and
molecules that have the specificity and potency of larger because of their advantages), more than 100 peptide-based
protein biologics, but are smaller in size and more accessi- drugs have already reached the market. Our analysis of
ble and cheaper to manufacture using chemical methods, structural data for a well-documented subset of these (6)
thus potentially combining some of the advantages of pro- shows that the majority of current peptide-based products
teins with those of small molecules. This article identifies a are at the smaller end of the size spectrum (8–10 amino
few selected classes of peptides that could be used to fill acids), although peptides up to 45 amino acids are currently
this gap, and in particular, we focus on the future potential on the market (Figure 2). Almost invariably, the current pep-
of cyclic peptides in the size range 5–50 amino acids. tide therapeutics are delivered via injection. In addition to the
Readers are referred to recent reviews of peptides in drug billion dollar peptide drugs listed above, some other exam-
design for coverage of the existing peptide pharmaceutical ples of success stories include the following: oxytocin (8 aa,
market (5,6) and to focused reviews (7–15) on specific labor), calcitonin (32 aa, hypercalcemia, osteoporosis), teri-
subclasses of therapeutic peptides. paratide (34 aa, parathyroid hormone analog, osteoporosis),
Fuzeon (36 aa, enfuvirtide, antiretroviral), corticotropin-
releasing hormone ⁄ factor (41 aa), and growth-hormone-
500 5000
Small 12 aa
MW (Da)
50 aa Biologics releasing hormone ⁄ factor (44 aa, lipodystrophy).
5 aa 20 aa >5000 Da
molecule
Mimics Grafts (eg Insulin,
drugs
$40 billion per year, or 10% of the ethical
pharmaceutical market. This market share is growing
much faster than that of other pharmaceuticals, and suc-
cess rates for bringing biologics to market are now about
OUTCOMES twice that of small molecule drugs. The paradigm shift in
of proteins but the stability and bioavailability
of small molecules interest by the pharmaceutical industry toward proteins is
exemplified by recent successes of recombinant proteins
Figure 1: Schematic illustration of the molecular weight (MW) gap (especially monoclonal antibodies) as blockbuster thera-
between conventional small molecule drugs (5000 Da). Peptides offer the potential to fill this gap if ways predictions for how they might compete with small mole-
of overcoming their intrinsically unfavorable biopharmaceutical cule drugs on the one hand, and biologics on the other,
properties can be developed. Two classes of peptides that are of
as well as with the range of other new technologies that
particular interest are mimics of secondary structure elements of
proteins as these are often involved in target interactions, and are described in other articles in this themed issue.
cyclic peptide scaffolds that can be used to accept a ’graft’ of a
bioactive peptide sequence, stabilizing it while retaining biological Exenatide (Byetta) is one of the most recent peptides to
activity. reach the market and is used in the treatment of type 2 dia-
138 Chem Biol Drug Des 2013; 81: 136–147Peptides in Drug Development
Table 2: Advantages and disadvantages of peptides as drugs One of the particular advantages of peptides from natural
sources, relative to small molecules from natural sources,
Advantages Disadvantages
is that peptides can be mined both directly (e.g., via phar-
High potency Poor metabolic stability macological screening and extraction from venoms, for
High selectivity Poor membrane permeability example), or detected indirectly from the encoded nucleic
Broad range of targets Poor oral bioavailability acids. A good example of this powerful combination of
Potentially lower toxicity than High production costs approaches is the CONCO project (http://www.conco.org),
small molecules designed to map the genome, transcriptome and prote-
Low accumulation in tissues Rapid clearance
ome of a venomous marine snail and potentially to exploit
High chemical and biological Sometimes poor solubility
diversity
the outcomes for human health. One outcome from the
Discoverable at peptide and ⁄ or project so far is a lead molecule, XEP-018, being devel-
nucleic acid levels oped for pain control and local anesthesia. We will return
to venoms with particular reference to disulfide-rich pep-
tides later, but first focus on mining peptides from a variety
betes (16). It is interesting to note that this peptide was of other natural sources, including bacteria, fungi and
derived originally from the saliva of a lizard, the Gila monster, plants, with the mining followed by chemical value-adding
thus illustrating that natural sources remain a powerful approaches to optimize biopharmaceutical properties.
source of peptide leads, just as they did for small molecule
drugs. This 39 amino acid peptide violates all rule-of-five
associated parameters (MW 4187, CLogP -21, HBD 58, Mine the Gap?
HBA 67, Rot bonds 135, tPSA 1780: MW = molecular
weight, HBD = hydrogen bond donors, HBA = hydrogen The 500–5000 Da gap shown in Figure 1 prompts the
bond acceptors, Rot bonds = rotatable bonds, tPSA = total question of ’do such molecules exist in nature’ and if so
polar surface area), but can be made orally active (F = 4%, why have they not been examined previously for their
mice) through biotinylation at Lys12 and Lys27 to enhance pharmaceutical potential. Of course, the answer is that
plasma protein binding (17,18). In fact, to date nearly all they do exist, and it has partly been the reluctance of the
orally active peptides have F = 0–5% oral bioavailability, pharmaceutical industry to engage in peptide-based drug
mainly at the low end of this scale. Nevertheless, exenatide development that has not seen this spectrum of molecular
has stimulated the development of several related mole- weight space more fully explored. But with the current
cules, just reaching the market or in clinical trials (16). changing environment, driven by the technological and
regulatory issues outlined in Table 1, the opportunity exists
We chose to feature exenatide as an example as it nicely to mine natural sources for bioactive peptides as drug
illustrates what we believe will be a continuing trend of ani- leads. In doing so, it is important to recognize the deficien-
mal venoms being a particularly rich source of peptide cies of peptides in terms of their biopharmaceutical prop-
leads. By their nature, venoms from predatory animals erties and perhaps design discovery approaches that will
must be potent and fast acting and require a degree of find molecules with intrinsically more favorable properties.
stability of their constituent peptides. These evolutionary
requirements have selected particular classes of peptides An early, and perhaps the best known, example of a natu-
that make excellent staring points in drug design (19). To ral peptide in this size range that has succeeded as a drug
illustrate this, Table 3 shows that there are currently six with a reasonable degree of oral bioavailability is cyclospo-
marketed compounds that are derived from animal ven- rin A (CSA) (20). Originally discovered (mined) for its anti-
oms (if we include Gila monster saliva as pseudovenom). fungal properties, it has been used widely as an
Of these, ziconotide (Prialt) was the first peptide-based immunosuppressant and contributed to revolutionizing
drug derived from a marine animal, but several others are organ transplant therapy. This 11-residue peptide has
in preclinical development. three key structural features that contribute to its favorable
Table 3: Examples of marketed peptide-based drugsa derived from animal venoms
Drug Size (aa) Target disease Pharmacological mechanism Year registered
Captopril (Capoten) 3 Hypertension ACE inhibitor 1982
Tirofiban (Aggrastat) 3 Anticoagulant Platelet inhibitor 1998
Epifibatide (Integrilin) 7 Acute coronary syndrome, Anticoagulant 1998
unstable angina
Bivalirudinrub (Angiomax) 20 Unstable angina Anticoagulant 2000
Ziconotide (Prialt) 26 Neuropathic pain N-type Ca channel blocker 2004
Exenatide (Byetta) 39 Type 2 diabetes GLP-1 receptor antagonist 2005
a
Includes examples derived from venom peptides but not actually peptides themselves.
Chem Biol Drug Des 2013; 81: 136–147 139Craik et al.
biopharmaceutical properties, namely its cyclic backbone using crystallographic and NMR techniques, there are a
that protects against proteolytic degradation and (along number of ’slowly inter-converting’ conformers that are in
with its hydrophobic side chains) buries polar groups in equilibrium in polar solvents such as DMSO, methanol or
the interior of the molecule, incorporation of seven N- methanol ⁄ water mixtures.
methyl groups that reduce the number of amide hydrogen
bond donors, and four intramolecular hydrogen bonds that A recent paper from the Lokey group (24) reported Com-
tie up the remaining four amide NH protons to reduce their pound 1, which has a physicochemical profile that would
hydrogen bonding potential for solvation by water. Future be viewed as lying outside of traditional oral drug space
mining of natural peptides will likely pay attention to with a molecular weight of >750 Da and multiple hydrogen
searching for compounds with similar attributes. bond donors and acceptors that upon initial inspection
would be expected to limit the ability of the compound to
A cyclic peptide backbone appears to be a particularly cross the gut wall. Compound 1 was profiled in a range of
valuable attribute, but is a difficult one to mine for, in vitro assays and progressed to an in vivo study, where
because MS-based proteomics studies are typically not its pharmacokinetic profile in rats was characterized by
good at sequencing cyclic peptides without prior lineariza- low clearance and a moderate ability to partition into tis-
tion. Furthermore, until recently, most natural cyclic pep- sue, leading to a terminal elimination half-life of 2.8 h. The
tides were thought to be biosynthesized via non-ribosomal absolute oral bioavailability (F) of 1 was determined to be
routes, as is the case for CSA, and this non-genetic route ~28%, an impressively high value for a peptide (24). It was
does not lend itself to screening at the nucleic acid level, the most bioavailable of a series of macrocycles designed
because no transcripts exist. However, over the last dec- with specific N-methylation patterns to simultaneously
ade, increasing numbers of ribosomally synthesized cyclic reduce the hydrogen bond donor count and to promote
peptides have been reported (21,22), thus opening up the intramolecular hydrogen bonding networks of the remain-
possibility in future of more rapidly detecting precursors ing protons and carbonyl lone pairs. These compounds
encoding cyclic peptides. Recent examples include a fam- were prepared as pharmacokinetic probes and had no
ily of ribosomally biosynthesized cyclic peptides from associated pharmacology described (24), but the study is
Caryophyllaceae plants that have the typical high stability nevertheless a seminal paper from an absorption stand-
of cyclic peptides (23). As noted above, having two possi- point. The ability to understand and predict the influence
ble approaches to mining is a definite advantage that pep- of intramolecular hydrogen bonding networks on cell per-
tides have over small molecule drugs, and even difficult meability is an exciting advance in the field.
(cyclic) peptides are now becoming amenable to two-
pronged mining approaches. Similarly, important advances in the understanding of
membrane permeability have come from the Kessler group
Just as the value of traditional mined products, like baux- (25), based on their earlier postulation that a combination
ite, iron ore or precious metals, is increased by subse- of macrocyclization and N-methylation of a peptide may
quent manufacturing (value-adding) steps, significant value be a general strategy to confer the combination of mem-
has been added to peptide-based drug design by recent brane permeability and resistance to proteolytic degrada-
studies from the Lokey and Kessler groups (24,25) that tion that is required to achieve oral bioavailability (28).
have examined the role of key structural features (e.g., Much of this thinking had also been influenced by the sys-
cyclization and N-methylation) on biopharmaceutical prop- tematic evaluation of CSA. Interestingly, CSA and the
erties, as well as earlier detailed analyses (26) linking the designed hexapeptide 1, as well as other related natural
effects of these and other conformational properties to products, are structurally populated with lipophilic amino
three dimensional structures and pharmacological activi-
ties. These studies provide valuable information on fea-
tures that enhance stability, cell permeability, and oral
O
bioavailability. For example, investigation of CSA and clo-
N
sely related analogs suggested that passive transcellular N
absorption is a characteristic of macrocycles that can H O
N
assume multiple, interconverting conformations with differ- O
O O N
ent populations in low and high dielectric environments H
(27). In the aqueous conditions of the gut, the conformer N
N OH
population will be different to the population in the low O
dielectric environment of the membrane interior. Thus,
membrane permeability is driven by a dominant conforma-
tion where intramolecular, transannular hydrogen bonding
Compound 1: An N-methylated cyclic peptide containing multiple
masks the overall hydrogen bonding potential and reduces hydrogen bond donors and acceptors, and a molecular weight
overall hydrophilicity, enabling CSA to cross the gut wall. >750 Da. Compound 1 was determined to have a terminal elimi-
The conformational flexibility of CSA has intrigued many nation half-life of 2.8 h and an absolute oral bioavailability of
laboratories, and in addition to the conformations identified ~28% (24).
140 Chem Biol Drug Des 2013; 81: 136–147Peptides in Drug Development
acid side chains, and it is worth noting that naturally a variety of experiments failed to demonstrate whether the
occurring, N-methylated cyclic peptides typically do not improvement was due to a transcellular transport mode or
contain charged and polar side chains. This identifies a an unexpected active transport mechanism. This research
possible design limitation in this space of peptidic macro- clearly demonstrates the evolution and development of
cycles. Yet, this observation suggests that there may be a robust in vitro transporter assays that need to take place
large unexplored opportunity for the design of peptidic in macrocyclic drug space to understand which in vitro
macrocycles with unnatural amino acids, which will provide systems can be used with greatest predictability of phar-
flexibility in testing a broad range of physicochemical prop- macokinetics in preclinical species or man.
erties such as logD and pKa. Thus, within this broad strat-
egy, the specific tactics that promote oral bioavailability Overall, these exemplar studies highlight the fact that the abil-
remain to be determined and it is clear that only a subset ity of chemists to design and synthesize peptides to explicitly
of this chemical space may be relevant. explore structure-property and structure-activity relationships
will be extremely valuable in developing the next generation
In other work from the Kessler group, a study on cyclic of peptide-based drug leads. In this process, medicinal
N-methylated somatostatin analogs related to the Veber– chemists will evolve to accommodate new skills and thinking.
Hirschmann peptide (Compound 2) generated a library of A sophisticated understanding of the synthetic challenges
30 compounds with varying degrees of methylation of the associated with peptides and peptide macrocycles, aided by
secondary amides contained in the starting macrocycle innovative methods for amino acid functional manipulation
(29). Extensive in vitro evaluation showed that specific and macrocyclization and enrichment of the pool of unnatural
methylation of D-Trp8, Lys9, and Ph11 gave rise to a large amino acids synthons, will be essential. In addition, research
enhancement in membrane permeability in a Caco-2 cell in this field will be clearly propelled by a deeper, physics-dri-
monolayer model, while other methylated derivatives gave ven understanding of conformation and its impact on perme-
no enhancement. This compound also displayed reason- ability, pharmacological activity, and overall disposition. A
able oral bioavailability in rat. The optimized peptide with greater understanding of ADME properties in this space will
regard to cell permeability was also the peptide most effi- be also essential. Physicochemical properties that affect per-
ciently cyclized using standard peptide coupling condi- meability, clearance and presumably influence elimination
tions. The authors suggested that the linear precursor routes are still not well understood. Similar to the small mole-
peptide exhibits a dynamic structure in solution while cule world, the creation of in silico and statistical models that
undergoing some preorganization to bring the N and C predict key ADME properties will be a major accelerator as
termini into close proximity to improve the efficiency of the well. Examples of the types of considerations that might
cyclization reaction. The optimized macrocycle showed no prove of further benefit in future medicinal ⁄ peptide chemistry
degradation after extensive incubation in rat serum and optimizations can be extrapolated from considering a pivotal
was stable in simulated gut media. Progression of the lead study by Veber et al. (30) who effectively extended Lipinski’s
analog to in vivo studies showed an oral bioavailability of rule-of-five after finding that reduced molecular flexibility [£10
F = 9% and a volume of distribution (VD) of 3.7 L ⁄ kg, with rotatable bonds (RotB), topological polar surface area
an elimination half-life of 74 min. The relatively high oral (tPSA < 140 Å2), and £12 hydrogen bond donors + accep-
bioavailability, and in particular the ability to cross the gut tors (HBD + HBA)] correlated with higher oral bioavailability
wall, was not fully explained and demonstrates the com- independent of molecular weight (MW) in 1100 small mole-
plexity of the pharmacokinetic profile. The high VD sug- cule drug candidates. Cell permeation increased with lower
gests that the compound can distribute effectively into polar surface area and fewer rotatable bonds but, surpris-
tissues and move out of the plasma compartment. The ingly, not always with higher lipophilicity (CLogP). It therefore
improvement in permeability was not fully understood, and seems reasonable to propose that peptides might be made
more orally bioavailable by: (i) conformationally constraining
them to reduce molecular flexibility, (ii) reducing solvent
Phe7 exposed HBD ⁄ HBA atoms by replacing them or masking
them with bulky groups, and (iii) forcing HBD ⁄ HBA atoms to
Trp8 hydrogen bond with one another. There has been no com-
O
Pro6 H prehensive or systematic evaluation of peptides of MW 500–
N 5000 Da to identify upper limits for each parameter (MW,
N
H RotB, HBA, HBD, PSA, ClogP) that determines cell perme-
N O O N
O O NH ability and oral bioavailability, and these studies are likely to
H H
N prove valuable feedback in future drug design efforts.
N NH2
Phe11 H Lys9
O
HO
Thr10 Bridging the Gap
Compound 2: Veber–Hirschmann peptide; MW 806.9 Da; The mining and structure-activity studies described in Sec-
CLogP 4.11. tion 3 are broadly applicable to a wide range of peptides
Chem Biol Drug Des 2013; 81: 136–147 141Craik et al.
but most emphasis to date has been on those smaller In the mimicry approach, beta strands have been identified
than 20 amino acids in size. This size range represents the as important structural elements recognized by enzymes
majority (75%) of existing marketed peptides (Figure 2), (e.g., proteases) and by other strands that form sheets
but in terms of future developments, one promising area (e.g., amyloids) and have been successfully mimicked by
will be to bridge the size gap up to 50 amino acids using both non-peptides and short constrained peptides. They
a variety of technologies, including mimicking elements of often contain cyclic tripeptides, heterocyclic or other
secondary structures in proteins (helices, turns, strands ⁄ - organic constraints inserted in a peptide sequence to
sheets) or grafting bioactive peptides onto stable scaffolds. maintain the peptide backbone in a linear saw-toothed
Both of these latter approaches offer the potential to capi- strand structure. The resulting constrained strand mimetics
talize on the largely untapped target space of protein–pro- (32) and sheet mimetics (31,41) are usually maintained in
tein interactions, through conformational constraints that this conformation when bound to proteins (42). Many have
reduce peptide flexibility, reduced peptide HBD ⁄ HBA shown highly potent and very selective enzyme inhibiting
atoms or reduced exposure to their solvation by water, or protein antagonizing properties, often leading to cell
and ⁄ or masking of HBD ⁄ HBA atoms by their hydrogen permeable and bioavailable compounds. Many are, how-
bonding with one another within a peptide. ever, usually small enough to match or only slightly exceed
the guiding rule-of-five parameters.
The first approach is based on the finding that many pro-
tein–protein interactions involve recognition of key ele- Helix mimics, in which a peptide is covalently constrained
ments of secondary structure, and these structural motifs (43) to adopt an alpha or other helical structure, have
can be mimicked in small, carefully designed, peptides to become possible by inserting (i) lactam (44,45) or hetero-
recapitulate protein-like biological activities. Examples cycle (46) bridges between amino acid side chains, (ii) a
include mimetics of beta strands ⁄ sheets (31,32), beta, hydrocarbon linker between amino acid side chains (47–
gamma and other turns (33), and alpha helices (34) (and 49), (iii) a carbon–carbon (50,51) or carbon–nitrogen (52)
references therein), and these are cartooned in Figure 3. bond replacement for an intramolecular hydrogen bond at
The second approach is based on discoveries of minipro- an end of a helix, or (iv) a metal ion clip (53,54). Downsiz-
tein scaffolds in nature that are particularly stable and ing proteins to bioactive, helix-constrained peptides has
amenable to sequence insertions, or ’grafting’ of bioactive afforded potent modulators of biological targets, including
peptides. Examples of these scaffolds include knottins transcription factors, oncoproteins like HDM2, viral fusion
(35,36) and cyclotides (7,37) from plants, lasso peptides helix bundles, BCL apoptosis proteins, GPCRs like ORL-1
from bacteria (38) and a variety of animal toxins, but most involved in pain transmission, Alzheimer’s notch protein,
notably the conotoxins (39,40) from marine cone snail ven- antibacterial pheromones, and HIV-binding RNA.
oms. The goal of this approach is to introduce a bioactive
peptide sequence into the stable framework, with the aim Turns are short or large loops, sometimes referred to as
of retaining the bioactivity of the target peptide, but reverse turns, in proteins and peptides, which have a ten-
enhancing its stability and bioavailability by capturing some dency to fold by forming intramolecular hydrogen bonds.
of the biopharmaceutical properties of the framework. Typically, such folding creates 10-membered (beta turns)
These two approaches are highly complementary, with the or 7-membered (gamma turns) hydrogen-bonded ’cycles’
mimicry approach optimized for peptides of up to 20 aa within a peptide sequence. These are classified by phi and
and the scaffolding approach bridging the gap up to the psi torsional angles as types I, I¢, II, II¢, III, III¢, IV–VI beta
50 aa entry point of conventional biologics. turns or classic and inverse gamma turns (55,56), although
there are also other types of turns in peptides. Beta turns
Turn Helix
Strand
Figure 2: Size distributions of peptide drugs on the market in
2010. The distribution reflects the sizes, in terms of number of
amino acids (aa) of 65 separate peptide products on the market
as reported by Vlieghe et al. (6). Note that, in some cases, differ- Figure 3: Elements of protein secondary structure successfully
ent derivatives of the same peptide are counted as separate prod- mimicked. The boxes indicate three common elements of second-
ucts if they are formulated differently, marketed by different ary structure discussed in the text, including helices (blue), strands
companies, or used for different disease indications. (yellow), and turns (green).
142 Chem Biol Drug Des 2013; 81: 136–147Peptides in Drug Development
are most common and defined by four, gamma turns by angiogenic agents for wound healing applications (68),
three, sequential residues in which the ends come into anti-angiogenic agents with the aim of reducing blood ves-
close proximity. Biological activity of short peptides can be sel growth in tumors (69), anti-infective agents against
enhanced by stabilizing such turns through cyclization foot-and-mouth disease virus (70), and orally active pep-
and ⁄ or incorporation of heterocyclic or organic constraints. tides against inflammatory pain (71). Although so far most
Resulting ’turn mimetics’ are designed either to preserve attention has been directed toward extracellular targets,
turn-defining phi and psi angles in peptide components or some of these frameworks offer the potential of delivering
to replace them altogether [reviewed in refs (33,57–60)]. bioactive epitopes to intracellular targets based on their
G-protein-coupled receptors have an especially striking ability to penetrate cells (72,73). This attribute gives these
tendency to recognize turns of protein ⁄ peptide ligands, peptides a distinct advantage over classic ’biologic’
and many agonists and antagonists have been developed agents, which are typically limited to extracellular targets.
based on mimicking such turns (61). Because turns and
loops account for >30% of protein structure, it is not sur- In some cases, disulfide-rich miniprotein scaffolds have
prising that peptide drugs have been developed around intrinsic activity that is of pharmaceutical relevance, as in
this concept of mimicking turns, especially the more pre- the case for example of MVIIA (ziconotide, Prialt) a 25
valent beta turns. amino acid cystine knot peptide from cone snail venom that
is used clinically for the treatment of neuropathic pain
By contrast with the protein surface mimic approach, the (74,75). Ziconotide is administered intrathecally, but the
scaffold approach to peptide-based drug design (7,62) gen- potential for oral delivery of conotoxin peptides was recently
erally involves larger peptides (20–50 aa) and two distinct realized with the development of an engineered cyclic ver-
regions of the resultant peptide can be identified – the core sion of another conotoxin, Vc1.1, that is orally active in a rat
scaffold and the inserted bioactive sequence, as illustrated model of neuropathic pain (76,77). This perhaps represents
in Figure 4. Disulfide-rich scaffolds have proven to be partic- a prophetic case for future developments where the lessons
ularly valuable and have applications not only as drugs, but learned from naturally stable cyclic peptides such as the
as pharmacological probes (39) or imaging agents (63–66). plant cyclotides can be used to re-engineer bioactive pep-
In keeping with the theme noted above of animal venoms tides to enhance their stability and oral activity.
and plant toxins being a source of stable disulfide-rich scaf-
folds, we will focus mainly on this class here.
Future Directions
In the first such example to produce a therapeutic lead,
Vita et al. (67) used a scorpion toxin scaffold to design a One important area for the future is the need to consider
grafted molecule that inhibited the CD4-gp120 interaction new sources of lead materials for drug development. Yeast
associated with HIV infection. In more recent studies, plant cell surface display methods, ribosomal and other biomi-
cyclotides have been used as frameworks to develop metic cyclic peptide synthesis methods all could be handy
A B C
Figure 4: Schematic illustration
of the miniprotein scaffold
approach to peptide-based drug
design. Panel A highlights poten-
tial sources of target epitopes,
from fragments of proteins, from
bioactive peptides, or from phage
display. Panel B schematically
illustrates a range of disulfide-rich
frameworks, including SFTI-1,
cyclotides and theta-defensins.
Panel C shows the bioactive
epitopes grafted into the stable
frameworks.
Chem Biol Drug Des 2013; 81: 136–147 143Craik et al.
in providing good starting points for hit discovery and opti- Research Council (NHMRC) Senior Principal Research Fel-
mization. lowships to DJC (APP1026501) and DPF (APP1027369).
Some key technical hurdles to the development of effec-
tive peptide-based therapeutics will need to be addressed Conflict of Interest
in the near future. First, the synthesis of small peptides
relies on expensive coupling reagents, resins and pro- DJC and DPF are inventors on patents relating to peptides
tected amino acids, so cheaper methods for their synthe- in drug development. SL and DP are employees of a phar-
sis and purification will need to be developed. This might maceutical company.
be achieved by either chemical synthesis or molecular biol-
ogy techniques (recombinant peptide expression). Second,
modifications will need to be devised for enhancing mem- References
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