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Current Topics in Medicinal Chemistry, 2013, 13, 821-836
821
Form and Function in Cyclic Peptide Natural Products: A Pharmacokinetic Perspective
Andrew T. Bockus, Cayla M. McEwen and R. Scott Lokey*
Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, USA
Abstract: The structural complexity of many natural products sets them apart from common synthetic drugs, allowing
them to access a biological target space that lies beyond the enzyme active sites and receptors targeted by conventional
small molecule drugs. Naturally occurring cyclic peptides, in particular, exhibit a wide variety of unusual and potent biological activities. Many of these compounds penetrate cells by passive diffusion and some, like the clinically important
drug cyclosporine A, are orally bioavailable. These natural products tend to have molecular weights and polar group
counts that put them outside the norm based on classic predictors of “drug-likeness”. Because of their size and complexity, cyclic peptides occupy a chemical “middle space” in drug discovery that may provide useful scaffolds for modulating
more challenging biological targets such as protein-protein interactions and allosteric binding sites. However, the relationship between structure and pharmacokinetic (PK) behavior, especially cell permeability and metabolic clearance, in cyclic
peptides has not been studied systematically, and the generality of cyclic peptides as orally bioavailable scaffolds remains
an open question. This review focuses on cyclic peptide natural products from a “structure-PK” perspective, outlining
what we know and don’t know about their properties in the hope of uncovering trends that might be useful in the design of
novel “rule-breaking” molecules.
Keywords: Cyclic peptides, Pharmacokinetics, Cell permeability, Lipinski’s rules, Cyclosporine A.
INTRODUCTION
High-throughput –omics technologies (e.g., shRNA and
expression profiling, deep sequencing, proteomics, metabolomics) have delivered vast datasets for biologists to
mine for new clues into the molecular basis of disease. One
result of these efforts has been the emergence of a new genseration of candidate drug targets. Unlike typical enzymes
and receptors, many of these potential targets are signaling
proteins and transcription factors whose functions are carried
out through protein-protein or protein-DNA interactions[1].
Since few of these biomolecules have endogenous lowmolecular weight ligands, finding traditional, drug-like small
molecules that can modulate their functions represents a major challenge ahead. Medicinal chemists are therefore
charged with the task of identifying scaffolds that are large
and complex enough to bind macromolecular interfaces and
non-canonical allosteric binding sites, but yet retain druglike membrane permeability and metabolic stability.
It is our view that cyclic peptides, especially those of
natural origin, offer strategic insights into this design problem. Many cyclic peptide natural products are biosynthesized
by non-ribosomal peptide synthetases (NRPSs), which, unlike the ribosome, often utilize as substrates both regular and
non-proteinogenic amino acids. NRPSs also often incorporate “tailoring” activities such as epimerization, Nmethylation, and heterocyclization[2], further increasing the
diversity of these natural products. And like other natural
*Address correspondence to this author at the Department of Chemistry and
Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064,
USA; Tel: 831-459-1307; Email: [email protected]
/13 $58.00+.00
products, especially those in the polyketide family, cyclic
peptides tend to have properties (e.g., MW, number of polar
atoms, total polar surface area) that put them outside conventional predictors of “drug-likeness” such as Lipinski’s “Rule
of Five”[3, 4]. Yet many of these compounds exhibit druglike properties, including the ability to penetrate biological
membranes. Some cyclic peptides, such as the important
immunosuppressive agent cyclosporine A (CSA), are in
clinical use as orally administered drugs.
Drug discovery often involves the iterative (or simultaneous) optimization of pharmacodynamics (PD) on the one
hand, related to target binding and specificity, and pharmacokinetics (PK) on the other, related to factors that impinge
on a molecule’s disposition and fate within the body, e.g.,
absorption, distribution, metabolism, and excretion (ADME).
Often the same structural changes that improve PD have a
negative impact on PK, and vice versa, driving the need for
reliable predictors in both arenas. For natural products, the
focus has historically centered on potency and specificity,
while PK is often taken for granted since most natural products were “designed” by natural selection to avoid metabolic
degradation and cross biological membranes, the same factors that impact PK in modern drug discovery.
In addition to membrane permeability, for a compound to
be orally bioavailable it must survive metabolism by proteases in the gut and first-pass gut and hepatic biliary excretion
and metabolism, especially by cytochrome P450 (CYP) enzymes[5]. The selectivity profiles of metabolic CYPs are
biased toward more hydrophobic compounds[6, 7], creating
another trade-off for the medicinal chemist to negotiate:
more hydrophobic compounds, while they may be more
© 2013 Bentham Science Publishers
822 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
membrane permeable, tend to be cleared more quickly by
CYPs in the liver, whereas compounds that are hydrophilic
enough to survive clearance may not be permeable enough to
reach circulation in the first place. As with permeability,
QSAR models of clearance are generally based on training
sets comprised of typical small molecules[7], and so predicting the clearance of large, “non-Lipinski” molecules remains
a challenge.
Cyclization in peptides eliminates charged termini, which
can favor both membrane permeability[8] and metabolic
stability[9]. Cyclization is also thought to minimize conformational entropy losses upon target binding, although some
studies have shown the impact of cyclization on binding entropy to be more complex than conventional wisdom suggests[10, 11]. Nevertheless, while cyclization may improve
the PK properties of peptides in general, the systematic design of orally bioavailable cyclic peptide therapeutics (e.g.
CSA) will require a deeper understanding of the impact of
structure on physicochemical properties in these non-“druglike” molecules. This in turn will require more direct and
systematic investigations into the PK properties of these remarkable compounds, including cell-based permeability and
absolute oral bioavailability studies. This review examines
cyclic peptide natural products from a PK perspective, with
an emphasis on those molecular properties and structural
motifs that might be emulated in the design of novel synthetic inhibitors against the next generation of protein targets.
Cyclic Peptide Natural Products Classified by Physical
Properties
Cyclic peptide natural products can be grouped into the
following categories based on a broad survey of their structures and known biological functions: 1) Highly charged
molecules (polycationic and polyanionic) that function
mostly as antimicrobial agents by disrupting bacterial membranes; 2) Non-polar cyclic peptides that contain mostly
lipophilic side chains and modifications to the amide backbone (e.g., N-methylation) and are likely to penetrate eukaryotic cells by passive diffusion; 3) Cyclic peptides of
mixed polarity, most of which are amphiphilic but are not
limited to microbial targets; 4) Cyclotides and cysteine-knot
proteins, which are small (2-8 kD) proteins with unusual
topologies that can show remarkable oral activity.
Within each of these categories, cyclic peptides can be
organized into three main sub-groups based on structure/topology: 1) homodetic, cyclized from head-to-tail; 2)
heterodetic, cyclized between side chains or from a side
chain to one of the termini; and 3) complex, comprised of a
mixture of homodetic and heterodetic linkages (Fig. 1).
Many heterodetic cyclic peptides have a “lariat” structure
formed by cyclization of the C-terminal carboxylic acid onto
a side chain, typically via a lactam linkage through a lysine
or ornithine residue, or a lactone linkage through serine or
threonine. Heterodetic cyclic peptides can also be capped on
their amino termini with a lipid tail of varying length and
composition. Compounds in the “complex” category include
bicyclic peptides and cyclic peptides with knot topologies.
Bockus et al.
O
NH
OH
H2N
O
O
H2N
O
NH
X
X = N, O
complex
heterodetic
homodetic
Fig. (1). The three main sub-groups of cyclic peptides based on
structure: a) homodetic, b) heterodetic, c) complex.
1. Cationic Antimicrobial Peptides
The cationic antimicrobial peptides (AMPs) comprise a
diverse group of compounds whose activities are associated
primarily with their ability to interact with and, in many
cases, form pores in bacterial membranes (Fig. 2). Most
AMPs are aphiphilic peptides that disrupt negatively charged
microbial membranes with varying selectivity depending on
the structure of the AMP. While some natural AMPs are
cyclic, many are linear peptides that adopt various secondary
structures upon their interaction with membranes. Despite
much evidence that AMPs exert their antimicrobial activity
by disrupting membranes, some cationic AMPs have potent
antimicrobial activity without disrupting membranes, and
some have activities that are associated with inhibition of
intracellular targets[12]. Nevertheless, these compounds are
not orally bioavailable and tend to show selectivity toward
bacterial membranes. Their structures and activities have
been reviewed elsewhere[12-14] and will not be taken up
further here.
a
Pro
Val
d-Phe
b
Orn
Leu
Orn
d-Phe
Val
O
O
N
H
OH
H
N
O
NH2
c
gramicidin S
Pro
O
H2N
polymyxin B
H
N
O
O
H2N
NH
N
H
NH2
N
H
O
O
NH
H
N
O
NH
NH2
HN
HN
O
NH2
O
HO
tachyplysin
H2N
d
Leu
Arg
Trp
Cys
Phe
Arg
Val
Cys
Tyr
Arg
HO2C
Arg
Cys
Arg
Arg
Tyr
Cys
Ile
Gly
Arg
Leu
Cys
Arg
Ile
HO2C
Arg
Cys
Val
Arg
S
S
S
S
Val
S
S
bactenecin
Val
Ile
Fig. (2). Antimicrobial Peptides a) gramicidin S, b) polymyxin B,
c) tachyplysin, d) bactenecin.
2. Non-Polar Cyclic Peptides
Cyclic peptide natural products that are comprised of
mostly neutral, lipophilic side chains and few if any polar
residues can be classified as “non-polar” cyclic peptides.
These cyclic peptides and depsipeptides exhibit a wide range
of biological activities, many of which are associated with
intracellular targets in eukaryotes (Table 1). Although the
Form and Function in Cyclic Peptide Natural Products
Table 1.
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
823
Cyclic Peptide Structural Classes.
Classification
Structural features
Mechanisms of membrane interactions
Biological activities
Charged
•cationic
More than one cationic residue, e.g., Arg or
Lys
Destabilize and/or form pores in anionic (usu- antimicrobial
ally bacterial) membranes
•anionic
More than one anionic residue, usually Asp or Interact with membranes by chelation of diva- antimicrobial
Glu
lent cations
Polar
Antimicrobial; various intracelluUp to one charged residue and/or a number of
Direct binding to specific phospholipids. For
lar mammalian targets
highly polar residues (e.g., Asn, Gln). Many
many in this category the mechanisms of memare heterodetic and contain a lipid appended to
brane permeability are poorly defined
the macrocycle
Nonpolar
Primarily comprised of aliphatic residues;
often contain multiple sites of N-methylation;
networks of intramolecular hydrogen bonds in
low dielectric media generate defined, lipophilic
conformations;
beta-hydroxylsubstituted residues common
Diverse activities in mammalian
Passive (transcellular) diffusion. Active trans- cells. Most known targets are
port observed in some cases. Few systems have intracellular.
been studied in detail with respect to membrane
permeability.
cell permeabilities of few of these compounds have been
investigated directly (e.g., by quantifying diffusion across
Caco-2 or other epithelial cell lines), many are assumed to be
cell permeable based on an observed correlation between
direct in vitro binding and/or activity data and activity in
cell-based assays. Because they do not interact with membranes electrostatically, these compounds are thought to
penetrate membranes via the same classical transcellular
route ascribed to most small molecule drugs. However, until
detailed in vitro studies are performed to evaluate their
mechanism(s) of cell penetration, such hypotheses remain
purely speculative. The archetype of the nonpolar cyclic peptides is the immunosuppressive natural product cyclosporine
A, a cyclic undecapeptide with a molecular weight of ~1200
that has an oral bioavailability in humans of 29% and is passively permeable[15]. The high molecular weights of these
compounds compared with traditional small molecule drugs
make them excellent model systems for studying the role of
structure in passive membrane permeability. Our lab has
investigated synthetic cyclic peptides inspired by these natural products to gain a better understanding of the chemical
features driving their unusual PK properties. We have shown
using simple model systems that the ability to form intramolecular hydrogen bonds in low dielectric media correlates with passive membrane diffusion[8, 16]. We have also
observed that N-methylation patterns that facilitate the formation of transannular hydrogen bonds are associated with
good passive permeability and oral bioavailability[17]. This
result is corroborated by many examples of cyclic peptide
natural products whose structures have been solved, either
by NMR in low dielectric solvents or in the solid state, or in
some cases both. Many of these peptides contain multiple Nmethyl and/or Pro residues, and most have extensive intramolecular hydrogen bond networks that sequester polar
amide groups in the low dielectric of the membrane. While
the total hydrogen bond donor and acceptor counts put most
compounds in this class out of compliance with Lipinski’s
Rule (<5 donors and <10 acceptors), the number of free,
non-hydrogen bonded donors and acceptors puts them well
within the acceptable range (Table 2). Although they contain
many rotatable bonds and have molecular weights in the
~800-1500 range, solution NMR data suggest that these
compounds are relatively rigid, especially in low dielectric
media. In this sense, their membrane permeability is consistent with the observation by Veber, et al., that rigidity is a
better predictor of bioavailability than molecular weight[18].
In that retrospective study, the number of rotatable bonds
(which has a roughly linear relationship with MW) was a
better predictor of bioavailability than MW. Although the
number of rotatable bonds in natural macrocycles may be
high, they remain relatively rigid due to constraints imposed
by cyclization and local nonbonded interactions.
Non-polar cyclic peptides are characterized by a predominance of aliphatic residues, the presence of D-amino
acids, and N-methylation of backbone amides. If they are
present, polar residues often form hydrogen bonds between
the side chain and backbone, for example in the cases of argyrin B, aureobasidin, and guangomide A (Fig. 3). Few if
any cyclic peptides in this category have ionizable side
chains, and they rarely contain highly polar neutral residues
such as Gln and Asn.
The cyclic peptides in this category whose crystal structures have been solved often contain one or more !-turns that
template various patterns of intramolecular hydrogen bonds.
These !-turns are defined by a transannular hydrogen bond
between the i and i + 3 residues, while the stereochemistry,
N-methylation status, and side chain functionality of the i +
1 and i + 2 residues provide the phi and psi restraints that
template the turn. Detailed reviews of various subtypes of !
824 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
Table 2.
Bockus et al.
Selected Cyclic Peptides, their Biological Activities, and Key Structural Features.
Cyclic Peptide
Mw
Target
in vitro
IC50 (nM)
EC50 in cells
(nM)
# Residues
# N-methyls
Total
Free
Donors
Acceptors
Donors
Acceptors
cyclosporine A [37, 38]
1203
Cyclophilin/
calcineurin
30
7-10
11
7
5
12
1
8
cyclomarin A[39, 40]
1043
ClpC1
Subunit of the
Caseinolytic
Protease
100
300
7
2
7
11
4
9
2500
argyrin[41, 42]
825
26S proteasome
100
2.8
8
1
8
9
2
3
HUN7293/
893
Sec61
30
10
7
3
3
8
0
5
luzopeptin[48, 49]
1427
dsDNA
53
7
10
4
8
22
0
14
didemnin B[50, 51]
1112
EF-1alpha;
inhibits protein synthesis
90
2.5
9
2
5
12
2
9
aureobasidin A[52]
1114
phosphatidylinositol:
ceramide
phosphoinositol transferase
0.2
90
9
4
4
10
0
6
YM-254890[53, 54]
959
Heterotrimeric Gaq;
inhibits GDPGTP exchange
50
0.1
8
3
5
12
0
8
CT8[43-47]
turns found in proteins[19, 20] and peptides[21], and the
residues that favor their formation, have been published.
Many of these turns contain Pro and/or Gly residues and Damino acids, all of which favor !-turns when present at both
the i + 1 or i + 2 positions. Both Pro and N-methyl residues
can adopt the cis-amide conformation, which, when located
at i + 2, induces the type VI !-turns found in many neutral
cyclic peptide natural products.
Homodetic Non-Polar Cyclic Peptides
The homodetic non-polar cyclic peptides can be further
classified into four major categories defined by the types of
residues and backbone modifications that they contain: regular (all-amide) peptides, depsipeptides, pattelamide-like peptides, and proline-rich peptides. Examples of regular nonpolar cyclic peptides are argyrin B, cyclomarin, and CSA
(Figs. 3 and 4). Most of the cyclic peptides in this category
contain multiple N-methyl residues, yet very few are permethylated. This trend is consistent with work in our lab
showing that permethylation can decrease membrane permeability relative to specific patterns of N-methylation that favor intramolecular hydrogen bonds[17]. Indeed, for those
peptides in this class whose structures are known, the vast
majority of non-N-methylated amides are involved in transannular hydrogen bonds. While this simple hypothesis is
consistent with many cyclic peptide structures (vide infra),
other work using similar model systems suggests a more
complex role of N-methylation in the active transport of cyclic peptides[22-25]. Recent reviews treat N-methylation in
peptides from synthetic and conformational perspectives[22,
26].
CSA is an 11-amino acid, homoetic non-polar cyclic peptide that contains seven N-methyl groups, a single D-amino
acid, and a non-proteinogenic prenylated Thr residue. CSA is
a clinically used immunosuppressive agent that binds the
prolyl cis-trans isomerase cyclophilin. The resulting
CSA/cyclophilin complex is a potent inhibitor of calcineurin,
Form and Function in Cyclic Peptide Natural Products
a
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
b
O
Ph
!e N
O
O
H
N
HO
O
O H
O
O
O
O
HN
O
N
H
HN
O
O
Cl selenamide
N
NH
H
d
O
N
H
N
N
!e
H
O
O
O
N
!e
O
O
O
HO
O
O
!e
N
N
H
N
R1 H
O
h
O
O
H
N
!e O
N
O
N
O
NH
O
O
f
Y = OH: didemnin B
Y = O: aplidin
O
O
O
O
N !e
H
N
O
O
O
!e
N
N
H
O
H
N
N
O
N
O
O
O
N
N
N
N
H
NH
O
O
HN
callynormine D
OH
N
H
O!e
O
destruxin E
O
N
H
N
RO
H
N
O
N
!e
O
O
N
H
O
O O
O O
O
N
O
H
O
O
H
N
N
H
HUN-7293
i
O!e
N
H
O
R2
O
O
"
O
O
O
O
N
!e
O
!e
O
S
O
O
OR
!e
N
HN
NH
O
O
N
O
O
O
NH
N
H
N!e
aureobasidin A
g
O
c
!e
N
O
N
NH
OH
!e
N
O
NH
argyrin B
e
O
O
H
O
H
N
N
H
NH
O HN
!e
O
N
H
O
O O
HN
O
!e
N
N
H
O
O
825
H
N
N
Me
N Me
O
guagomide A
cyclomarin
Fig. (3). Selected nonpolar cyclic peptide natural products showing intramolecular hydrogen bonding observed in X-ray crystal structures. a)
selenamide, b) argyrin B35, 36, c) callynormine D d) aureobasidin A46, e) didemnin B44, 45, f) destruxin B, g) HUN-7293, h) cyclomarin33,
34, i)
a
b
!e
N
N
H
O O
!e N
O
H
N
H
O
OH
!e
N
O
!e
N
O
!e
N
N
!e O
!e
O
H
N
!e N
O
N
!e
O
N
H
O
H
N
O
O
O
HO
O
O
O
N
O
N
N
N!e
N
H
OAc
O
N
!e
O
O
AcO
H
N
O
N
O
OH
O
O
N
O
HN
HO
O
NH
N
N
O
O
N
!e
O
!e N
O
O
O
N
N
H
H
O
H
N
N
!e
O
O
!e
N
!eN
OAc
AcO
N!e
O
O
O
O
N
O
!eN
N
!e
O!e
O
!e
N
O
!e
N
N
HO
NH
O
O
HO
d
N
HO
!e
N
H
O!e
c
H
O
N
N
N
H
HN
O
O
OH
N
OH
N
OH
O
O
!eO
O!e
e
R
O!e
O
O
O
N
H
N
O
OH
!e
O
HN
N
O
!e
O
O
!eN
O
O
OH
R'
O
O
H
N
f
R'
N
H
O
HN
R
O!e
O
O
O
N
O
O
O
!e N
O
NH
!e
R'
R'
O
O
O
H
N
!eN
N
H
O
O
O
Fig. (4). Structures of cyclic peptides in free (a, c, e) and target-bound (b, d, f) states. a,b) cyclosporine A31, 32; c,d) luzopeptin42, 43; e)
FR900359 (R = CH3); f) YM254890 [47, 48] (R = H); Intramolecular hydrogen bonds indicated in orange dotted lines and intermolecular
hydrogen bonds indicated in blue dotted lines.
826 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
a central phosphatase involved in T-cell activation[27].
Thus, CSA has the unusual function of inducing an inhibitory protein-protein interaction. The structure of CSA has
been determined both in the free state, by X-ray crystallography and NMR[28, 29], and in the ternary complex with
cyclophilin and calcineurin[30]. In the free state, in both the
crystal structure and the NMR solution structure in CDCl3,
CSA adopts a rectangular conformation characterized by
three transannular hydrogen bonds and an external, "-turn
forming hydrogen bond (Fig. 4a). In contrast, in the crystal
structure of the ternary complex with cyclophilin and calcineurin, CSA adopts a more open conformation in which
many of the polar groups point outward, forming intermolecular hydrogen bonds with both protein partners (Fig.
4b)[30]. Interestingly, the non-polar conformation of CSA
observed in CDCl3 was also significantly populated in 50%
aqueous methanol[31]. This type of conformational flexibility may be common to nonpolar cyclic peptides and has been
identified in at least two other cases (see below). Whether
this behavior contributes to the unexpected passive permeability of this class has not been examined directly.
The homodetic non-polar cyclic depsipeptides have at
least one depsi- (i.e., lactone) linkage and include compounds such as destruxin B, aureobasidin A, HUN7293/CT08, and guangomide A (Fig. 3). Whether these lactone linkages serve a critical role in the pharmacokinetic
properties of these molecules has not been studied systematically, although their prevalence among natural cyclic peptides of disparate origins suggests that the lactone, like Nmethylation, may serve an important role in enhancing the
permeability and/or stability of these compounds.
A well-known cyclic peptide natural product is the K+
ionophore valinomycin, a cell-permeable and cytotoxic cyclic depsipeptide that acts as a mitochondrial uncoupler by
facilitating the passive diffusion of potassium ions across the
mitochondrial inner membrane (Fig. 5). The apo-form of
valinomycin crystallized from octane shows a rectangular
geometry in which all of the amide NH groups are involved
in trans-annular hydrogen bonds. These hydrogen bonds define #-turns between the lactic acid C=O groups and the
valine NH groups at i and i + 3, respectively (Fig. 5b)[32].
In complex with K+, the molecule rearranges and adopts a
toroidal geometry using a different arrangement of # turns in
which six of the C=O groups point toward the K+ in the center of the torus (Fig. 5c)[33]. In both the apo-form and the K+
complex, all six of the NH groups are involved in transannular hydrogen bonds, but the two structures have very different H-bonding arrangements. In yet another conformation
(crystallized from DMSO in the absence of K+) that might be
considered a model for its aqueous, apo state, valinomycin
adopts a flat, triangular conformation in which three of the
amide N-H groups are solvent-exposed, while the remaining
three N-H groups point inward forming type II #-turns (Fig.
5d).
The KD between valinomycin and K+ is 1-3 mM in aqueous solution[34], significantly higher than environmental
levels but 10-fold lower than the concentration of K+ in the
mitochondrion, suggesting that in order to function as an
uncoupler, valinomycin must be cell permeable in both its
apo- and K+-complexed forms. That it is able to adopt lipo-
Bockus et al.
philic conformations in both complexed and uncomplexed
forms points toward a mechanism of passive diffusion, while
the “open” conformer may account for its water solubility in
the apo form. Indeed, valinomycin dissolves in solvents
ranging in polarity from water to hexane[34].
Heterodetic Non-Polar Cyclic Peptides
Many non-polar cyclic peptides have heterodetic linkages
and include compounds such as coibamide A, didemnin B,
YM254890, selenamide, luzopeptin, and callynormine A.
Didemnin B (Fig. 3e) is a non-polar lariat cyclic peptide that
potently inhibits protein synthesis by binding to the eukaryotic translation elongation factor EF-1! [35-37]. It has lownM activity in cells and was the first marine natural product
to enter human clinical trials. Although clinical trials of
didemnin B were discontinued due to toxicity and low efficacy, the nearly identical compound aplidin (Fig. 3, also
called plitidepsin) has shown promise in phase II/III clinical
trials against a variety of cancers[38, 39]. Aplidin has been
the subject of in vitro metabolism[40] and in vivo i.v. pharmacokinetics studies and has lower plasma half-life[41] (44
h vs. 0.14 h) and lower cardiotoxicity[42] than didemnin B.
No direct cell permeability or oral bioavailability data have
been reported for didemnin B or aplidin.
a
O
O
O
O
H
N
O
O
O
HN
NH
O
valinomycin
O
O
HN
O
O
O
HN
O
NH
O
HN
O
d
NH
O
O
HN
NH
NH
S
O
HN
NH
O
H
N
O
b
NH
O
O
O
#
O
c
O
NH
O HN
NH O
O
K+
O
NH
HN
HN
O
OS
O
HN
O
HN
O
HN
O
S
Fig. (5). a) Chemical structure of valinomycin; b-d) schematic diagrams of X-ray structures showing hydrogen bonding patterns.
Valinomycin crystallized from non-polar solvent in the absence (b)
and presence (c) of K+ and (d) crystallized from DMSO in the absence of K+.
Another heterodetic lariat peptide, callynormine A (MW
1188), consists of 11 amino acids with an unusual !-# dehy-
Form and Function in Cyclic Peptide Natural Products
drodiaminopropionic acid group linking the cyclic octapeptide portion with the short tripeptide tail (Fig. 3c). While
callynormine contains no N-methyl residues, the crystal
structure shows multiple hydrogen bonds both within the
ring and between the ring and the tail[43]. The lariat depsipeptide coibamide (MW 1288) also contains 11 amino acids, with 7 residues in the ring and 4 in the tail, and shows
low nM cytotoxicity in various human cancer cell lines. Coibamide exhibited an unusual COMPARE fingerprint in the
NCI60 cell line screen, suggesting that it has a unique biological target or MOA. In contrast to callynormine A, coibamide is extensively N-methylated both in the tail and in
the cyclic portion. Although its three-dimensional structure
is unknown, its bioactivity and N-methylation pattern are
consistent with cell permeability by the same mechanism(s)
associated with other members of this family.
Luzopeptin is a heterodetic cyclic peptide that contains
two pendant aromatic 3-hydroxy-6-methoxyquinaldic acid
groups. It has C2 symmetry and binds to DNA by intercalating both chromophores in a staple-like fashion with the macrocycle sitting in the major groove. In both the X-ray crystal
structure[44] and the NMR structure in CDCl3[45], the free
compound has a collapsed, rectangular structure with intramolecular hydrogen bonds between the Gly NH and the
sarcosine carbonyl groups, and between the OH groups of
the hydroxyvalines to their own carbonyls. In the DNAbound form, luzopeptin adopts a square conformation in
which the Gly NH groups and the hydroxyvaline carbonyls
form hydrogen bonds with bases in the major groove[46,
47].
YM-254890 is another potently bioactive heterodetic cyclic peptide that was first identified as an inhibitor of ADPinduced platelet aggregation[48]. This compound was subsequently shown to inhibit signaling through G!q, a subfamily
of the heterotrimeric guanine nucleotide-binding proteins (G
proteins) that transduce signals from ligand-activated Gprotein coupled receptors (GPCRs). An activated GPCR
stimulates the GDP-to-GTP exchange in a G! protein inside
the cell, causing the dissociation of G! from its associated
G#" subunit. The free G! and G#" subunits each bind and
activate various downstream effectors, which, for the G!q
subtype, is phospholipase C. The cyclic peptide YM-254890
binds to G!q and prevents the exchange of GDP for GTP,
locking the G!q subunit in its “off” state[49]. The X-ray
crystal structure (of FR900359, a close structural analog of
YM-254890)[50] reveals a complex network of intramolecular hydrogen bonds involving all four free amide NH groups
and the –OH group of the #-hydroxyleucine residue (Fig.
4e). The molecule undergoes a major conformational rearrangement upon target binding such that, in the bound conformation, most of the polar groups point away from the ring
forming hydrogen bonds with the protein, with a new intramolecular hydrogen bond formed within the macrocycle
(Fig. 4f)[49]. Both luzopeptin and YM-254890 are presumed
to be cell permeable based on their nM potencies in both in
vitro and in cell-based assays and the congruence between
their cellular phenotypes and in vitro activities, although
their cell permeabilities have not been measured directly.
Could the favorable PK properties such as good permeability and low clearance of the non-polar cyclic peptides
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
827
relate in general to their ability to equilibrate between open,
hydrophilic conformers in water and closed, hydrophobic
conformers in the membrane? For example, if CSA could be
locked into its low-dielectric conformation so that it could
not equilibrate into its “open” conformer in aqueous solution, what impact would it have on clearance, permeability,
and solubility? How general and/or important is conformational flexibility in determining the ADME properties of
neutral cyclic peptides? The impact of such conformational
dynamics on permeability and solubility has not been investigated using well-controled model systems, suggesting that
more systematic studies are needed in this area.
One study that may shed light on the relationship between structure and cell permeability found that the permeability of CSA in Caco-2 increased by two orders of magnitude over the temperature range from 5 to 37˚C[51]. Equilibrium sedimentation studies showed a similarly dramatic increase in the partial specific volume of CSA over the same
range, and a corresponding decrease in its aqueous solubility.
Importantly, these authors found that the equilibrium distribution of conformers in aqueous solution remained constant
over the same temperature range, suggesting that the observed increase in permeability was not due to a corresponding increase in the relative abundance of the internally hydrogen bonded conformer at the higher temperature. Like
CSA, the permeabilities of salicylic acid and propranolol
(two other drugs that penetrate membranes by passive diffusion) also increased with increasing temperature, although
for these drugs the effect was an order of magnitude less
pronounced than that of CSA. These data suggest that the
classical, entropy-driven hydrophobic effect drives the energetics of membrane permeability for CSA. Could the ability
to adopt open, hydrophilic conformers in aqueous solution
serve to prevent aggregation in large, non-polar cyclic peptides like CSA? Since non-specific, hydrophobic aggregation
is presumably also entropically driven, any advantage gained
with an increase in non-polar surface area would be counteracted by a tendency to aggregate. Equilibration among
“open” hydrophilic conformers would therefore disfavor
aggregation in large, non-polar solutes by decreasing the
relative concentration of the “lipophilic” conformer compared to more water-soluble ones. Furthermore, how does
the size-dependent diffusion coefficient, a factor that predicts
decreasing permeability with increasing cross-sectional
area[52, 53], factor into the permeability equation in opposition to the classical hydrophobic effect? Applying similar
experimental techniques to other cyclic peptides could help
tease apart the contributions of each of these factors in determining the membrane permeability of large macrocycles.
Patellamide-Like Cyclic Peptides
While most nonpolar cyclic peptides are biosynthesized
by NRPSs, the patellamide-like cyclic peptides are derived
from ribosomally synthesized sequences of alternating lipophilic and Ser, Thr, or Cys residues. These hydroxyl- and
sulfhydryl-containing residues are precursors of the oxazoline and thiazoline rings that characterize this class of
natural products, formal products of the cyclodehydration
between the side chain -OH or -SH groups and the neighboring i - 1 backbone carbonyl group. Oxidation of the oxazoline and thiazoline rings leads to the corresponding aro-
828 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
matic oxazoles and thiazoles. Examples of the pattelamidelike cyclic peptides include lissoclinamide 7, patellamide D,
and ascidiacyclamide (Fig. 6). Some of these, such as the
patellamides and ascidiacyclamide, are comprised exclusively of these backbone-cyclized residues, while others,
such as trunkamide, are hybrids and contain one or more
unmodified residues. While the pure patellamide-like peptides rarely contain N-methyl groups, consistent with their
ribosomal origin, they generally exhibit multiple intramolecular hydrogen bonds, either between the 5membered ring nitrogen lone pair and the neighboring N-H
group, or, in some cases, between NH groups and C=O
groups within the molecule. Patellamides and ascidiacyclamide can adopt a square conformation in which the NH
groups point into the center of the ring, or a twisted “Figure
eight” conformation in which the thiazole rings are stacked
and the structure is stabilized by transannular hydrogen
bonds. Which of these conformations a particular patellamide adopts appears to be determined by the nature of the Rgroups on the ! carbons, with the square conformation being
preferred by C2-symmetric arrangements of R-groups and
the twisted conformation favored by asymmetric arrangements of R-groups. Although there was one report that lissoclinamide 7 exhibits nM cytotoxicity in some cell lines[54],
this degree of toxicity was not recapitulated in a screen by
the NCI[55]. Other peptides in this family have failed to
show appreciable cytotoxicity or any other significant biological activity, leading to the speculation that they act as
multivalent cation transporters or play some other role in the
metabolic maintenance of the producing organism.
a
R4
O
R%
O
S
N
H
N
N
H
N
S
R2
N
S
R1
O
O
N
H
N
H
N
O
HN
O
N
O
HN
N
H
N
Ph
O
S
Ph
H
N
O
N
S
O
O
N
S
H
N
O
ascidiacyclmide
Conjoined NRPS and polyketide synthase (PKS) modules yield a diverse class of macrocycles comprised of both
polyketide and polypeptide fragments[59-61]. These compounds display impressive backbone variety and complexity,
pushing beyond the limits of the 20 proteinogenic amino
acids (and modifications thereof) by incorporating
polyketide fragments of varying lengths and functionality.
Such scaffolds are biosynthesized in modular fashion by
hybrid PK-NRPS Claisen-like mechanisms forming backbone alkene and #-hydroxy functionality. These molecules
commonly possess a polypeptide hemisphere and a
polyketide hemisphere, the latter consisting of a carbon
chain with multiple stereocenters defined by pendant methyl
and hydroxyl groups and endocyclic double bonds.
OH
phakellistatin 8
O
H
N
N
N
H
O
N
O
N
O
O
O
O
O
N
H
NH
NH
H
N
N
O
Fig. (7). Proline-rich cyclic peptide phakellistatin 8.
d
O
O
N
O
patellamide D
lissoclinamide 7
c
N
N
O
Hybrid Polyketide-Polypeptide Macrocycles
O
NH
O
HN
methyl residues, although many of them have extensive networks of internal hydrogen bonds. These peptides are associated with various bioactivities, although, similar to the patellamide-like peptides, their potencies are moderate, at least
in the assays in which they have been screened to date.
O
O
N
NH
O
b
R$
Bockus et al.
NH
NH
O
O
N
H
O O
O
N
HN
N
S
trunkamide
Fig. (6). Patellamide like cyclic peptides, a) lissoclinamide 7, b)
patellamide D, c) ascidiacyclmide, d) trunkamide
Proline-Rich Cyclic Peptides
The phakellistatins[56-58] (Fig. 7) are examples of homodetic proline-rich cyclic peptides. These rarely contain N-
Apratoxin A (Fig. 8a) is one such 25-member hybrid
polyketide-polypeptide macrocycle. Isolated from the marine
cyanobacterium Lyngbya majuscula, this secondary metabolite exhibits potent activity against human solid tumor cell
lines (KB IC50 = 0.52 nM, LoVo IC50 = 0.36 nM) and has
shown marginal activity in vivo against colon and mammary
cancer[62]. More recent studies have shown that apratoxin A
modulates cotranslational translocation[63], although other
evidence points toward a mechanism involving association
with Hsp/Hsc70 and subsequent stimulation of chaperonemediated autophagy of specific client proteins[64]. No intramolecular hydrogen bonding was observed via CDCl3
NMR and subsequent molecular dynamics minimization[62].
Jasplakinolide (Fig. 8b) is a 19-member polyketide cyclodepsipeptide hybrid isolated from the marine sponge
Jaspis. The backbone is composed of a #-Tyr, N-Me bromoTrp, and Ala tripeptide coupled to a 12-carbon alkenecontaining polyketide fragment cyclized through an ester
linkage. The natural product exhibits anti-proliferative activity in human cell lines[65, 66] and is one of the few cell
permeable small molecules known to stabilize F-actin[67],
binding competitively at the phalloidin site with a Kd of 15
nM[68]. The solution and crystal structures display no intramolecular hydrogen bonding[69], although the skeleton is
quite flexible, capable of adopting several conformations as
Form and Function in Cyclic Peptide Natural Products
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
a result of the long carbon polyketide fragment[70]. Jasplakinolide has long been presumed to be cell permeable
based on the correspondance between its actin-stabilizing
activity in vitro and its dramatic effect on the cytoskeleton in
living cells. Its cell permeabilty was confirmed directly,
however, in a recent report that used synthetic fluorescent
probes of jasplakinolide to image the actin cytoskeleton in
live cells[71].
The complex skeletons of hybrid polyketide-polypeptide
macrocycles permit the molecules to adopt conformations
that are not available to strictly peptide-based scaffolds. The
lack of intramolecular hydrogen bonding in the few molecules of this class whose 3-D structures have been characterized suggests that perhaps the hydrophobic nature of the
polyketide segment is able to compensate for the polarity
imparted by the non-N-methylated amides. However, it
should be noted that the mechanism of membrane penetration of these compounds has not been investigated explicitly.
a
b
OH
S
N
apratoxin A
HN
O
O
N
O
O
NH
Br
O
N
OH
O
O
O
O
jaspakinolide
N
NH
HN
O
O
O
N
Fig. (8). Examples of cell-permeable hybrid polypeptide-polyketide
macrocycles a) apratoxin A, b) jaspakinolide.
3. Cyclic Peptides of Mixed Polarity
The various membrane-disrupting and membranepenetrating mechanisms attributed to the amphiphilic antimicrobial peptides are primarily physical in nature, that is, their
MOAs do not depend on inhibition of specific macromolecules. In contrast, there is a class of cyclic peptide natural
products that are amphiphilic and yet exhibit activities in
mammalian cells that point toward intracellular targets and
PK profiles that suggest passive membrane diffusion. These
cyclic peptides of mixed polarity encompass a variety of
structural types and biological activities. For example, kahalilide F[72](KF; Fig. 9a), is a 13-amino acid cyclic lariat
peptide (C-terminal-to-Thr linked) that is potently cytotoxic
in human cells (EC50 ~100 nM) with good selectivity for
tumor cells vs. primary human cells[73]. Although its biological activity has been the subject of intense study[73-76],
the target and mechanism of action (MOA) remain obscure.
The cyclic portion of KF contains 6 residues and is neutral except for a single Orn residue, with a relatively short, 6carbon aliphatic cap at the N-terminus of the 7-residue tail.
While the mechanism of internalization of kahalalide F is
unknown, its rapid effect on endosome morphology suggests
that its target may be associated with intracellular membrane
compartments[76]. Fluorescent[77] and gold nanoparticle[78] conjugates of KF were localized to endosomal compartments, and KF itself causes dramatic swelling of
829
lysosomes[76] followed by severe disruption of organelle
architecture and cell death via a non-apoptotic mechanism[73]. KF was also stable to proteolysis and oxidative
metabolism in vitro in the presence of pooled human plasma
and liver microsomes[79], consistent with its in vivo stability.
Human PK studies[80-83] showed a rapid clearance from
plasma following i.v. infusion, with a steady state volume of
distribution (Vss) of ~7 L/kg, indicative of low distribution in
tissues. Whether this Vss value is a function of poor cell permeability or high plasma protein binding was not determined.
Perhaps reflecting the deleterious effect of highly polar
substituents on passive membrane diffusion, many polar cyclic peptides have a lipid appendage. Examples in this group
include largamide, pseudodesmin A, and the papuamides
(Fig. 9b-f). Pseudodesmin A is a member of a family of “cyclolipeptides” (CLPs) derived from Pseudomonas spp. characterized by a lipid tail appended to an oligopeptide macrocycle cyclized through a depsi-linkage to a Thr side
chain[84]. The crystal structures of several members of this
class have been solved, including pseudodesmin A[85],
WLIP[86], pseudophomin[87], amphisin[88], and tensin[89],
and all adopt a similar conformation in which one half of the
macrocycle is folded into a left-handed !-helix stabilized by
a network of intramolecular hydrogen bonds (Fig. 9d). The
lipid tail is extended along one side of the macrocycle with
the lipophilic residues, while the hydrophilic residues project
from the opposite side of the macrocycle, creating an amphiphilic geometry (Fig. 9d, e). Overall, the biological activity of these compounds in various antimicrobial screens is
mild to moderate[84], and little has been reported on their
effects, if any, on mammalian cells. Further, the mechanism(s) by which these molecules interact with and/or penetrate membranes has not been studied directly.
Papuamide A protects cells against HIV-induced toxicity
by interfering with viral entry, with an EC50 of ~100
nM[90]. The very similar papuamide B (Fig. 9f) was shown
to interact with membranes in S. cerevisiae by specific binding to phosphatidylserine (PS)[91], although PS binding
could not be linked to the anti-viral activity of either
papuamide A or B[90].
Thiostrepton (Fig. 9g), and the related molecule siomycin
A, are antibiotics that inhibit prokaryotic translation[92], but
are also selectively cytotoxic to transformed eukaryotic cells.
Siomycin A induced apoptosis in transformed, but not in
normal lung fibroblasts, and it selectively killed breast cancer cells through inhibition of FOXM1 signaling[93]. Siomycin A also inhibited tumor growth from glioblastoma
stem cells through down-regulation of maternal embryonic
leucine zipper kinase (MELK)[94]. More recent studies
showed that thiostrepton and siomycin A are both proteasome inhibitors and that this activity is responsible for their
ability to decrease FOXM1 levels and selectively kill certain
cancer cells[95, 96]. However, another group reported recently that the down-regulation of FOXM1 in malignant
mesothelioma cells is not related to proteasome inhibition,
but rather correlates with the ability of thiostrepton to disable
the mitochondrial anti-oxidant pathway by covalently dimer-
830 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
a
kahalilide F
O
O
H
N
N
H
O
O
H
N
N
H
OH
HN
NH
OH
f
O
O
OH
NH
O
N
H
H
N
O
OH
H
N
NH
O
O
O
N
H
O
H2 N
O
NH
O
O
HN
O
OH
HN
O
O
O
OH
e
X-ray crystal structure
of pseudodesmin A
molecular surface of pseudodesmin A
OH
NH
back
front
g
OH
O
O
HN
N
H
O
O
papuamide B
H
N
O
H
N
N
H
HN
d
O
O
N
H
O
OH
thiostrepton
O
NH
OH
HO
HN
N
H
NH
HN
NH2
O
O
O
O
largamide
O
NH2
NH
NH
O
O
O
O
O
N
O
O
N
H
O
O
O
N
H
NH
H
N
O
OH
H
N
O
O
N
H
O
b
NH
O
c
pseudodesmin A
O
H
N
N
O
O
O
O
H2N
h
Bockus et al.
O
X-ray crystal structure of
microsin J25 (backbone
atoms only)
O
H2 N
O
N
H
N
H
N
NH
O
NH
O
O
O
OH
S
N
NH HN
O
O
N
H
N
S
O
NH
N
N
HO
OH
O
HN
N
S
S
HN
H
N
S
N
O
O
HO
Fig. (9). Heterodetic polar cyclic peptides a) kahalilide F (KF), b) largamide, c) pseudodesmin A d) X-ray crystal structure of pseudodesmin
A shown as stick diagram e) front and back views of molecular surface based on pseudodesmin A crystal structure; color coded by hydrophilicity: purple = hydrophilic; green = lipophilic; white = neutral; f) papuamide B, g) thiostrepton, h) X-ray crystal structure of microcin
J25; side chains have been ommitted for clarity.
izing the protein peroxiredoxin 3 (PRX3) through thiostrepton’s multiple dehydroalanine residues[97].
Despite the conflicting hypotheses regarding the MOA of
thiostrepton, its target(s) is/are likely intracellular. The Xray[98] and solution NMR[99] structures of thiostrepton
show some intramolecular hydrogen bonding, but also a significant number of solvent-exposed polar groups, suggesting
that thiostrepton is unlikely to traverse membranes by a simple passive diffusion-type mechanism. However, the lack of
cationic residues suggests its mechanism of membrane penetration is distinct from the pore-forming and membranedisrupting mechanisms shared by the classical amphiphilic
antimicrobial peptides.
Microcin J25 is a lariat “protoknot” peptide with a lactam
linkage formed between the N-terminal Gly and an internal
Glu side chain (Fig. 9h). It inhibits bacterial transcription by
blocking the nucleotide uptake channel of bacterial RNA
polymerase[100, 101], and has also been shown to increase
superoxide production in bacteria. Microcin J25 also acts as
a mitochondrial uncoupler and stimulates cytochrome C release from isolated, intact mammalian mitochondria[102,
103], although it has minimal apoptotic activity in human
cells in culture. In this remarkable structure, the “tail” portion of microcin J25 is threaded through and sterically
trapped within the ring, forming a lasso topology [104, 105].
The 21-residue compound is mostly hydrophobic and has
numerous intramolecular hydrogen bonds, with a single His
residue that forms an intramolecular side-chain-to-backbone
hydrogen bond. It interacts with artificial liposome preparations, showing selectivity for zwitterionic over negatively
charged phospholipids, and transports small ions across the
membrane without causing membrane disruption[106, 107].
The interaction of microcin J25 with membranes has also
been studied using Langmuir monolayers and was shown to
be entropically favorable, suggesting that the classic hydrophobic effect may drive the thermodynamics of its partitioning into membranes[108].
Form and Function in Cyclic Peptide Natural Products
4. Cyclotides and Cysteine-Knot Proteins
Cyclotides[109] are backbone-cyclized “microproteins”
that contain multiple disulfide bonds and a conserved multiloop structure[110, 111]. Kalata B1, a plant-derived cyclotide with 29 amino acids and three disulfide bonds (Fig.
10a), was discovered as the active constituent of a tea made
from Oldenlandia affinis, used by midwives in Central Africa to stimulate uterine contractions[112]. Remarkably,
kalata B1 is orally active, although its in vivo PK in animals
has not been reported. It is therefore difficult to discern
whether the oral activity of kalata B1 is due to good absorption, or rather is achieved despite poor absorption, for example, because of very high affinity for its pharmacological
target. A classic example of this phenomenon is the clinical
drug desmopressin, an orally delivered derivative of the cyclic peptide vasopressin that is used to treat diabetes insipidus, nocturia, and various coagulation disorders. Desmopressin has an absolute oral bioavailability of only
0.1%[113] and yet shows oral efficacy at doses as low as 100
!g in adults. Such potent oral activity in the context of poor
oral bioavailability can occur when a ligand has a very slow
dissociation rate from its receptor, allowing the receptor to
become fully occupied even though steady-state levels of the
drug in serum remain very low[114].
In studies using mammalian cell cultures, kalata B1
formed aggregates on the cell exterior. In the same study, a
structurally similar cyclotide, MCoTI-II, was shown to penetrate cells by macropinocytosis[115], an energy-dependent
but receptor-independent mechanism of internalization
known to operate with poly-Arg and other cell penetrating
peptides such as Tat[116]. Nonetheless, solution NMR studies investigating the interaction of kalata B1 and other cyclotides with micelles have revealed that the cyclic peptides
interact with membranes in a specific geometry that depends
on the 3-dimensional arrangement of hydrophobic residues
among the three loops[117, 118]. The cell permeability of
cyclotides has been successfully applied to the design of
orally active cyclotides with novel functions by grafting bioactive peptide sequences into cyclotide loops, suggesting that
these proteins may be general scaffolds with which to design
orally active molecules based on any bioactive peptide of
interest[119-121]. Despite the intriguing oral activity of
these compounds, however, their absolute oral bioavailability has not been reported.
Another disulfide-rich protein that has interesting pharmacokinetic properties is the soybean-derived 8 kD Bowman-Birk inhibitor (BBI; Fig. 10b)[122]. BBI is not backbone-cyclized, although like the cyclotides it has a knot-like
topology[123, 124]. BBI suppressed radiation-induced transformation of pluripotent stem cells at sub-nM concentrations[125], and when fed to mice prevented chemicalinduced adenocarcinoma[126]. Indeed, BBI was widely distributed in tissues within 3 hours after oral administration[127]. It retained full protease inhibitory activity after
oral delivery and purification from serum and internal organs, indicating that in addition to its permeability it survived first pass metabolism intact and had both low metabolic clearance and minimal proteolytic degradation. However, whether BBI penetrates membranes by passive diffusion or some other mechanism (e.g. by receptor-mediated
Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
831
endocytosis) remains unknown, and its absolute oral biovailability has not been reported.
While the mechanism(s) of membrane permeation of the
cysteine knot microproteins has not been firmly established,
at first glance it would seem unlikely that these molecules
penetrate cells by passive diffusion. If, however, cysteine
knot microproteins such as kalata B1 and BBI prove to exhibit passive membrane permeability in a cell-free membrane
permeability system, it will force us to re-evaluate the molecular weight barrier typically associated with passive
membrane diffusion. These proteins are highly rigid, stabilized by C-to-N cyclization and multiple internal disulfide
bonds, which allows them to adopt unusual folds in which
the polar groups are buried (forming complex networks of
hydrogen bonds) and multiple hydrophobic residues are exposed to solvent. Could the rigidity of these molecules coupled with their relatively large hydrophobic surfaces allow
them to pass through membranes by a mechanism more akin
to passive diffusion than the sort of direct, electrostatic interaction ascribed to the cationic antimicrobial peptides? NMR
evidence showing the specific interaction of kalata B1 and
other cyclotides with micelles[117, 118] points toward the
possibility of passive diffusion for these molecules, although
more studies are needed to establish a detailed mechanism of
penetration.
a
b
kalata B1
Bowman-Birk trypsin
Inhibitor
Fig. (10). a) cyclotide Kalata B1 and b) Bowman-Birk trypsin inhibitor from soybean.
DISCUSSION
The cell permeability associated with many relatively
large cyclic peptide natural products runs counter to “druglikeness” predictors applied to typical small molecules.
These natural products thus provide excellent model systems
with which to study ADME properties of large macrocycles,
and perhaps for large, “non-Lipinski” molecules in general.
832 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 7
The observation that molecules like CSA and others can be
cell permeable begs the question: What is the relationship
between molecular weight and membrane permeability? If
the number of rotatable bonds is indeed a better predictor of
permeability than MW, this supports the notion of an entropic barrier to permeability that increases with increasing
size. For example, transit through the ordered phospholipid
bilayer may involve the freezing out of bond rotations with
an associated entropic liability, while structurally rigid molecules are preorganized enough to avoid such an entropic
cost. While the retrospective study by Veber et al.[18], using
a database of known drugs and their associated permeability
supports this hypothesis, thermodynamic measurements using controlled model systems would provide an orthogonal
means of testing the impact of entropy on permeability and
its connection to molecular weight and/or rigidity. Current
predictive models of permeability that take size into account[52] might benefit from measurements obtained on
such “outlier” systems as these large cyclic peptides.
CSA has been by far the most intensively studied “nonpolar” cyclic peptide and is the only such molecule for which
we have extensive pharmacokinetic data in humans. Although it is tempting to infer general principles based on
CSA, its PK properties may not be generalizable to other
cyclic peptides. For example, although there is strong evidence that CSA penetrates epithelial cells by passive diffusion[15], it is also a substrate for the P-glycoprotein (Pgp)
efflux pump and is actively transported from epithelial cells
in the basolateral-to-apical direction[128]. Other non-polar
cyclic peptides are also known P-glycoprotein substrates
(and, like CSA, capable of reversing the multi-drug resistance due to Pgp overexpression)[129], although it is unclear
to what extent this observation can be further generalized.
Further, the oral bioavailability and volume of distribution of
CSA are highly variable in the population [130], a function
of patient-to-patient variability in activities and expression
levels of Pgp as well as CYP3a, the major CYP involved in
CSA metabolism[131, 132]. Whether this degree of variability holds for other non-polar cyclic peptides remains to be
seen.
While we are beginning to learn about the connections
between structure, molecular weight, and membrane permeability in cyclic peptides, we have only begun to scratch the
surface in terms of the interplay of factors that determine PK
in these large, functionally rich molecules. Indeed, few cyclic peptide natural products have been subjected to direct
cell permeability measurements, much less in vivo PK studies. These studies would benefit not only from the traditional
techniques used in this area (e.g. PAMPA and Caco-2 permeability), but also from the biophysical techniques that
have been applied to the study of cationic antimicrobial peptides using defined, cell-free membrane systems. These include solid state NMR using 19F and 15N probes[133], attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR)[134] and surface plasmon resonance
(SPR)[135]. While these studies have shed light on the detailed interactions between pore-forming peptides and biological membranes, their application to the study of passive
membrane diffusion by cyclic peptides has been limited. A
more detailed biophysical model of passive membrane diffusion would likely benefit from an investigation into the
Bockus et al.
structure-permeability relationship in these larger, “nonLipinski” molecules.
Chemists tend to assume that natural products were designed by evolution to carry out functions related primarily
to defense or predation, although evidence is scant either in
support of or against this hypothesis. Nonetheless, it would
explain why natural products tend to target essential and
highly conserved biological processes and structures such as
protein synthesis, the cytoskeleton, and critical organelles,
and similarly why natural products continue to be the logical
source of leads in antimicrobial and anticancer drug discovery. This hypothesis has also led to the view that natural
products may not be the best choice for leads against most
other human disease targets, since humans did not co-evolve
with the organisms that produce them. Indeed, a systemslevel study of the protein targets of natural products revealed
that they tend to be more highly connected pathway “hubs”
than human disease-related targets in general[136].
Yet, while evolution may have selected natural products
that inhibit a relatively narrow range of human disease targets, the realm of PK may represent a nexus where the constraints driving both natural product evolution and drug design converge. In terms of natural selection, the ability to
cross biological membranes and avoid metabolic degradation, both oxidative and proteolytic, are likely to be just as
important as binding affinity in the evolution of natural
products. Large cyclic peptides, in particular, seem to have
evolved tricks that allow them to push the PK envelope,
maximizing size and complexity (toward greater potency)
while maintaining good stability and membrane permeability. Thus, the design of combinatorial libraries of “natural
product-like” molecules, which attempt to recapitulate the
diversity and complexity of natural products without the target limitations set by natural selection, can perhaps be informed by the structural features shared by natural products
which favor permeability and stability. For cyclic peptides,
these features include general trends in side chain functionality, and also specific patterns of backbone stereochemistry
and N-methylation, which permit or even stabilize membrane
permeable conformations. Indeed, it is likely that cyclic peptide natural products will continue to inform our understanding of the relationship between structure and pharmacokinetics, especially as we attempt to design new molecules in the
outer reaches of “drug-like” chemical space, for some time
to come.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts of interest.
ACKNOWLEDGEMENTS
Declared none.
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