Screening bicyclic peptide libraries for protein-protein interaction inhibitors: Discovery of a tumor necrosis factor-α Antagonist

Article abstract of DOI:10.1021/ja405106u

Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well-defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions. Screening of a bicyclic peptide library against tumor necrosis factor-α (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions.

Full text of DOI:10.1021/ja405106u

Article
pubs.acs.org/JACS
Screening Bicyclic Peptide Libraries for Protein−Protein Interaction
Inhibitors: Discovery of a Tumor Necrosis Factor‑α Antagonist
Wenlong Lian,†,§ Punit Upadhyaya,†,§ Curran A. Rhodes,† Yusen Liu,‡ and Dehua Pei*,†
†Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210,
United States
‡Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics,
The Ohio State University College of Medicine, 700 Children’s Drive, Columbus, Ohio 43205, United States
S
* Supporting Information
ABSTRACT: Protein−protein interactions represent a new
class of exciting but challenging drug targets, because their
large, flat binding sites lack well-defined pockets for small
molecules to bind. We report here a methodology for chemical
synthesis and screening of large combinatorial libraries of
bicyclic peptides displayed on rigid small-molecule scaffolds.
With planar trimesic acid as the scaffold, the resulting bicyclic
peptides are effective for binding to protein surfaces such as the
interfaces of protein−protein interactions. Screening of a bicyclic
peptide library against tumor necrosis factor-α (TNFα) identified
a potent antagonist that inhibits the TNFα−TNFα receptor interaction and protects cells from TNFα-induced cell death.
Bicyclic peptides of this type may provide a general solution for inhibition of protein−protein interactions.
properties of small molecules such as membrane permeability.22−24
Compared to protein drugs, macrocycles have greater metabolic
stability, less likelihood of eliciting an immune response, and
INTRODUCTION
■
Protein−protein interactions (PPIs) are of central importance
in essentially all biochemical pathways, including those involved
lower cost of production. To further rigidify the structures and
in disease processes. PPIs therefore represent a large class of
improve the binding affinity/specificity and metabolic stability,
new, exciting drug targets.1 However, PPIs are considered the
prototypical “undruggable” or “challenging” targets for the
bicyclic peptides and peptoids have also been generated.25−32
Since rational design of macrocyclic inhibitors against PPIs
conventional small-molecule approach, because PPIs usually
is difficult, a popular approach has involved synthesizing and
involve large, flat interfaces, with which small molecules usually
do not make enough points of contact to impart high affinity or
specificity. For some of these PPIs, small-molecule inhibitors
screening large libraries of bicyclic peptides and peptoids. To
date, bicyclic peptide libraries have only been synthesized
ribosomally by phage or mRNA display and are largely limited to
have been successfully developed by targeting the so-called “hot
spots” at the interaction interface.1−4 A more general approach is
proteinogenic amino acids (and certain unnatural α-L-amino
acids) as building blocks.27−29
to develop specific antibodies against the PPI interface.5−7
Tumor necrosis factor-α (TNFα) is a pleiotropic inflamma-
Nonimmunoglobulin protein scaffolds have also been engi-
tory cytokine of a variety of functions, many of which are not yet
neered into specific binders to target proteins through library
fully understood.33 TNFα is responsible for cachexia, a wasting in
screening and/or in vitro evolution.8−11 Antibodies and protein
patients with chronic diseases such as cancer and tuberculosis,34
binders possess large binding surfaces of their own and are
and is implicated in the development of septic shock and multi-
capable of making multiple contacts with a target surface (e.g.,
organ failure in severely infected patients.35 It is also responsible
those involved in PPIs). Unfortunately, protein-based drugs are
impermeable to the mammalian cell membrane; as such they are
generally limited to targeting extracellular proteins and are not
for numerous chronic inflammatory disorders such as rheuma-
toid arthritis, ankylosing spondylitis, inflammatory bowel disease,
psoriasis, hidradenitis suppurativa, and refractory asthma.36
These disorders are currently treated with protein inhibitors,
including monoclonal antibodies infliximab (Remicade), adalimumab
(Humira), or certolizumab pegol (Cimzia), and a circulating receptor
fusion protein etanercept (Enbrel). These proteins bind specifically to
TNFα and prevent its interaction with TNFα receptors (TNFRs).
orally available. Recently, others12−18 and we19,20 have begun a
third approach by generating macrocyclic compounds (e.g.,
cyclic peptides and peptidomimetics) as PPI inhibitors. These
macrocycles typically have molecular weights between 500 and
2000 and occupy a largely untapped therapeutic space, often
referred to as the “middle space”.21 Because of their relatively
large sizes and ability to make multiple points of contact with
a flat surface, macrocycles effectively compete with proteins for
binding to flat surfaces and yet retain many of the pharmacokinetic
Received: May 21, 2013
© XXXX American Chemical Society
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Figure 1. Synthesis of bicyclic peptide library. Reagents and conditions: (a) Standard Fmoc/HATU chemistry; (b) soak in water; (c) 0.4 equiv of Fmoc-
OSu in Et2O/CH2Cl2; (d) di-tert-butyl dicarbonate; (e) piperidine; (f) 4-hydroxybenzoic acid/HBTU/HOBT; (g) Fmoc-β-Ala-OH/DIC; (h) 50%
TFA in DCM; (i) split-and-pool synthesis by Fmoc/HATU chemistry; (j) 2% TFA in DCM (6×); (k) Fmoc-OSu/DIPEA in DCM; (l) Pd(PPh3)4; (m)
diallyl protected trimesic acid/HATU; (n) PyBOP/HOBT/DIPEA; (o) modified reagent K.
These biologic drugs are administered by in-hospital intravenous
injections. Considerable efforts have been made over the past two
decades todevelop small-molecule inhibitors againstTNFα, which
have thepotentialtobeadministeredorally. However, these efforts
have so far led to only a few weak inhibitors.37−42 In this work, we
developed a general methodology for chemical synthesis and
screening of large combinatorial libraries of bicyclic peptides
displayed on rigid small-molecule scaffolds. Chemical synthesis
permitted the incorporation of unnatural amino acids (e.g.,
D-amino acids) and potentially nonpeptidic building blocks into
the bicyclic molecules, increasing their structural diversity and
metabolic stability. Screening of a bicyclic peptide library against
TNFα identified a compound that inhibits the TNFα−TNFR
interaction and protects cells from TNFα-induced cell death.
surfaces. To test the validity of this approach, we designed a
bicyclic peptide library by “wrapping” a peptide sequence of
6−10 random residues around a trimesoyl group (Figure 1). Pep-
tide cyclization was mediated by the formation of three amide
bonds between trimesic acid and the N-terminal amine, the side
chain of a C-terminal L-2,3-diaminopropionic acid (Dap), and
the side chain of a fixed lysine within the random region.
The resulting bicyclic peptides contained 3−5 random residues
in each ring. The random sequence was constructed with a
24-amino acid set that included 10 proteinogenic amino acids
[Ala, Arg, Asp, Gln, Gly, His, Ile, Ser, Trp, and Tyr], 4 non-
proteinogenic α-L-amino acids [L-4-fluorophenylalanine (Fpa),
L-norleucine (Nle), L-ornithine (Orn), and L-phenylglycine
(Phg)], and 10 α-D-amino acids [D-2-naphthylalanine (D-Nal),
D-Ala, D-Asn, D-Glu, D-Leu, D-Lys, D-Phe, D-Pro, D-Thr, and
D-Val]. These building blocks were selected on the basis of their
structural diversity, metabolic stability, and commercial avail-
ability. Although not included here, nonpeptidic building blocks
are also compatible with our library synthesis and decoding
method.19 This library has a theoretical diversity of 6.6 × 1013. In
practice, the library size is limited by the amount of resin that can
be conveniently employed and typically on the order of 107 (vide
infra). Despite the fact that only a small fraction of all possible
structures can be synthesized, we felt that it is critical to sample a
large structural space. Once an active compound is identified, its
affinity and specificity for the target protein may be improved by
synthesizing and screening a second-generation library consist-
ing of analogues of the initial hit. Inclusion of the unnatural
amino acids increases the structural diversity and metabolic
RESULTS AND DISCUSSION
■
Design and Synthesis of Bicyclic Peptide Library. Anti-
bodies recognize specific antigens by utilizing six small loops,
called the “complementarity determining regions”. By grafting
two or more flexible loops onto rigid protein scaffolds, other
investigators have engineered protein binders of antibody-like
affinity and specificity.8−11 We envisioned that displaying pep-
tidic loops on rigid small-molecule scaffolds should also generate
molecules that rival antibodies for binding affinity and specificity.
To develop inhibitors against PPIs, we chose a planar structure as
the scaffold, which should bias the resulting bicyclic peptides
toward an overall “planar” (as opposed to globular) shape. An
overall planar geometry would maximize the surface area of the
molecules and therefore their ability to interact with flat protein
B
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Figure 2. Scheme showing the steps involved in peptide library screening against TNFα.
stability of the library compounds but necessitates chemical
synthesis of the library.
were removed, and the surface peptides were cyclized by treat-
ment with benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyBOP). The peptides inside the beads
were unaffected by the cyclization procedure due to lack of the
Dap residue and remained in the linear form to serve as encoding
tags. Note that macromolecular targets (e.g., proteins) cannot
diffuse into the bead interior, and thus the linear encoding
peptides do not interfere with library screening. The symmetry of
the trimesoyl unit ensured that a single bicyclic product was
formed on each bead.
Library Screening against TNFα. The bicyclic peptide
library was subjected to four rounds of screening against
recombinant TNFα that contained an N-terminal ybbR tag48
(MVLDSLEFIASKL) and had been specifically labeled at the
ybbR tag with a biotin or fluorescent dye molecule. During the
first round, 100 mg of the bicyclic peptide library (∼3 × 105
beads) was incubated with biotinylated TNFα (0.8 μM) and
streptavidin-coated magnetic particles (Figure 2). The resulting
magnetic beads (∼400 beads) were isolated from the library by
magnetic sorting,49,50 during which the positive beads were
attracted to the wall while the negative beads settled to the
bottom of the container. The ∼400 beads were washed,
incubated again with the biotinylated TNFα (1.5 μM), and
subjected to a second round of screening using an on-bead
enzyme-linked assay and a streptavidin−alkaline phosphatase
(SA-AP) conjugate. Binding of TNFα to a bead recruited SA-AP
to the bead surface and,upon the addition of 5-bromo-4-chloro-
3-indolyl phosphate (BCIP), produced a turquoise-colored
precipitate on that bead. This procedure resulted in 150 intensely
colored beads, which were manually isolated with a micropipet
and incubated with Texas-red-labeled TNFα (0.3 μM). The
44 most fluorescent beads were selected under a fluorescence
microscope.
The main challenge associated with screening chemically
synthesized bicyclic peptide libraries is the structural determi-
nation of hit compounds. To overcome this difficulty, we
synthesized the bicyclic peptide library in the one bead−two
compound (OBTC) format on 2.0 g of TentaGel microbeads
(90 μm; ∼100 pmol peptide/bead; 2.86 × 106 beads/g). Each
library bead was topologically segregated into two different
layers, with the outer layer displaying a unique bicyclic peptide
and the inner layer containing the corresponding linear peptide
as an encoding tag (Figure 1). This was achieved by quickly
suspending wet beads in 1:1 (v/v) DCM/Et2O containing
0.5 equiv of N-(9-fluorenylethoxycarbonyloxy)succinimide
(Fmoc-OSu).43,44 Because the organic solvent is immiscible
with water, only peptides on the bead surface were exposed to
and reacted with Fmoc-OSu. The beads were washed with DMF,
and the remaining free N-terminal amines inside the beads were
protected with a Boc group. After removal of the Fmoc group,
a p-hydroxymethylbenzoic acid (Hmb) linker was added (for
selective release of the bicyclic peptide), followed by the addition
of β-Ala, L-propargylglycine (Pra), two β-Ala, and Fmoc-L-
Dap(Alloc)-OH. The Pra residue serves as a handle for selective
labeling of the bicyclic peptide via click chemistry (vide infra).
The Dap residue permits attachment of the bicyclic peptide to
the solid support as well as providing a side chain for peptide
cyclization. The N-terminal Boc group was then removed from
the inner peptides by treatment with trifluoroacetic acid (TFA),
and an arginine residue was added to provide a fixed positive
charge, which facilitates later peptide sequencing by mass
spectrometry. The random region was synthesized by the split-
and-pool method,45−47 and an Nε-4-methoxytrityl (Mmt)-
protected lysine was added in the middle of the random
sequence to provide a side-chain amine for peptide cyclization.
Following completion of the linear peptide synthesis, the Mmt
group was removed using 2% TFA in DCM and replaced with
an Fmoc group (the Mmt group was partially removed
during deprotection of the Alloc group). The Alloc group on the
C-terminal Dap was removed by treatment with Pd(PPh3)4, and
the exposed side chain amine was acylated with diallyl trimesic
acid. Finally, the allyl (on the trimesoyl moiety) and Fmoc
protecting groups (on the N-terminus and the lysine side chain)
Finally, the 44 beads were treated with tetramethylrhodamine
(TMR) azide in the presence of Cu(I), resulting in selective
labeling of the bicyclic peptides at the Pra residue (Figures 2 and
3a). The beads were then placed into individual microcentrifuge
tubes (1 bead/tube), and the TMR-labeled bicyclic peptide was
released from each bead by treatment with 0.1 M NaOH, which
selectively hydrolyzed the Hmb ester linkage associated with
the bicyclic peptide. After neutralization, the released bicyclic
peptide from each bead was tested for binding to TNFα in
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Figure 3. Solution-phase screening of initial hits (fourth round). (a) Selective labeling of bicyclic peptides with tetramethylrhodamine (TMR) and their
release from individual beads by base hydrolysis. (b) Evaluation of the 44 released bicyclic peptides for binding to TNFα in solution by fluorescent
anisotropy using a fixed concentration of TNFα (5 μM) and TMR-labeled bicyclic peptide (100 nM).
solution by fluorescence anisotropy (FA).51,52 Each of the
44 bicyclic peptides (∼100 nM) was incubated with 5 μM TNFα,
and the 12 bicyclic peptides that showed ≥15% anisotropy
increase (relative to the control without TNFα) (Figure 3b)
were further analyzed at varying concentrations of TNFα
(0−18 μM) to determine their dissociation constants (KD). Six
peptides (beads No. 1, 16, 22, 24, 36, and 41) had KD values
ranging from 0.8 to 7.8 μM (Figure S1 in Supporting Information
[SI]), while the other peptides showed no significant TNFα
binding (bead No. 18, 19, 28, 29, 31, and 44). Next, for the 6
binding peptides, their corresponding beads containing the linear
encoding peptides were retrieved from the microcentrifuge tubes
and subjected to partial Edman degradation-mass spectrometry
(PED-MS) analysis.53 Two of the beads (hits No. 1 and 36) produced
mass spectra of sufficient quality, from which unambiguous, complete
sequences of bicyclo(Phg-Tyr-D-Ala-Lys-Tyr-D-Phe-Gly-D-Lys-
His-Dap) and bicyclo(Ala-D-Phe-Trp-D-Thr-Gln-Lys-Nle-
D-Leu-Ala-His-Dap) were obtained (Figure 4a and Figure S2 in SI).
These compounds are named as anticachexins C1 and C2 there-
after, respectively.
interactions (i.e., simultaneous interaction of a single TNFα
molecule with two or more resin-bound bicyclic peptides) and
high avidity.54 False negatives are also possible as a result of
several factors (e.g., poor aqueous solubility of a bicyclic peptide,
inefficient release of a bicyclic peptide from resin by 0.1 M NaOH
due to its strong noncovalent binding to the hydrophobic
TentaGel resin, and/or strong binding of a bicyclic peptide to
bovine serum albumin which was present in all FA assays).
Elimination of these false negative compounds at this stage is
actually desirable, as they are likely very hydrophobic and
may bind nonspecifically to many proteins. This highlights the
importance of our library design, which permits selective release
of the bicyclic peptide and therefore solution-phase binding
analysis and avoids the need to individually resynthesize all 44
initial hits.
Binding Affinity and Specificity of Hit Compounds for
TNFα. Anticachexins C1 and C2 and the linear and monocyclic
variants of anticachexin C1 were resynthesized with a fluorescein
isothiocyanate (FITC) label (Figure S3 in SI), purified by HPLC,
and assayed against TNFα by FA analysis. Anticachexin C1
and C2 bound to TNFα with KD values of 0.45 and 1.6 μM,
respectively (Figure S3 in SI). The linear C1 variant exhibited
weak binding to TNFα (KD > 10 μM), whereas the monocyclic
peptide showed no significant binding affinity. Bicyclo(Arg-Arg-
Arg-Arg-Nal-Phe-Dap-Ser-D-Val-Pro-pTyr-His-Dap), a control
peptide unrelated to TNFα, also showed no detectable binding
The fact that only a relatively small number of the hits derived
from on-bead screening (6 out of 44 beads) showed strong
binding to TNFα in solution suggests that most of the initial hits
were weak binders or false positives, a problem commonly
associated with on-bead screening. Most likely, the high ligand
density on the library beads (∼100 mM) resulted in multidentate
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and a GST−BRCT fusion protein. Anticachexin C1 showed
weak binding to BSA (KD ≈ 34 μM), but not to any of the other
proteins, while anticachexin C2 was less selective and showed
substantial binding to BSA and GST−SH2 proteins (KD values of
7.4 and 1.5 μM, respectively; Figure S4 in SI). Finally, unlabeled
anticachexins C1 and C2 inhibited the binding of FITC-labeled
anticachexin C2 to TNFα in a concentration-dependent manner
(IC50 values of ∼1 and ∼4 μM, respectively) (Figure S5 in SI),
suggesting that both compounds bind to the same (or overl-
apping) site on TNFα. Because of its higher affinity and specificity
for TNFα, anticachexin C1 was selected for further biological tests.
Inhibition of TNFα−TNFR Interaction by Anticachexin
C1. TNFα signaling begins with the binding of the TNFα trimer
to the extracellular domain of TNFR1, triggering the release of
an inhibitory protein, silencer of death domains, from the intra-
cellular domain of TNFR1.33 To test whether anticachexin C1
inhibits the interaction between TNFα and TNFR1, biotinylated
TNFα was immobilized onto a Neutravidin-coated 96-well
microtiter plate. The plate was incubated with 0.5 nM
horseradish peroxidase (HRP)-conjugated TNFR1 in the
presence of varying concentrations of anticachexin C1. After
the wells were washed, the amount of HRP−TNFR1 bound to
each well was quantitated by ELISA.55 Anticachexin C1 inhibited
the TNFα−TNFR1 interaction in a concentration-dependent
manner, with an IC50 value of 3.1 0.3 μM (Figure 4b).
Protection against TNFα-Induced Cell Death. The ability
of anticachexin C1 to protect cells against TNFα-induced cell
death was tested with cultured WEHI-13VAR fibroblasts, which
are highly sensitive to TNFα in the presence of actinomycin-D.56
The cells were treated with a fixed concentration of TNFα
(0.04 ng/mL) and varying concentrations of anticachexin C1
(0−25 μM), and the fraction of live cells was quantitated by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. Anticachexin C1 protected the cells from
TNFα-induced cell death in a concentration-dependent manner,
whereas the corresponding monocyclic and linear peptides did
not (Figure 4c). As expected, the control bicyclic peptide that
does not bind TNFα had no protective effect. The MTT assay
was also conducted at a fixed concentration of anticachexin C1
(50 μM) but varying concentrations of TNFα (0−250 ng/mL).
TNFα exhibited an LD50 value of 0.46 ng/mL in the absence of
TNFα inhibitor; in the presence of 50 μM anticachexin C1, the
LD50 value was shifted to 1.8 ng/mL (Figure S6 in SI).
CONCLUSION
■
In this study, we report the first chemical synthesis and screening
of a large combinatorial library of bicyclic peptides against a
macromolecular target of biomedical significance. Compared to
previous methods for bicyclic peptide library synthesis,27−29
which involve ribosomal peptide synthesis followed by chemical
cyclization, our method has the advantage that it allows the
incorporation of any unnatural amino acid or nonpeptidic
building blocks, greatly increasing the structural diversity and
metabolic stability of the cyclic peptides. Chemical synthesis also
allows for the use of orthogonal protecting groups, which in turn
permits more “forcing” reaction conditions to drive the desired
cyclization reaction to completion and prevents any undesired
cyclization reaction from occurring. We demonstrate that bicyclic
peptides displayed on a rigid planar scaffold are effective for
binding to protein surfaces such as PPI interfaces. With a KD value
of 0.45 μM, anticachexin C1 is the most potent nonprotein TNFα
inhibitor reported to date. The bicyclic peptide library may be
readily screened against other protein and nucleic acid targets.
Figure 4. (a) Structure of anticachexin C1. (b) Inhibition of TNFα−
TNFR1 interaction by anticachexin C1. The absorbance values on the y
axis, which reflect the amount of TNFR1 bound to immobilized TNFα
in the presence of increasing concentrations of anticachexin C1,
are relative to that in the absence of peptide inhibitor (100%). (c)
Protection of WEHI-13VAR cells against TNFα 0.04 ng/mL-induced
cell death by anticachexin C1 (0−25 μM). The absorbance values on the
y axis, which reflect the number of live cells, are relative to that of DMSO
control (no TNFα, no peptide).
to TNFα. These results demonstrate that both the peptide
sequence and the overall bicyclic structure are critical for binding
to the target protein. To determine whether anticachexin C1 and
C2 are specific ligands of TNFα, we tested them for binding to
five arbitrarily selected proteins, including bovine serum albumin
(BSA), a glutathione-S-transferase-PLCγ SH2 domain fusion
(GST−SH2), protein phosphatase PTP1B, HIV capsid protein,
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(23) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem. Res.
2008, 41, 1331−1342.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and additional data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(24) Qian, Z.; Liu, T.; Liu, Y.-Y.; Briesewitz, R.; Barrios, A. M.; Jhiang,
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AUTHOR INFORMATION
■
Corresponding Author
pei.3@osu.edu
(28) Chen, S.; Morales-Sanfrutos, J.; Angelini, A.; Cutting, B.; Heinis,
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Author Contributions
§W.L. and P.U. contributed equally.
(30) Timmerman, P.; Barderas, R.; Desmet, J.; Altschuh, D.; Shochat,
Notes
S.; Hollestelle, M. J.; Hoppener, J. W. M.; Monasterio, A.; Casal, J. L.;
̈
Meloen, R. H. J. Biol. Chem. 2009, 284, 34126−34134.
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Institutes
of Health (GM062820 and CA312855).
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