Identification, Characterization, and Optimization of Integrin αvβ6-Targeting Peptides from a One-Bead One-Compound (OBOC) Library: Towards the development of positron emission tomography (PET) imaging agents

Article abstract of DOI:10.3390/molecules24020309

The current translation of peptides identified through the one-bead one-compound (OBOC) technology into positron emission tomography (PET) imaging agents is a slow process, with a major delay between ligand identification and subsequent lead optimization.

Full text of DOI:10.3390/molecules24020309

molecules
Article
Identification, Characterization, and Optimization of
Integrin α β -Targeting Peptides from a One-Bead
v 6
One-Compound (OBOC) Library: Towards the
Development of Positron Emission Tomography (PET)
Imaging Agents
Yng (Sarah) C. Tang 1 , Ryan A. Davis 2 , Tanushree Ganguly 1 and Julie L. Sutcliffe 1,2,3,4,
*
1
Department of Internal Medicine, Division of Hematology/Oncology, University of California, Davis and
Sacramento, CA 95817, USA; yngtang@ucdavis.edu (Y.C.T.); tganguly@ucdavis.edu (T.G.)
Department of Biomedical Engineering, University of California, Davis, CA 95616, USA;
rydavis@ucdavis.edu
Center for Molecular and Genomic Imaging, University of California, Davis, CA 95616, USA
Radiochemistry Research and Training Facility, University of California, Davis, Sacramento, CA 95817, USA
Correspondence: jlsutcliffe@ucdavis.edu; Tel.: +1-916-734-5536
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 December 2018; Accepted: 10 January 2019; Published: 16 January 2019
Abstract: The current translation of peptides identified through the one-bead one-compound (OBOC)
technology into positron emission tomography (PET) imaging agents is a slow process, with a major
delay between ligand identification and subsequent lead optimization. This work aims to streamline
the development process of 18F-peptide based PET imaging agents to target the integrin α β6. By
v
directly identify α β6–targeting peptides from a 9-mer 4-fluorobenzoyl peptide library using the
v
on-bead two-color (OBTC) cell-screening assay, a total of 185 peptide beads were identified and 5
beads sequenced for further evaluation. The lead peptide
25% stable in serum at 1 h) was further modified at the N-, C-, and bi-termini. C-terminal PEGylation
increased the metabolic stability (>95% stable), but decreased binding affinity (IC50 = 3.7 M) was
noted. C-terminal extension (1i, VGDLTYLKK(FB)KVART) significantly increased binding affinity for
integrin α β6 (IC50 = 0.021 0.002 M), binding selectivity for α β6-expressing cells (3.1 0.8:1), and
the serum stability (>99% stable). Our results demonstrate the challenges in optimizing OBOC-derived
peptides, indicate both termini of are sensitive to modifications, and show that further modification
of 1 is necessary to demonstrate utility as an 18F-peptide imaging agent.
1
(VGDLTYLKK(FB), IC50 = 0.45
±
0.06 µM,
±
1 µ
±
µ
±
v
v
1
Keywords: one-bead one-compound (OBOC); integrin α β6; library screening; positron emission
v
tomography (PET); Fluorine-18; solid-phase radiolabeling; affinity; selectivity
1. Introduction
The expanding use of positron emission tomography (PET) imaging in oncology to aid in early
detection, more accurate diagnosis, and patient treatment stratification has led to an increased demand
of novel peptide-based molecular imaging agents [
several reasons, including favorable pharmacokinetic properties, such as high target affinity, good
tumor penetration, rapid non-specific clearance, and minimal immunogenicity [ ]. Additionally, once
peptide sequences are identified, they can be readily synthesized to high purity and are amenable
to chemical modifications to alter affinity, charge, hydrophobicity, solubility, and stability [ ]. The
1]. Peptides are attractive as imaging agents for
2
3
biological half-life of peptides ideally matches with many clinically used radionuclides, such as the
short-lived positron emitting fluorine-18 (t1/2 = 109.7 min) that possesses favorable nuclear properties
Molecules 2019, 24, 309; doi:10.3390/molecules24020309
www.mdpi.com/journal/molecules

Molecules 2019, 24, 309
2 of 16
(e.g., 97% positron emission), and the ease of production with high yield and molar activity for PET
imaging [4].
Combinatorial libraries, such as the one-bead one-compound (OBOC) and phage display, have
provided indispensable tools for rapid identification of peptides for a given biological target, especially
those expressed on the cell surface [
on solid-support beads, which can be screened against either purified proteins or whole cells to
identify receptor-specific peptides [ ]. We, and others, have utilized the OBOC approach to identify
peptides for the development of PET imaging agents [ ]. However, to date, only a handful of
OBOC-derived peptides, notably the α5β1–targeting OA02 peptide [ ] and the α4β1-targeting LLP2A
peptidomimetic [ 10], have been evaluated for preclinical in vivo imaging. Clearly, the current
5]. OBOC libraries display randomized peptide sequences
6
6,7
8
9
,
translation of OBOC-derived peptides from bench to bedside remains a slow process [11].
To ensure target-specificity of the identified peptides, on-bead library screening requires multiple
rounds of screening to eliminate false positive (non-specific binders) [12]. This stringent and iterative
screening of OBOC libraries for target-selective peptides is a lengthy, time-consuming, and inefficient
process. For OBOC-derived peptides to be utilized as PET imaging agents, a positron-emitting isotope,
such as fluorine-18, needs to be attached to the peptide. The introduction of the radioisotope, typically
via a prosthetic group post-identification from the OBOC library, can have a negative impact on
the pharmacokinetic properties and binding affinity of the original peptide, as it adds mass, alters
hydrophobicity, and changes the charge of the peptide [13]. In addition, once a peptide sequence
has been successfully identified, it typically requires further optimization before it can be used as
an imaging agent, as peptides are susceptible to proteolytic degradation in serum in vivo, and are
often eliminated rapidly from the system [14]. Therefore, chemical modifications of the OBOC-derived
peptides are carried out to improve their in vivo stability to make them suitable for PET imaging.
In this work, we proposed to address some of the aforementioned limitations and describe the
developmental process for the identification and optimization of integrin α β6-targeting peptide
v
PET imaging agents, using OBOC and on-bead two-color (OBTC) technologies. The integrin α β
v
6
is an epithelial-specific cell surface receptor that is undetectable in healthy adult epithelium, but
is upregulated in many aggressive cancers, such as ovarian, colon, pancreatic, and breast, and
its expression level has been linked to poor prognosis, making it an interesting target for both
diagnostic and therapeutic agents [15]. Notable examples of integrin α β6-targeting peptides that
v
have been used as imaging agents include the A20FMDV2 [16] and Cystine Knot R01 peptides [17].
Both peptides contain the known α β6-targeting motif (DLXXL), originally identified from phage
v
display [18], and they have been radiolabeled with 18F and 64Cu for in vivo imaging in small animal
models [7,16,17,19,20]. In a proof of concept effort, Gagnon et al. reported the use of combinatorial
libraries, solid-phase radiolabeling, and high throughput in vivo screening to evaluate an OBOC library
featuring the DLXXL motif, and discovered 42 lead peptides in 11 consecutive days [
identified lead peptides still required substantial optimization.
7]. However, the
Herein, we designed an OBOC library containing the known integrin α β6-binding motif
v
(DLXXL) [18] and the nonradioactive substitute of the commonly used 18F-radiolabeling prosthetic
group, 4-fluorobenzoic acid ([19F]FBA) at the C-terminal lysine (XXDLXXLXK([19F]FB, 9-mer).
To facilitate the direct identification of integrin α β6-binding peptides in a single step, an OBTC
v
cell screen assay [21], utilizing transfected fluorescent DX3puroβ6mCherry (α β6+, red) and
v
DX3puroEmGFP (α β −, green) cell lines, was investigated. Competitive binding ELISAs with
v
6
purified integrins (α β , α β , α β , α β6, and α β8) were performed to evaluate the binding profiles
v v v v
1 3 5
v
of lead peptides. Furthermore, efforts focused on optimizing the most promising peptide via N-,
C-, and bi-terminal chemical modifications such as methylation, acetylation, 4-fluorobenzoylation,
and PEGylation, as well as C-terminal extension for improved binding affinity, binding selectivity,
and metabolic stability. Peptides were radiolabeled on solid-phase using 4-[18F]fluorobenzoic acid
([18F]FBA) for evaluation of cell binding and serum stability.

Molecules 2019, 24, 309
3 of 16
2. Results
2.1. Design and Synthesis of 4-Fluorobenzoyl OBOC Library
Using standard solid-phase peptide synthesis (SPPS) and the split-mix approach, the 9-mer
peptide library of sequence “XXDLXXLXK([19F]FB)” was built containing the integrin α β6-targeting
v
motif (DLXXL) and the nonradioactive [19F]FBA, coupled to the sidechain of the built-in C-terminal
lysine (K) at gram scale to yield approxmately 2.5 million unique sequences (theoretical permutation
of 2.47 × 106 peptides) (Scheme 1).
TentaGel resin
C
B
A
B
B
B
A
A
A
C
C
C
Library
O
O
H
20% piperidine in DMF
Fmoc-Lys(Alloc)-OH
HATU, DIPEA, DMF
O
N
H2N
H2N
N
N
H
H
O
Tentagel
C
B
B
A
C
A
HN
O
NH(Alloc)
C
C
A
A
A
A
B
C
B
B
B
O
B
A
C
C
Library = XXDLXXLX
O
HN
1.4-fluorobenzoic acid
HATU, DIPEA, DMF
2. 20% piperidine in DMF
F
Pd(PPh3)4, PhSiH3, DCM
Fmoc-XXDLXXLXK
Alloc
Fmoc-XXDLXXLXK
NH2
H
H2N
XXDLXXLX
N
3. TFA:H2O: EDT:TIPS
(94.5:2.5:2.5:1)
N
H
O
~2.5 million beads
Scheme 1. Design and synthesis of integrin α β -targeting 4-fluorobenzoyl-peptide library.
v
6
2.2. On-Bead Two-Color (OBTC) Fluorescent Cell Screening Assay
Dx3puroβ mCherry (α β +, red) and DX3puroEmGFP (α β −, green) cells (500,000 total) were
v
v
6
6
6
premixed in a petri dish for an hour prior to incubating with library beads (150,000) (Figure 1). A total
of 185 beads were manually identified as integrin α β6-specific (i.e., beads that were coated exclusively
v
with the red cells (α β6+)) and were isolated from the assay over 5 days. The first 5 beads isolated
v
from the assay (
1
–5) were sequenced by Edman degradation and their sequences are shown in Table 1.
v6-selective
185 beads
(+)
Manual bead isolation
(+/−)
nonspecific
150,000 library beads
DX3puro6mCherry (v6 +)
DX3puroEmGFP (v6 −)
Figure 1. Schematic representation of the On-Bead Two-Color (OBTC) cell screening utilizing fluorescent
DX3puroβ mCherry (α β +, red) and DX3puroEmGFP (α β −, green) cells.
v
v
6
6
6

Molecules 2019, 24, 309
4 of 16
Table 1. ELISA data showing the relative binding affinity and selectivity of lead peptides measured by
IC50 values (n = 3).
IC50 (µM)
α β
Sequence
ID
α β
v
α β
v
α β
v
α β
v
8
v
1
3
5
6
1
2
3
4
5
VGDLTYLKK(FB)
RGDLMKLAK(FB)
RGDLADLRK(FB)
GIDLTSLTK(FB)
RGDLRELAK(FB)
>100
0.9 ± 0.12
0.6 ± 0.15
ND
>100
>100
>100
>100
ND
0.45 ± 0.06
0.95 ± 0.17
0.42 ± 0.07
>100
5.3 ± 0.14
2.6 ± 0.35
0.2 ± 0.02
ND
2.2 ± 0.83
0.5 ± 0.08
>100
1.9 ± 0.3
0.3 ± 0.02
>100
0.89 ± 0.14
2.6 ± 0.29
ND = not determined.
2.3. On-Bead Validation of the Identified Peptide Sequences
In the mixed cell assay, all peptides showed good binding to the DX3puroβ6mCherry (α β6+,
v
red) cells and minimal cross binding to DX3puroEmGFP (α β −, green) cells, except
4
. In the single
v
6
cell assay,
whereas
to DX3puroEmGFP (α β −, green) cells in the single cell assay.
1
showed high selectivity for α β6+, red cells with little binding to α β −, green cells,
v
v
6
4
showed no binding to either cell line (Figure 2). 2, 3, and 5 also showed noticeable binding
v
6
Figure 2. Representative pictures of
DX3puroEmGFP (α β −, green); and (
1
–
5
incubated with (
a) DX3puroβ6mCherry (α β6+, red); (b)
v
c) mixed DX3puroβ6mCherry (α β6+, red), and DX3puroEmGFP
v
v
6
(α β −, green) cells 3 h post-incubation.
v
6
2.4. Competitive Binding ELISAs
As summarized in Table 1, all lead peptides, except for
4
, showed low micromolar IC50 values
was the most
M), demonstrating a selectivity ratio of >200:1 when
α β3, and α β5), and with some binding to integrin α β
for α β6, i.e., moderate to good binding to purified integrin α β6. In summary,
1
v
v
selective for integrin α β6 (IC50 = 0.45
±
0.06 µ
v
compared to the other integrins (α β
,
v
v
v
v
8
1
(IC50 = 5.3 ± 0.14 µM, 20:1). The peptides containing the RGD motif (
affinity for integrin α β6 (IC50 = 0.95 0.17, 0.42 0.07, and 0.89 0.14
poor selectivity towards other integrins (α β α β3, and α β8, except for α β (>100:1)). Meanwhile,
2
,
3, and
5) all showed comparable
±
±
±
µM, respectively), however, with
v
,
v
v
v
v
5
1
4
consistently showed little to no binding to integrins α β6 and α β3, only achieving an IC50 > 100 µM.
v v

Molecules 2019, 24, 309
5 of 16
2.5. Modifications of 1
Peptide
were explored, including moving the 4-fluorobenzoyl moiety from the C-terminal lysine to the
N-terminus (1a, IC50 = 0.81 0.09 M), N-terminal acetylation (1b, IC50 > 100 M), N-terminal
methylation (1c, IC50 > 5 M), N-terminal PEGylation (1d, IC50 > 100 M), and containing both the
C- and N-terminal 4-fluorobenzoylation (1e, IC50 = 0.84 0.36 M). Modification of the C-terminus
was also done via PEGylation (1f, IC50 = 3.7 M). In addition, the C-terminal PEGylated
peptides were also synthesized as the N-acetylated (1g, IC50 > 100 M) and N-fluorobenzoylated
was also further extended by five amino acids,
1 was selected for further optimization (Table 2). First, a series of N-terminal modifications
±
µ
µ
µ
µ
±
µ
±
1 µ
µ
(1h, IC50 = 1.47 ± 0.23 µM) analogs. The C-terminus of
KVART, (1i, IC50 = 0.021 ± 0.002 µM).
1
Table 2. ELISA data showing the relative binding affinity of derivatives of
by IC50 values (n = 3).
1 to integrin α β measured
v
6
ID
Sequence
IC50 (µM) α β
v
6
1 *
1a *
1b
VGDLTYLKK(FB)
FB-VGDLTYLKK
Ac-VGDLTYLKK(FB)
0.45 ± 0.06
0.81 ± 0.09
>100
1c
1d
1e
1f *
1g
Me-VGDLTYLKK(FB)
PEG12-VGDLTYLKK(FB)
FB-VGDLTYLKK(FB)
VGDLTYLKK(FB)-PEG28
Ac-VGDLTYLKK(FB)-PEG28
FB-VGDLTYLKK(FB)-PEG28
VGDLTYLKK(FB)KVART
>5
>100
0.84 ± 0.36
3.7 ± 1
>100
1h *
1i *
1.47 ± 0.23
0.021 ± 0.002
Note: * peptides that were 18F-radiolabeled for cell binding and serum stability studies.
2.6. Radiochemical Synthesis of 18F-Radiolabeled Peptides
All radiolabeled peptides were obtained with molar activity of >37 GBq/
purity of >99% after semi-prep RP-HPLC purification, except for (Table 3). Peptide
µ
mol and radiochemical
2
2
exhibited
decomposition and formation of a radioactive by-product (22%) preceding the product peak (78%), as
observed by analytical radio-RP-HPLC, even after semi-prep RP-HPLC purification (Figure S3). This
by-product increased over time and as such 2 was not further evaluated.
Table 3. Analytical data of all 18F-radiolabeled peptides evaluated in cell binding and serum
stability experiments.
ID
Sequence
dc RCY (%)
RCP (%)
Retention Time (min)
[
[
[
[
18F]1
18F]2
18F]3
18F]5
VGDLTYLKK([18F]FB)
RGDLMKLAK([18F]FB)
RGDLADLRK([18F]FB)
RGDLRELAK([18F]FB)
5.4 ± 1.2
>99
78
14.2
13.6
12.5
12.7
15.7
16.7
19.8
12.8
4
13.4
>99
>99
>99
>95
>99
>99
13.2 ± 10.1
[
18F]1a
[
18F]FB-VGDLTYLKK
6.5
[
18F]1f
18F]1h
18F]1i
VGDLTYLKK([18F]FB)-PEG28
FB-VGDLTYLKK([18F]FB)-PEG28
VGDLTYLKK([18F]FB)KVART
12.8
10.9
2.8
[
[
2.7. In Vitro Evaluation of 18F-Radiolabeled Peptides
2.7.1. In Vitro Cell Binding of 18F-Radiolabeled Peptides
The control peptide [18F]FB-PEG28-A20FMDV2-K16R-PEG28 showed a typical binding level to
DX3puroβ6 α β6+) cells (77.1
0.1%) with minimal binding to DX3puro (α β −) cells (1.5 ± 0.1%)
(
±
,
v
v
6

Molecules 2019, 24, 309
6 of 16
confirming the integrity of the binding assay (Figure 3) [22]. The % binding of [18F]1
,
[18F]3, and
[18F]5 to DX3puroβ6
(
α β6+) cells was 12.8
±
1.9%, 7.7
±
3.8%, 6.4
±
0.1%, respectively. The
2.2%, 5.3 2.8%,
v
% binding of [18F]1
,
[18F]3, and [18F]5 to DX3puro (α β −) cells was 11.7
±
±
v
6
and 5.9 ± 0.8%, respectively. Unfortunately, all three peptides exhibited non-favorable selectivity
towards the DX3puroβ6 (α β6+) cells, as demonstrated by low DX3puroβ6/DX3puro binding ratios
v
([18F]1 = 1.1 ± 0.1:1
,
[18F]3 = 1.4 ± 0.2:1, and [18F]5 = 1.1
±
0.1:1). Further, [18F]1a [18F]1f, and [18F]1h
,
all showed less than 2% binding to both DX3puroβ6
(
α β6+) and DX3puro (α β −) cell lines, and
v
v
6
[18F]1i demonstrated 5.0
±
0.5% binding to DX3puroβ6
(α β6+) cells with a significant improvement
v
in binding selectivity ratio of 3.1 ± 0.8:1 (p < 0.05).
Total Binding at 60 min
100
80
DX3puro6 (v6+)
DX3puro (v6-)
***
60
40
20
15
10
5
***
0
i
f
1
3
5
a
h
1
]
]
]
]
1
]
1
1
]
8
8
8
F
[
]
F
F
8
8
F
[
F
1
1
1
8
8
F
F
1
1
1
1
[
[
[
[
[
Figure 3. In vitro binding results of
[
18F]1
,
[
18F]3
,
[
18F]5
,
[
18F]1a
, [ , [ , [
18F]1f 18F]1h 18F]1i and the control
peptide after 60 min incubation with either DX3puroβ6
*** paired Student t-test gives p < 0.05.
(
v
α β6+) or DX3puro (α β −) cell line (n = 3).
v
6
2.7.2. Serum Stability of 18F-Radiolabeled Peptides
As summarized in Table 4, [18F]1 remained 25% intact, but both [18F]3 and [18F]5 exhibited 100%
degradation at 1 h post-incubation in mouse serum.
Table 4. Summary of the metabolic stability of 18F-radiolabeled lead peptides evaluated in mouse
serum 1 h post-incubation.
ID
Sequence
% Intact (1 h)
[
[
[
18F]1
18F]3
18F]5
VGDLTYLKK([18F]FB)
RGDLMKLAK([18F]FB)
RGDLADLRK([18F]FB)
25
0
0
2.7.3. Serum Stability of 18F-Radiolabeled Modified Derivatives of 1
As indicated by radio-RP-HPLC, upon incubating with mouse serum for 1 h [18F]1a was completely
metabolized; however, both [18F]1f and [18F]1h were >95% intact (Figure 4a–c), and [18F]1i remained
>99% intact (Figure 4d).

Molecules 2019, 24, 309
7 of 16
Figure 4. Radio-RP-HPLC chromatograms of the parent peptides (
and (
18F]1i (magenta) and their corresponding metabolites after 60 min incubating with mouse
serum (red).
a) [ b) [ c) [ ;
18F]1a; ( 18F]1f; ( 18F]1h
d) [

Molecules 2019, 24, 309
8 of 16
2.8. Evaluation of Secondary Structure of 1, 1i, and the Control Peptide Using Circular Dichroism (CD)
Spectroscopy
CD data revealed that all three peptides exhibited random coil conformation in CD buffer (red).
However, α-helices were observed when 30% 2,2,2-trifluoroethanol (TFE) was added to CD buffer for
1i (Figure 5b, black curve) and the control peptide (Figure 5c, black curve), whereas the parent peptide
1 did not form the helix under the same condition (Figure 5a).
Figure 5. Circular dichroism (CD) spectra of (
a) the parent peptide 1; (b) 1i; and (c) the control peptide
in aqueous buffer (red) and in 30% TFE (black).
3. Discussion
The OBOC technology has been successfully utilized to identify cancer-targeting peptides [
yet the approach remains painstakingly slow, with clear delays between on-bead ligand identification
and subsequent optimization to a potential imaging agent [ 11]. The aim of this work was to combine
6–8],
7,
multiple strategies to streamline the development of integrin α β6-targeting peptide-based imaging
v
agents for PET. This included (1) OBOC chemistry to produce large peptide libraries, (2) elimination of
setbacks due to post-screening modifications of peptides by incorporating 4-[19F]fluorobenzoic acid
(FBA) into the peptide library design, (3) utilizing the OBTC fluorescent cell screening assay to expedite
the on-bead library screening process (combine three rounds of screening into a single step), and (4)
structural-based modifications to optimize OBOC-derived peptides for improved serum stability and
pharmacokinetics properties.
The incorporation of a prosthetic group or a chelator into a peptide sequence post-identification
from a library can often have a negative impact on the binding properties [13]. To avoid this, the
OBOC library was designed to contain the nonradioactive prosthetic group [19F]FBA pre-attached
at the C-terminal lysine’s side chain. The placement of the prosthetic group at the C-terminus was
designed to allow for Edman sequencing of the beads identified as selective binders to the cells that
express integrin α β6. In addition, the library contained the α β6-targeting motif DLXXL, a motif
v
v
originally discovered from a phage display library as an important binding sequence for the integrin
α β 18 23]. Stringent and iterative screening of OBOC libraries for target-selective peptides is lengthy,
[
,
v
6
time-consuming, and not highly efficient. Traditionally, the OBOC libraries are screened against either
purified proteins or whole cells, and positive beads (i.e., bound to protein or cells) are visualized under
a microscope, and manually isolated [24]. To ensure target-specificity of the peptides, this process is
often carried out in multiple rounds [24], with multiple washing cycles between subsequent rounds
of screening, which often leads to broken or damaged beads. To streamline the screening, the OBTC
cell-screening assay developed by Udagamasooriya et al. was adopted [21]. Using a mixed pool of
two-color fluorescent cells differing only in the expression of integrin α β6, beads that were recognized
v
by the DX3puroβ6mCherry (α β6+, red) cells were directly retrieved from the assay in one round
v
of screening. This approach resulted in the rapid identification of 185 α β6-positive beads from the
v
entire 4-fluorobenzoyl peptide library (~2.5 million compounds), which otherwise would be difficult to
achieve using the traditional screening approach. Conventionally, sequence determination of on-resin

Molecules 2019, 24, 309
9 of 16
peptide is commonly carried out with Edman degradation [25
–
27]; however, this method is rather
time consuming (hours to a day to sequence one peptide) and expensive (~$300/peptide) [28]. With
these limitations in mind for the purpose of this work only, the first five beads identified from the first
aliquot of screening were sequenced by Edman degradation for further evaluations.
Of the five beads sequenced, three contained the three amino acids RGD sequence, a motif known
to mediate cell adhesion and binds to
αv integrins, including integrin α β [29]. The remaining two
v
6
sequences did not share any homology and had the unique VGD and GID motifs. Qualitative on-bead
cell binding with DX3puroβ6mCherry (α β6+, red) cells confirmed binding of four of the five peptides.
v
The three RGD containing peptides also showed some interactions with the negative cell line, which
was likely due to recognition of the RGD motif by other RGD-binding integrins also expressed on the
DX3puroEmGFP (α β −, green) cell line. This was corroborated by the ELISA data, where binding to
v
6
all
line or
picking process.
Based on the initial in vitro data,
αv-integrins except for α β was observed. The GID containing peptide did not bind to either cell
v
5
α
v-integrins by ELISA, and was possibly an outlier picked by mistake during the manual bead
1
(VGDLTYLKK(FB)) was selected for further optimization.
Modifications at the N-terminus with various chemical moieties, including aliphatic (e.g., acetyl,
methyl, PEG12) and aromatic (e.g., FBA) groups, PEGylation (e.g., PEG28) at the C-terminus, as well as
a combination of both, were performed. Unfortunately, none of the modifications resulted in a new
analog with a better binding affinity or selectivity for the integrin α β6, as indicated by the ELISA and
v
cell binding data. Interestingly, the N-terminus of the peptide appeared to be more tolerant towards
modifications with the aromatic FBA than the aliphatic methyl, acetyl, and PEG moieties, which all
showed poor binding (IC50 > 5–100 µM). These results suggest that the presence of the FB at the
N-terminus of the peptide could possibly have favorable interactions with the protein residues in an
existing binding pocket. A similar observation has been recently reported for a series of 18F-radiolabeled
phosphoamidate peptidomimetic inhibitors for prostate specific membrane antigen (PSMA), where the
FB ring of one of the analogues in the series engaged in an “arene-binding” interaction with the surface
of the protein, and thus contributing to its high affinity in vitro and in vivo
[30]. Future molecular
docking studies or co-crystallization of these peptides with integrin α β are necessary to provide
v
6
better understanding of the peptide-integrin interaction.
Although peptides possess many favorable pharmacokinetics properties, such as high target
affinity, good tumor penetration, and rapid non-specific clearance, they are prone to proteolytic
degradation in vivo. PEGylation has been a widely adopted strategy to improve the pharmacokinetics
properties of both small molecules and biopharmaceuticals (e.g., peptides, proteins, and antibodies,
etc.) [31]. Based on our previous experience, the addition of PEG moieties to the N- and C-termini
of the highly α β6-selective A20FMDV2 peptide successfully prolonged the biological half-life,
v
improved metabolic stability and enhanced tumor retention of the [18F]FB-PEG28-A20FMDV2,
[18F]FB-(PEG28)2-A20FMDV2, and [18F]FB-PEG28-A20FMDV2-PEG28 in human tumor xenograft
models [16
,
19
,
20]. Of the numerous peptides evaluated,
1
1
demonstrated the highest serum stability.
was explored. Our results demonstrated
To further improve the stability, C-terminal PEGylation of
that C-terminal PEGylation of the peptide (with and without N-terminal FBA, 1h, and 1f, respectively)
indeed did significantly improve the metabolic stability of the peptide, with >95% intact peptide
observed at 1 h post-incubation in mouse serum. Despite having excellent serum stability, both
analogs showed significant reduction in binding affinity (<2% binding to DX3puroβ6 (α β6+) cells).
v
Our data suggests that the introduction of a PEG moiety at the C-terminus of the peptide could
effectively shield the peptide from proteases, which is consistent with the trend observed for our
[18F]FB-A20FMDV2-PEG28 (76% intact at 1 h) [19], comparing to the first generation [18F]FB-A20FMDV2
(0% intact) [16]. However, the introduction of the relatively large PEG moiety (MW = 1.5 kDa) to the
relatively short peptide (MW = 1.1 kDa) resulted in a drastic loss in binding of 1f and 1h to integrin α β
.
v
6
A possible explanation is that the relatively large PEG chain length could have imposed some steric
shielding effect on the peptide (i.e., PEG wrap around and shield the binding domain from interacting

Molecules 2019, 24, 309
10 of 16
with the receptor), and in turn negatively affected the binding affinity of the resulting PEGylated
peptides. This steric shielding effect of PEG on drug conjugates was previously reported by Mu et al.
in a study investigating the impact of PEG chain length and PEGylation site on the bioactivity of the
therapeutic protein Staphylokinase, using both experimental assays and computational simulations [32].
Future work entails the investigation of C-terminal PEGylation of
further test this hypothesis.
1 with shorter PEG chain length to
Since
terminus of the peptide could negatively affect the binding affinity due to the close proximity to the
binding domain. Furthermore, a co-crystal structure of the integrin α β with pro-TGF 3 peptide
revealed that integrin α β not only recognizes the RGD motif but also binds preferentially to the
1 (VGDLTYLKK(FB)) is a relatively small peptide, it is possible that modifications at either
β
v
6
v
6
LXXL/I motif that folds into an amphipathic
α-helix, fitting into a hydrophobic pocket composed solely
from residues of the β6 subunit [33]. As reported previously, the α β6-binding A20FMDV2 peptide has
v
been confirmed to have a post-RGD
α-helix, contributing to its high affinity and selectivity for integrin
α β 34]. Additionally, other α β6-selective peptides reported in the literature, such as the R01
[
v
v
6
peptide [17] and the H2009.1 peptide [23], are relatively larger, composing of 30- and 20-amino acids,
respectively. To that end, we suspected that the lack of binding affinity and selectivity of our peptide
(9-mer) to integrin α β6 could be attributed to its inability to form a stabilized -helix to fit the binding
to improve its binding affinity and
selectivity for integrin α β6. Indeed, extension of the C-terminus of with the KVART sequence from
the known integrin α β -binding A20FMDV2 (NAVPNLRGDLQVLAQKVART) resulted in a 14-mer
1
α
v
pocket tightly. This led us to explore extending the C-terminus of
1
1
v
v
6
peptide with improved binding affinity and a 3-fold increase in binding selectivity for DX3puroβ6
α β6+) cells, and excellent serum stability (>99% intact at 1 h). The CD results demonstrated that both
(
v
the control A20FDMV2 and the 14-mer 1i peptides could form an
the parent peptide could not under the same condition, which supported our hypothesis on the
importance of peptide secondary structure on binding affinity and selectivity for integrin α β .
α-helix in 30% TFE/buffer, while
1
v
6
4. Materials and Methods
General Experimental Information. All the Fmoc-protected amino acids were purchased from GL
Biochem (Shanghai, China). TentaGel S NH2 resin (90 m) was purchased from RAPP Polymere GmbH
µ
(Tuebingen, Germany) and NovaSyn TGR resin was purchased from NovaBiochem (Burlington, MA,
USA). All other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) and Fluka (Mexico
City, Mexico), unless otherwise specified. A reverse-phase high performance liquid chromatography
(RP-HPLC) Beckman Coulter Gold system (Brea, CA, USA) was used for peptide characterization
(Phenomenex Jupiter 4
µ Proteo 90 Å column, Torrance, CA, USA) and purification (Phenomenex
Jupiter 10 C18 300 Å column). The mobile phase used was 0.05% trifluoroacetic acid (TFA) in water
µ
(v/v; solvent A) and 100% acetonitrile (solvent B); solvent B isocratic 9% for 2 min followed by a
linear gradient to 81% over 30 min at a flow rate of 1.5 mL/min (analytical RP-HPLC) and 3 mL/min
(semi-prep RP-HPLC). All peptides were characterized using a UltraFlextreme (Bruker, Billerica, MA,
USA) Matrix-assisted laser desorption-ionization time of flight/time of flight (MALDI TOF/TOF) mass
spectrometer with a matrix of sinapinic acid or α-cyano-4-hydroxycinnamic acid.
4.1. OBOC Library Synthesis
The peptide library was assembled on TentaGel resin using the “split-mix” technique and
standard Fmoc-based solid phase peptide synthesis (SPPS), utilizing all l-amino acids except l-cysteine.
Briefly, the resin was first divided into 19 aliquots; each was coupled with a single amino acid
(3 equiv.) using O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate
(HATU, 2.7 equiv.) as a coupling agent and N,N-diisopropylethylamine (DIPEA, 3 equiv.) as a base in
N,N-dimethylformamide (DMF) at room temperature for 1.5 h. At the end of each coupling, the resin was
re-pooled, mixed, and split for the next coupling step. Fluorenylmethyloxycarbonyl (Fmoc)-protecting
group was removed with 20% piperidine in DMF, and allyloxycarbonyl (Alloc)-protecting group was

Molecules 2019, 24, 309
11 of 16
removed with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 0.5 equiv.), and phenylsilane
(PhSiH3, 20 equiv.) in dichloromethane (DCM). Global deprotection was achieved with trifluoroacetic
acid (TFA) mixture with triisopropylsilane (TIPS) and water (95:2.5:2.5 v/v/v) for 3 h. Finally, the resin
was washed thoroughly with DMF, water, and methanol (10
until screening.
×
each) and stored in 70% ethanol at 4 ◦C
4.2. Peptide Synthesis
All peptides were synthesized manually on NovaSyn TGR resin (0.25 mmol/g, Burlington, MA,
USA) using standard Fmoc-based SPPS yielding C-terminal amides. Starting with N- -Fmoc-N- -1-
α
ε
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-l-lysine (Fmoc-Lys(ivDde)-OH), orthogonal
protecting group for site-specific labeling, each amino acid was coupled using HATU (2.7 equiv.) as a
coupling agent and DIPEA (3 equiv.) as a base in DMF at room temperature for 1.5 h. The removal of
the Fmoc- and ivDde-protecting groups was facilitated with 20% piperidine in DMF and 2% hydrazine
in DMF, respectively. Peptides were cleaved off the resin using a mixture of TFA, water, and TIPS
(95:2.5:2.5 v/v/v) for 3 h. The resin was washed with fresh TFA and the filtrate was collected then
evaporated under reduced pressure. Crude peptides were precipitated in diethyl ether and extracted
from the water/ether bilayer. All crude peptides were purified on the semi-prep HPLC. The purified
peptides were analyzed on the analytical HPLC and characterized by MALDI-TOF/MS.
4.3. Cell Lines
DX3Puro (α β −) and DX3Puroβ6
(α β6+) have been described previously [35,36] and were a
v
generous gift from Dr. John Marshall. To generate DX3puroEmGFP and DX3puroβ6mCherry, DX3Puro
and DX3Puroβ6 cells were transduced with lentivirus carrying EmGFP or mCherry (under control of
v
6
an EF1
Transduced cells were cultured at ultra-low density in Dulbecco’s Modified Eagle Medium (DMEM,
Gibco, Waltham, MA, USA), supplemented with 10% FBS and blasticidin (10
g/mL) at 37 ◦C in the
α promoter) and the blasticidin resistance gene (under control of a hybrid SV40/EM7 promoter).
µ
presence of 5% CO2 for 21 days. Clonal populations of DX3puroEmGFP and DX3puroβ6mCherry
were then isolated, expanded, and screened for fluorescence, and in the case of DX3puroβ6mCherry,
retention of ITGβ6 expression. Integrin expression was determined by flow cytometry.
4.4. Flow Cytometry
Subconfluent cells were washed with PBS and harvested with Trypsin-EDTA (0.25%, Phenol-red,
Gibco). Cells were treated once with PBS containing 0.1% BSA and 0.1% sodium azide (wash buffer),
counted by trypan blue exclusion and resuspended at 6
treated with primary antibody clone 10D5 (Millipore Sigma, Burlington, MA, USA, at 10
serum free DMEM, 50
L) for 60 min at 4 ◦C and washed twice with wash buffer (WB). AlexaFluor488
goat anti-mouse secondary antibody (ThermoFisher, Waltham, MA, USA, 1:50 dilution in serum free
DMEM, 50
L) was applied to the cells for 30 min at 4 ◦C. Cells were washed twice, resuspended in
×
106 cells/mL. Aliquots of cells (50
µ
L) were
µg/mL in
µ
µ
0.5 mL WB, and scanned on the LSRFortesa Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ,
USA) and analyzed using FACSDiva software (8.02, San Jose, CA, USA), acquiring 1 × 104 events.
4.5. Library Screening
4.5.1. Library Beads Preparation
Prior to screening with cells, the beads were thoroughly washed to remove all traces of ethanol.
An aliquot of beads (200
over vacuum with deionized water (10
followed by washing with PBS (5×), and suspended in PBS for 15 min.
µL, equivalent to approximately 150,000 beads) was dried overnight, washed
×
), allowed to swell in water for 2 to 4 h at room temperature,

Molecules 2019, 24, 309
12 of 16
4.5.2. OBTC Fluorescent Cell Screening
DX3puroEmGFP (α β −) and DX3puroβ6mCherry (α β6+) cells were cultured in DMEM
v
v
6
supplemented with 10% FBS, 1% PSG, and blasticidin (10
µ
g/mL) as adherent monolayers at 37 ◦C in
the presence of 5% CO2 in a humidified incubator for 3 days. At 80% confluency, cells were harvested,
counted, and re-suspended in 10% fetal bovine serum (FBS) phenol red–free DMEM for screening.
Briefly, cells (500,000 cells/cell line) were mixed in 20 mL phenol red-free DMEM in a non-treated
tissue culture petri dish (100 mm; Spectrum Chemicals and Laborat◦ory Products) for 1 h. Library
beads (150,000) were added to the mixed cells and incubated at 37 C with gentle mixing for 3 h.
After incubation, the beads were analyzed under a fluorescent microscope. Beads that were >80%
covered in DX3puroβ6mCherry cells (α β6-specific) were manually selected with a micropipette. The
v
α β -specific beads isolated from the assay were sequenced using Edman degradation.
v
6
4.6. Competitive Binding ELISAs
Competitive binding ELISAs were performed to evaluate the binding affinity of each
peptide relative to each integrin’s biotinylated natural ligand (transforming growth factor beta 1
latency-associated peptide or TGF
β1-LAP (G&P Biosciences, Santa Clara, CA, USA) for integrins α β
v
6
and α β8, fibronectin (Thermo Fisher, Waltham, MA, USA) for integrin α β1, and vitronectin (Thermo
v
v
Fisher) for integrins α β3 and α β5), as previously described [22]. In brief, anti-
α
v antibody (P2W7,
v
v
5
µ
g/mL, Abcam) was plated (50
µ
L/well) at 37 ◦C for 1 h, washed with PBS (3
×
), and blocked overnight
with blocking buffer (300
washing with PBS (3 ). Purified integrin (R&D Systems, Minneapolis, MN, USA) in conjugate buffer
(50 L/well, 20mM Tris, 1mM MnCl2, 150 mM NaCl, 0.1% Tween, 1% BSA in water) was then added
to each well, incubated at 37 ◦C for 1 h, followed by washing using wash buffer (20 mM Tris, 1 mM
MnCl2, 150 mM NaCl, 0.1% Tween in water, 3 ). Serial dilutions of each peptide (10 M–10 pM or 100
M–100 pM in conjugate buffer) and each integrin’s respective biotinylated natural ligand was added
µL/well, 5% bovine serum albumin (BSA), 1% Tween 20, in PBS), followed by
×
µ
×
µ
µ
to each well (a total volume of 50 µ
L/well). The plate was incubated at 37 ◦C for 1 h, then washed with
wash buffer (3
well (50
L/well), incubated at 37 ◦C for 1 h, and then washed with wash buffer (3
HRP was detected with 3,30,5,50-tetramethylbenzidine (50
L/well, Promega, Madison, WI, USA) for
10–15 min at room temperature. The reaction was stopped by adding 1N H2SO4 (50 L/well) and the
×
). A 1:1000 dilution of ExtrAvidin Horseradish Peroxidase (HRP) was added to each
µ
×
). The ExtrAvidin
µ
µ
absorbance was measured in a Multiscan Ascent plate reader (Thermo Fisher) at 450 nm. Half-maximal
inhibitory concentration (IC50) of peptides was determined fitting to sigmoidal dose-response model
in GraphPad Prism 5.0 (San Diego, CA, USA).
4.7. Radiochemical Synthesis
All peptides were radiolabeled with [18F]fluorobenzoic acid ([18F]FBA) on resin following
our well-established solid-phase radiolabeling protocols [16,20,
37]. Briefly, [18F]fluoride was
reacted with ethyl-4-(trimethylammonium triflate)benzoate in the presence of 4,7,13,16,21,24-hexaoxa-
1,10-diazabicyclo [8.8.8]hexacosane (KryptofixTM [K2.2.2], (Sigma Aldrich, St. Louis, MO, USA) and
potassium carbonate at 100 ◦C in dimethylsulfoxide (DMSO) for 15 min, followed by saponification of
the ethyl ester protect group to yield [18F]FBA. The [18F]FBA was re-dissolved in DMF and coupled
to the peptide (~5 mg resin) using HATU (5 mg)/DIPEA (10 µ
L). The 18F-radiolabeled peptide on
resin was cleaved and globally deprotected with trifluoroacetic acid/triisopropylsilane/water 95:2.5:2.5
(v/v/v) mixture for 30 min at room temperature. The crude peptide was purified by semi-prep
radio-RP-HPLC (C18-column) and formulated in PBS for in vitro assays. The purity and molar
activity of the 18F-radiolabeled peptide was confirmed via analytical radio-RP-HPLC. Identity of
the 18F-radiolabeled peptide was confirmed by co-injection of the 19F-peptide, and all analytical
radio-RP-HPLC analysis was performed for each 18F-radiolabeled peptide prior to use.

Molecules 2019, 24, 309
13 of 16
4.8. The 18F-Radiolabeled Peptide Cell Binding Assay
Cell binding assay was performed as previously described [19
,
20]. In brief, each cell line was
suspended at a concentration of 75
(3.75 L) was added to 1.5 mL microcentrifuge tubes previously blocked with 5%
106 cells in 50
BSA in PBS for 10 min at room temperature. The 18F-radiolabeled peptide was prepared in a stock
×
106 cells/mL in serum-free medium and aliquots of each cell line
×
µ
solution of 148 kBq in 1 mL PBS (pH 7.2). Aliquots of each 18F-radiolabeled peptide (50
µL, 7.4 kBq
per sample) were added to each cell sample and incubated at 37 ◦C for 1 h with frequent suspension.
After incubation, each sample was centrifuged and the supernatant was collected. The cell pellet
was washed with 500
µL PBS, centrifuged, and the supernatant was combined with the previously
collected supernatant samples. The cell pellet was resuspended in 600
µL PBS and transferred to
gamma counter tubes. All pellet and supernatant samples were measured in a Wizard 1470 gamma
counter (Perkin Elmer, Waltham, MA, USA). Cell binding percentage for each cell line was calculated
as the radioactivity associated, with pellet divided by the total radioactivity associated with both the
pellet and supernatant.
4.9. Serum Stability Assay
Peptide stability was evaluated in mouse serum as previously described [19
formulated 18F-radiolabeled peptide (~3.7 MBq in PBS) was incubated with mouse serum (500
Sigma Aldrich, St. Louis, MO) at 37 ◦C for 1 h. An aliquot of 18F-radiolabeled peptide in mouse serum
(100 L) was taken at 1 h, mixed with absolute ethanol (500
L, 4 ◦C), and centrifuged to precipitate
,20]. In brief,
µL,
µ
µ
serum protein. The supernatant was diluted with HPLC solvent A before radio-HPLC analysis to
determine the fraction of intact 18F-radiolabeled peptide.
4.10. Circular Dichroism (CD) Spectroscopy
CD spectroscopy was performed on a Jasco J-810 spectropolarimeter equipped with a Jasco
CDF-426S Peltier set to 25 ◦C (Easton, MD, USA). Lyophilized peptide was diluted to 0.2 mg/mL in
buffer (final concentration was 25 mM phosphate + 100 mM NaF in PBS, pH 7.4), with or without 30%
TFE (v/v), placed in a quartz cuvette (1 mm), and after extensive purging with nitrogen, scanned in the
region 190–260 nm (scan speed was 20 nm/min). An average of five scans were baseline-subtracted
(buffer, 25 mM phosphate + 100 mM NaF in PBS).
4.11. Data and Statistical Analysis
Statistical analysis and comparisons between groups were performed using the student t test.
A p value of 0.05 or less was considered statistically significant.
5. Conclusions
In conclusion, this work utilized the OBOC technology, applied the OBTC fluorescent cell
screening assay, and provided a framework for optimization of OBOC-derived peptides to streamline
the development of 18F-peptide based imaging agents for PET. To expedite the development of peptide
identified from OBOC library into useful imaging agents, we eliminated the need of post-screening
modification by designing a 4-fluorobenzoyl peptide library, improved the traditional on-bead manual
library screening using OBTC fluorescent cell-screening, and further improved the binding affinity,
selectivity, and serum stability of the lead peptide using numerous strategies, including N-, C-, and
bi-terminal modifications. Chemical modifications for the optimization of an identified peptide
sequence have many challenges, as the outcome of each modification will depend on the peptide
sequence and the target receptor, and their behavior cannot be predicted. It is fair to conclude that
optimizing multiple parameters (i.e., binding affinity, selectivity, and serum stability) of a peptide
identified from a library to be used as an imaging agent remains a daunting task.

Molecules 2019, 24, 309
14 of 16
Supplementary Materials: The following are available online. Table S1: Analytical data of synthetic peptides
showing retention time, purity, and mass confirmation; Table S2: Analytical Radio-RP-HPLC chromatogram
confirming the purity of [18F]peptides; Figure S1: Analytical RP-HPLC chromatograms confirming the purity of
synthetic peptides; Figure S2: Mass analysis of synthetic peptides using MALDI/MS; Figure S3: Radio-RP-HPLC
of [18F]peptides showing the radioactive PMT trace (red) and their co-injected [19F]peptide standards (green
UV220nm).
Author Contributions: Conceptualization, Y.C.T., J.L.S.; methodology, Y.C.T., R.A.D., and T.G.; validation,
Y.C.T., R.A.D., and T.G.; formal analysis, Y.C.T.; investigation, Y.C.T., R.A.D., and T.G.; visualization, Y.C.T.;
writing—original draft preparation, Y.C.T.; writing—review and editing, Y.C.T., T.G., J.L.S.; supervision, J.L.S.;
funding acquisition, J.L.S.
Funding: This research was funded in part by the National Institute of Health RO1CA199725-01 and the Office of
Science, United States Department of Energy Grant: DE-SC0008385.
Acknowledgments: The authors acknowledge David Boucher for generating the fluorescent cell pairs used in the
library screening and on-bead cell binding experiments, Dave Kukis and Joshua Waltenburg at the Center for
Molecular and Genomic Imaging at UC Davis for providing Fluorine-18 isotope for experiments, and Madhu
Budamagunta at the Department of Biochemistry and Molecular Medicine for running the CD experiments.
Conflicts of Interest: Yng (Sarah) C. Tang and Julie L. Sutcliffe are named inventors of intellectual property related
to this data. Julie L. Sutcliffe is the founder and stockholder of Luminance Biosciences, Inc.
References
1.
Sun, X.; Li, Y.; Liu, T.; Li, Z.; Zhang, X.; Chen, X. Peptide-based imaging agents for cancer detection. Adv.
Drug Deliv. Rev. 2017, 110–111, 38–51. [CrossRef] [PubMed]
2.
Diao, L.; Meibohm, B. Pharmacokinetics and pharmacokinetic-pharmacodynamic correlations of therapeutic
peptides. Clin. Pharmacokinet. 2013, 52, 855–868. [CrossRef]
3.
4.
Di, L. Strategic approaches to optimizing peptide ADME properties. AAPS J. 2015, 17, 134–143. [CrossRef]
Richter, S.; Wuest, F. 18F-Labeled Peptides: The Future Is Bright. Molecules 2014, 19, 20536–20556. [CrossRef]
[PubMed]
5.
6.
7.
8.
Gray, B.P.; Brown, K.C. Combinatorial peptide libraries: Mining for cell-binding peptides. Chem. Rev. 2014
114, 1020–1081. [CrossRef] [PubMed]
,
Aina, O.H.; Liu, R.; Sutcliffe, J.L.; Marik, J.; Pan, C.X.; Lam, K.S. From combinatorial chemistry to
cancer-targeting peptides. Mol. Pharm. 2007, 4, 631–651. [CrossRef] [PubMed]
Gagnon, M.K.; Hausner, S.H.; Marik, J.; Abbey, C.K.; Marshall, J.F.; Sutcliffe, J.L. High-throughput in vivo
screening of targeted molecular imaging agents. PNAS 2009, 106, 17904–17909. [CrossRef]
Aina, O.H.; Marik, J.; Gandour-Edwards, R.; Lam, K.S. Near-infrared optical imaging of ovarian cancer
xenografts with novel alpha 3-integrin binding peptide “OA02”. Mol. Imaging 2005, 4, 439–447. [CrossRef]
[PubMed]
9.
Jiang, M.; Ferdani, R.; Shokeen, M.; Anderson, C.J. Comparison of two cross-bridged macrocyclic chelators
for the evaluation of 64Cu-labeled-LLP2A, a peptidomimetic ligand targeting VLA-4-positive tumors. Nucl.
Med. Biol. 2013, 40, 245–251. [CrossRef]
10. Walker, D.; Li, Y.; Roxin, A.; Schaffer, P.; Adam, M.J.; Perrin, D.M. Facile synthesis and 18F-radiolabeling of
alpha4beta1-specific LLP2A-aryltrifluoroborate peptidomimetic conjugates. Bioorg. Med. Chem. Lett. 2016
26, 5126–5131. [CrossRef]
,
11. Hu, L.Y.; Kelly, K.A.; Sutcliffe, J.L. High-Throughput Approaches to the Development of Molecular Imaging
Agents. Mol. Imaging Biol. 2017, 19, 163–182. [CrossRef] [PubMed]
12. Aina, O.H.; Marik, J.; Liu, R.; Lau, D.H.; Lam, K.S. Identification of novel targeting peptides for human
ovarian cancer cells using “one-bead one-compound” combinatorial libraries. Mol. Cancer Ther. 2005, 4,
806–813. [CrossRef] [PubMed]
13. Blasi, F.; Oliveira, B.L.; Rietz, T.A.; Rotile, N.J.; Day, H.; Looby, R.J.; Ay, I.; Caravan, P. Effect of Chelate Type
and Radioisotope on the Imaging Efficacy of 4 Fibrin-Specific PET Probes. J. Nucl. Med. 2014, 55, 1157–1163.
[CrossRef]
14. Otvos, L., Jr.; Wade, J.D. Current challenges in peptide-based drug discovery. Front. Chem. 2014, 2. [CrossRef]
[PubMed]
15. Bandyopadhyay, A.; Raghavan, S. Defining the role of integrin alphavbeta6 in cancer. Curr. Drug Targets
2009, 10, 645–652. [CrossRef] [PubMed]

Molecules 2019, 24, 309
15 of 16
16. Hausner, S.H.; DiCara, D.; Marik, J.; Marshall, J.F.; Sutcliffe, J.L. Use of a peptide derived from foot-and-mouth
disease virus for the noninvasive imaging of human cancer: Generation and evaluation of 4-[18F]fluorobenzoyl
A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography.
Cancer Res. 2007, 67, 7833–7840. [CrossRef]
17. Kimura, R.H.; Teed, R.; Hackel, B.J. Pharmacokinetically stabilized cystine knot peptides that bind
alpha-v-beta-6 interin with single-digit nanomolar affinities for detection of pancreatic cancer. Clin.
Cancer Res. 2012, 18, 839–849. [CrossRef] [PubMed]
18. Kraft, S.; Diefenbach, B.; Mehta, R.; Jonczyk, A.; Luckenbach, G.A.; Goodman, S.L. Definition of an unexpected
ligand recognition motif for alphav beta6 integrin. J. Biol. Chem. 1999, 274, 1979–1985. [CrossRef]
19. Hausner, S.H.; Abbey, C.K.; Bold, R.J.; Gagnon, M.K.; Marik, J.; Marshall, J.F.; Stanecki, C.E.; Sutcliffe, J.L.
Targeted in vivo imaging of integrin α β with an improved radiotracer and its relevance in pancreatic
v
6
tumor model. Cancer Res. 2009, 69, 5843–5850. [CrossRef]
20. Hausner, S.H.; Bauer, N.; Hu, L.; Knight, L.; Sutcliffe, J.L. The effect of bi-terminal PEGylation of an
integrin α β6-targeted 18F-peptide on pharmacokinetics and tumor uptake. J. Nucl. Med. 2015, 56, 784–790.
v
[CrossRef]
21. Udugamasooriya, D.G.; Kodadek, T. On-Bead Two-Color (OBTC) Cell Screen for Direct Identification of
Highly Selective Cell Surface Receptor Ligands. Curr. Protoc. Chem. Biol. 2012, 4, 35–48. [PubMed]
22. Hausner, S.H.; Bold, R.J.; Cheuy, L.Y.; Chew, H.K.; Daly, M.E.; Davis, R.A.; Foster, C.C.; Kim, E.J.; Sutcliffe, J.L.
Preclinical development and first-in-human imaging of the integrin α β with [18F]α β6-Binding Peptide in
v
v
6
metastatic carcinoma. Clin. Cancer Res. 2018. [CrossRef] [PubMed]
23. Elayadi, A.N.; Samli, K.N.; Prudkin, L.; Liu, Y.H.; Bian, A.; Xie, X.J.; Wistuba, I.I.; Roth, J.A.; McGuire, M.J.;
Brown, K.C. A peptide selected by biopanning identifies the integrin alphavbeta6 as a prognostic biomarker
for nonsmall cell lung cancer. Cancer Res. 2007, 67, 5889–5895. [CrossRef]
24. Lau, D.H.; Guo, L.L.; Liu, R.W.; Song, A.M.; Shao, C.K.; Lam, K.S. Identifying peptide ligands for cell
surface receptors using cell-growth-on-bead assay and one-bead one-compound combinatorial library.
Biotechnol. Lett. 2002, 24, 497–500. [CrossRef]
25. Edman, P. Method for determination for the amino acid sequence in peptides. Acta Chem. Scand. 1950, 4,
283–293. [CrossRef]
26. Hu, Y.J.; Wei, Y.M.; Zhou, Y.; Rajagopalan, P.T.R.; Pei, D.H. Determination of substrate specificity for
peptide deformylase through the screening of a combinatorial peptide library. Biochemistry 1999, 32, 643–650.
[CrossRef] [PubMed]
27. Cheung, Y.W.; Bell, C.A.; Balasubramanian, S. A combinatorial approach to identifying protein tyrosine
phosphatase substrates from a phosphotyrosine peptide library. J. Am. Chem. Soc. 1997, 119, 9568–9569.
[CrossRef]
28. Thakkar, A.; Cohen, A.S.; Connolly, M.D.; Zuckermann, R.N.; Pei, D. High-throughput sequencing of
peptoids and peptide-peptoid hybrids by partial Edman degradation and mass spectrometry. J. Comb. Sci.
2009, 11, 294–302. [CrossRef]
29. Ruoslahti, E. RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 1996, 12, 697–715.
[CrossRef]
30. Dannoon, S.; Ganguly, T.; Cahaya, H.; Geruntho, J.J.; Galliher, M.S.; Beyer, S.K.; Choy, C.J.; Hopkins, M.R.;
Regan, M.; Blecha, J.E.; et al. Structure-Activity Relationship of 18F-Labeled Phosphoramidate Peptidomimetic
Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate
Cancer. J. Med. Chem. 2016, 59, 5684–5694. [CrossRef]
31. Turecek, P.L.; Bossard, M.J.; Schoetens, F.; Ivens, I.A. PEGylation of biopharmaceuticals: A review of chemistry
and nonclinical safety information of approved drugs. J. Pharm. Sci. 2016, 105, 460–475. [CrossRef] [PubMed]
32. Mu, Q.; Hu, T.; Yu, J. Molecular insight into the steric shielding effect of PEG on the conjugated staphylokinase:
Biochemical characterization and molecular dynamics simulation. PLoS ONE 2013, 8, e68559. [CrossRef]
[PubMed]
33. Dong, X.; Hudson, N.E.; Lu, C.; Springer, T.A. Structural determinants of integrin beta-subunit specificity for
latent TGF-beta. Nat. Struct. Mol. Biol. 2014, 21, 1091–1096. [CrossRef]
34. DiCara, D.; Rapisarda, C.; Sutcliffe, J.L.; Violette, S.M.; Weinreb, P.H.; Hart, I.R.; Howard, M.J.; Marshall, J.F.
Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands. J. Biol. Chem.
2007, 282, 9657–9665. [CrossRef]

Molecules 2019, 24, 309
16 of 16
35. Albino, A.P.; Lloyd, K.O.; Houghton, A.N.; Oettgen, H.F.; Old, L.J. Heterogeneity in surface antigen and
glycoprotein expression of cell lines derived from different melanoma metastases of the same patient.
Implications for the study of tumor antigens. J. Exp. Med. 1981, 154, 1764–1778. [CrossRef]
36. Dalvi, N.; Thomas, G.J.; Marshall, J.F.; Morgan, M.; Bass, R.; Ellis, V.; Speight, P.M.; Whawell, S.A. Modulation
of the urokinase-type plasminogen activator receptor by the beta6 integrin subunit. Biochem. Biophys. Res.
Commun. 2004, 317, 92–99. [CrossRef] [PubMed]
37. Davis, R.A.; Lau, K.; Hausner, S.H.; Sutcliffe, J.L. Solid-phase synthesis and fluorine-18 radiolabeling of
cycloRGDyK. Org. Biomol. Chem. 2016, 14, 8659–8663. [CrossRef]
Sample Availability: Samples of all compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).