Incorporation of Fluorine into an OBOC Peptide Library by Copper-Free Click Chemistry toward the Discovery of PET Imaging Agents

Article abstract of DOI:10.1021/acscombsci.9b00146

A one-bead one-compound (OBOC) library of peptide-based imaging agents was developed where a ¹⁹F-containing moiety was added onto the N-Terminus of octamer peptides through copper-free click chemistry prior to screening of the library. This created a library of complete imaging agents that was screened against CXCR4, a receptor of interest for cancer imaging. The screen directly resulted in the discovery of a peptide-based imaging agent with an IC₅₀ of 138 μM. This proof-of-concept study describes a new type of OBOC peptide library design, where hits discovered from screening can be easily translated into their fluorine-18 counterpart for PET imaging without loss of affinity.

Full text of DOI:10.1021/acscombsci.9b00146

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Letter
Incorporation of Fluorine into an OBOC Peptide Library by Copper-
Free Click Chemistry toward the Discovery of PET Imaging Agents
Emily Murrell and Leonard G. Luyt*
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ABSTRACT: A one-bead one-compound (OBOC) library of peptide-based
imaging agents was developed where a 19F-containing moiety was added onto
the N-terminus of octamer peptides through copper-free click chemistry prior
to screening of the library. This created a library of complete imaging agents
that was screened against CXCR4, a receptor of interest for cancer imaging.
The screen directly resulted in the discovery of a peptide-based imaging agent
with an IC50 of 138 μM. This proof-of-concept study describes a new type of
OBOC peptide library design, where hits discovered from screening can be
easily translated into their fluorine-18 counterpart for PET imaging without
loss of affinity.
KEYWORDS: OBOC, fluorine-18, click chemistry, molecular imaging, CXCR4
In this work, we aimed to develop an entirely randomized
OBOC library containing an N-terminal functionalized
fluoride-containing moiety in hopes of producing imaging
agents where the portion containing the imaging isotope would
be involved in receptor-binding itself. Copper-free click
chemistry by strain-promoted alkyne−azide cycloaddition
(SPAAC)15 is an increasingly popular technique for incorpo-
rating radioisotopes into biomolecular targeting agents due to
its quick and mild reaction conditions.16−18 The design of the
library was such that the eventual use of cyclooctyne-modified
peptides and an azide-containing 18F-prosthetic group would
allow for efficient and simple radiolabeling to produce the 18F-
imaging agent for PET imaging.
ew peptide-based imaging agents can be developed by
Nrational design from known target-specific peptides or
through combinatorial methods. The synthesis of one-bead
one-compound (OBOC) libraries is a combinatorial method
introduced by Lam et al. in 19911 that involves split-and-mix
synthesis to create millions of candidates from which peptide−
receptor interactions can be discovered. Although the screen-
ing of classic OBOC libraries produces target-specific
peptides,2−6 post-identification modification of these peptides
to convert them into imaging agents often decreases binding
affinity and creates the necessity for further modifications.7−11
However, few ventures have been made into making OBOC
libraries of imaging agents.12−14
The chemokine receptor CXCR4 has been implicated as a
receptor of interest in a wide variety of cancers, especially in
advanced and metastatic stages.19 Current peptide-based 18F
PET imaging agents that target CXCR4 have been based on
the CXCR4-antagonist peptide T140, which was developed
from the natural peptide polyphemusin II.20 Unfortunately,
many of these imaging agents display poor pharmacokinetics
and biodistribution in vivo.21−24 New CXCR4-binding
peptide-based imaging agents could be discovered through
combinatorial methods.
Fluorine-18 is a preferred radioisotope for positron emission
tomography (PET) imaging due to its half-life and availability;
as such, incorporating a fluorine-19 isotopologue of a fluorine-
18 prosthetic group into each entity of an OBOC library would
create a library of complete potential imaging agents. This
allows the portion of the imaging agent responsible for the
imaging signal to be incorporated into the binding pocket of
the target protein during affinity studies. Hereby, peptide
sequences where this imaging portion would prevent binding
would be excluded before further synthesis and complex
binding assays. Conversely, peptides that bind better with the
imaging portion present compared to the unmodified peptide
are now included in further studies. This enables high-
throughput screening of millions of potential imaging agents.
Tang et al. recently created a focused OBOC peptide library
containing both the αvβ6-targeting motif and a C-terminal 4-
fluorobenzoyl group for imaging and had success in
discovering lead peptides toward developing new 18F PET
agents.14
Our unique combinatorial library was screened on-resin
against a U87 cell line that highly expresses CXCR4; beads that
showed binding to the cells were isolated, and the sequences of
Received: August 8, 2019
Revised: January 21, 2020
Published: February 3, 2020
https://dx.doi.org/10.1021/acscombsci.9b00146
© XXXX American Chemical Society
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the respective peptide portions were identified by MALDI
tandem mass spectrometry (MS/MS). This demonstrates a
methodology where novel fluorine-containing peptide-based
imaging agents can be discovered with straightforward
translation into 18F PET imaging agents for CXCR4-expressing
cancer.
paraformaldehyde, and excess paraformaldehyde was quenched
with 2 M glycine. Library beads were then recombined, media
was decanted, and the beads were resuspended in PBS.
Beads were sorted in aliquots using a Complex Object
Parametric Analyzer and Sorter (COPAS, Union Biometrica)27
through two screening steps. Initially, all beads were collected
that expressed any threshold of green fluorescence (dye λex/em
= 492/517 nm), which resulted in narrowing the library hits to
approximately 2500 beads. This pool, however, can include
false positive beads that exhibit autofluorescence of the
Tentagel bead. These beads were then subjected to a second
round of sorting by the COPAS with the green fluorescence
threshold set to a higher limit (Figure 2). Hit beads during this
The cyclooctyne moiety chosen for the construction of our
library was azadibenzocyclooctyne acid (ADIBO−COOH) 1,
which was synthesized according to the methods of Chadwick
et al.25 We chose to employ a PEG-based azide-containing 18F-
prosthetic group in order to offer some hydrophilicity to
balance the large hydrophobic cyclooctyne. The fluorine-19
analogue 2 was synthesized and used in the construction of our
library. An entirely randomized octamer OBOC library was
synthesized on 1 g of Tentagel S resin equipped with a
photocleavable linker ((3-amino-3-(2-nitro-phenyl)propionic
acid) (ANP)). This allows protecting groups to be removed
while the peptides stay attached to the resin beads; yet, the
peptides can be later removed from the resin for sequencing. A
total of 17 natural amino acids (excluding cysteine,
methionine, and isoleucine) were used in the split-and-mix
process, after which the cyclooctatriazole portion 3 was
coupled onto the N-terminus of the combined library (Figure
1). The result was a pool of about 1.5 million potential imaging
Figure 2. Confocal fluorescence images of (A) library pool after
incubation with U87.CD4.CXCR4 cells tagged with CellTracker
Green CMFDA; (B) library beads after initial screen showing some
hit beads with various degrees of cell coverage; (C) isolated hit library
bead in a well of a 96-well plate after second sorting round; (D)
isolated false positive bead displaying high autofluorescence.
step were sorted individually into 96-well plates, and around
200 beads were collected. Visualization of the wells was
performed under fluorescence microscopy to remove any
remaining false positive beads that were incorrectly identified
as hits during the automated COPAS sort due to high levels of
bead autofluorescence (Figure 2).
Remaining beads were swollen in water and placed under a
UV lamp for 2 h in order to cleave peptides off the beads. The
supernatant was then subjected to MALDI MS/MS for peptide
sequence deconvolution (Figure 3).
This step of the process proved challenging as some wells
produced no observable MS product (possibly due to
insolubility, incomplete cleavage from the bead, etc.) or
incomplete fragmentation in MS/MS leading to unsolvable
sequences (a known issue in this deconvolution technique).28
In addition, the near isobaric amino acids lysine and glutamine
were both used in this library to promote diversity, but the
corresponding residue mass was often observed in MS/MS
spectra, which lead to multiple possible iterations of the hit
from a single bead. An unexpected issue that arose was the
high frequency of fragmentation between the N-terminal
amino acid and the cyclooctatriazole portion (at b0), causing
less intense fragmentation patterns along the rest of the
peptide backbone. In the end, hit sequences that could be fully
identified (Table S1) were resynthesized on Tentagel resin,
including all possible combinations with Lys/Gln. Unsurpris-
ingly, due to the negatively charged nature of the CXCR4-
binding pocket,29 many hit sequences contained multiple
positively charged residues, which could result in nonspecific
binding of these peptides to cells. Evaluation of each hit
sequence using bulk beads and two cell lines that differentially
express CXCR4 allowed for a final determination of peptide
sequences legitimately showing interaction with CXCR4.
Peptides on resin were incubated with a single sequence in
individual wells with either fluorescently tagged
U87.CXCR4.CD4 cells or U87.CD4 cells to serve as a control.
Figure 1. Synthesis of an octamer library on Tentagel resin using
ADIBO−COOH 1 and a PEG-based azide-containing fluoride 2. X is
any natural amino acid excluding cysteine, methionine, and isoleucine.
Only one regioisomer of the library constructs is shown for simplicity.
agent candidates, in which there is likely no redundancy of
peptide sequences (less than 0.1% of potential diversity
explored).
The deprotected library beads were subjected to screening
against U87.CD4.CXCR426 cells that had been preincubated
with a green CellTracker dye (ThermoFisher) to afford a way
to visualize and sort the beads. The entire library was divided
into multiple wells and screened; in each well, approximately
500 000 cells were incubated with 20 000 beads in 3 mL of
media for 1 h at 37 °C. After incubation, some wells were
visualized under fluorescence microscopy to confirm inter-
actions between the cells and beads. Receptor−peptide
interactions between the cells and beads were fixed with 4%
B
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Figure 3. MALDI MS/MS deconvolution of a peptide hit from library screen, showing overlapping b and y series of fragments and demonstrating
high fragmentation at b0. The isobaric amino acids lysine (K) and glutamine (Q) are both possibilities for mass fragments of ∼128 Da.
Wells were imaged by fluorescent microscopy to reconfirm
CXCR4 affinity and to select for peptides with CXCR4
selectivity. This step serves to remove any remaining false
positives and to identify peptides that had nonspecific or off-
target binding to the cells. Figure 4 displays an example where
peptides were subjected to a competitive binding assay to
prove receptor affinity using U87.CD4.CXCR4 cells and [125I]-
SDF-1α as the radioligand. The lead compound from this
target affinity assay was identified as F-PEG2-ADIBO-
YKFKRLWP-NH2 and has an IC50 of 138 μM. To determine
whether the imaging moiety included within this peptide
structure was essential for binding to CXCR4, we also
performed a binding assay of the peptide H-YKFKRLWP-
NH2, which indicated that the unadorned peptide had the
same binding affinity as the modified peptide (Figure S5). This
suggests that the rather bulky portion containing the imaging
component likely lies outside the binding pocket. While
previously reported 18F-peptide-based imaging agents for
CXCR4 have higher affinities, in the nanomolar range, they
are primarily based on the same polyphemusin peptide
sequence and often have comparable drawbacks when it
comes to in vivo behavior such as nonspecific binding to red
blood cells and/or high liver and kidney uptake.21−23 This
OBOC library produced a new sequence that can serve as a
starting point for further development toward novel CXCR4-
targeted imaging agents.
Overall, we have developed an OBOC approach as a
combinatorial method to produce a large-scale library of
peptide imaging agent candidates. This can be useful for
screening against any target of interest for imaging purposes.
Our library conveniently includes fluorine-19 in place of our
chosen imaging isotope fluorine-18 through copper-free click
chemistry in order to produce hits that can be easily translated
into radiolabeled imaging agents. Although the discovery of
imaging agents from this method still proves to be laborious
and fraught with challenges, the screening steps directly
produced a lead imaging agent with micromolar affinity.
Modifications based on this sequence could be explored to
further improve the affinity for CXCR4. Exciting new
advancements in technologies for tracking synthetic histories
of solid-phase syntheses with DNA tags or RFID micro-
transponders are highly complementary to this sort of
application and will remove the sequencing bottleneck
Figure 4. On-bead screen of potential hits synthesized on Tentagel of
the sequence F-PEG2-ADIBO-YXFXRLWP-NH2 where X = Q or K.
This screen allows for confirmation of “hit” sequence when isobaric
amino acids are involved and also screens for selectivity to the target.
the four possible iterations of the “hit” sequence shown in
Figure 2 were synthesized separately on Tentagel resin. Of the
combinations, only one sequence (−YKFKRLWP−) showed
good affinity to the CXCR4-expressing cells as well as strong
selectivity for CXCR4 compared to the control cell line (cells
in the top right image are in the background and not adherent
to the beads). The other sequences show either low affinity,
poor selectivity, or both.
CXCR4-selective peptides were then resynthesized on Rink
Amide resin and purified as the C-terminal amide. These
C
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encountered, which would result in higher numbers of imaging
agent hits to study.30,31 Improvements in the methodology to
address some of the challenges encountered in development of
this library will be explored in future generations of OBOC
imaging agent libraries. Focused library synthesis would aid in
the throughput of sequence deconvolution when part of the
sequence is known as well as likely produce higher affinity
hits.5,14 Other library conformations where the imaging moiety
is dispersed through the peptide sequences could also be
explored to promote discovery of imaging agents where the
imaging moiety is integrated into the receptor-binding pocket.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscombsci.9b00146.
Synthetic procedures and characterization as well as
further screening details and confocal microscopy images
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
Leonard G. Luyt − Department of Chemistry and Departments
of Oncology and Medical Imaging, University of Western
Ontario, London, Ontario N6A 5B7, Canada; London
Regional Cancer Program, Lawson Health Research Institute,
London, Ontario N6A 4L6, Canada; orcid.org/0000-0002-
0941-4731; Email: lluyt@uwo.ca
Author
Emily Murrell − Department of Chemistry, University of
Western Ontario, London, Ontario N6A 5B7, Canada
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscombsci.9b00146
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Financial assistance was gratefully received from the Natural
Sciences and Engineering Research Council (NSERC) of
Canada. The following reagents were obtained through the
NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH:
U87.CD4.CXCR4, U87.CD4 from Dr. HongKui Deng and Dr.
Dan R. Littman. We would also like to thank the Western
MALDI MS Facility and the Molecular Imaging Collaborative
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ABBREVIATIONS
■
ADIBO, azadibenzocyclooctyne; CXCR4, chemokine receptor
4; MALDI, matrix-assisted laser desorption ionization; MS/
MS, tandem mass spectrometry; OBOC, one-bead one-
compound; PET, positron emission tomography; SPAAC,
strain-promoted alkyne−azide cycloaddition
D
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