A dual optical and nuclear imaging reagent for peptide labelling via disulfide bridging

Article abstract of DOI:10.1039/c5ob01468a

We report a concise approach to a multimodal imaging reagent for peptide labelling via disulfide bridging. The reagent is constructed using a one pot, three component, [3 + 2] cycloaddition of a fluorescent azide with a dithiomaleimide-alkyne, with concom

Full text of DOI:10.1039/c5ob01468a

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A dual optical and nuclear imaging reagent for
peptide labelling via disulfide bridging†
Cite this: DOI: 10.1039/c5ob01468a
Sally A. Fletcher,a Pak Kwan Brian Sin,b Muriel Nobles,c Erik Årstad,a,b Andrew Tinkerc
Received 17th July 2015,
Accepted 17th August 2015
and James R. Baker*a
DOI: 10.1039/c5ob01468a
www.rsc.org/obc
We report a concise approach to a multimodal imaging reagent for tissue, but are limited by low resolution. Once the diseased
peptide labelling via disulfide bridging. The reagent is constructed region has been located, high resolution imaging of the tumour
using a one pot, three component, [3 + 2] cycloaddition of a fluor- can be carried out by highly sensitive optical methods.3,4
escent azide with a dithiomaleimide-alkyne, with concomitant
Peptides present themselves as an attractive targeting
incorporation of 125I. The dithiomaleimide handle then enables site moiety in clinical applications due to their small size, good
selective conjugation to a disulfide bond of a peptide whilst retain- stability and ease of synthesis and characterisation.5 This com-
ing the key structural bridging functionality, as exemplified on the bined with their high specificity and affinity for their targets
therapeutic peptide octreotide.
has led to their successful application in clinical diagnostics
and therapeutics.5 Octreotide, a long-acting analogue of the
hormone somatostatin, is a cyclic octapeptide with high
affinity for the somatostatin receptor.6 Many neuroendocrine
tumours are reported to over-express one subtype of this
family of receptor, predominantly subtype 2 (SSTR2).7 With its
high potency and specificity for the SSTR2, radiolabelled
octreotide has been employed for imaging of neuroendocrine
tumours in patients for many years.8 Recently, Edwards et al.
reported on a derivative of octreotide which incorporates a che-
lating group at the N-terminus for binding either 177Lu or 64Cu
(for imaging by PET or SPECT) and the fluorescent dye cypate
(for optical imaging) conjugated at the C-terminus.4 Excellent
correlation in the biodistribution of the MOMIA by optical and
nuclear imaging techniques was observed.
Employing two complimentary imaging techniques synergisti-
cally can allow for more accurate staging and localisation of
tumours and is a concept being explored in cancer diagnosis.
Administering two separate imaging agents to patients carries
problems however, as both will have differing pharmacokinetic
properties to consider and the toxic effects of each component
may well be additive. To circumvent this, strategies have been
explored to combine multiple imaging agents into one con-
struct, such as in micelles and nanoparticles.1 However, these
carry their own drawbacks, as such large molecules have inher-
ently slow diffusion rates into the target tissue and rapid
uptake and excretion by the liver.2
A recent concept is the incorporation of desired reporter
groups into one molecule, to create a monomolecular multi-
modal imaging agent (MOMIA). MOMIAs contain two or more
imaging agents in a 1 : 1 signalling ratio, with each agent pro-
viding different but complimentary information.3 Coupling
optical imaging with nuclear techniques such as positron emis-
sion tomography (PET) or single photon emission computed
tomography (SPECT) allows for the strengths of the individual
methods to be combined. For instance, PET and SPECT allow
whole body imaging and detection of tumours within deep
We have previously reported on the application of a new
class of maleimide reagent, referred to as next generation malei-
mides (NGMs, see Scheme 1), for the rapid and cysteine-selec-
tive modification of peptides and proteins.9 Most recently, the
use of these reagents in the effective re-bridging of the two
cysteine residues of a reduced disulfide bond has been
aDepartment of Chemistry, University College London, 20 Gordon St, London, UK.
E-mail: j.r.baker@ucl.ac.uk; Tel: +44 (0) 2076792653
bDivision of Medicine, University College London, Rayne Building, 5 University
Street, London, WC1E 6JF, UK
cBarts and The London, Queen Mary’s School of Medicine and Dentistry,
Charterhouse Square, London, UK
†Electronic supplementary information (ESI) available: Experimental procedures
and full spectroscopic data. See DOI: 10.1039/c5ob01468a
Scheme 1 A platform for MOMIA construction by disulfide bridging.
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exploited; in this way, cystines can become tenable as sites for coupled inwardly rectifying K+ (GIRK) channel family. The
conjugation as the structural integrity conferred by the disulfide somatostatin receptor is known to couple to GIRK channels;
bond is retained.10 This disulfide re-bridging strategy has activation of the receptor leads to subsequent activation of the
proved effective in the functionalisation of peptides, proteins, GIRK channel by means of G protein signalling.16 To model
antibody fragments and full antibodies.10,11 By careful design of this system, a Human Embryonic Kidney (HEK) 293 cell line
the maleimide reagents for a desired application, a range of stably expressing the GIRK1/2a channel subtype was transi-
variants can be synthesised that differ in activity or cargo.10–12
ently transfected with the SSTR2. The GIRK1/2a subtype was
We envisaged that multi-functionalised NGMs could chosen as it is known to predominate in neuroendocrine cells,
provide a versatile platform for the construction of peptide providing the most physiologically relevant system.17 Current
and protein-derived MOMIAs (Scheme 1), as well as theranos- activation by inwards flux of K+ through GIRK1/2a channels
tic agents, by allowing a variety of imaging reporter groups and upon application of modified octreotide 2 was monitored by
drug constructs to be combined. Such reagents would offer a whole-cell patch clamp experiments. This was carried out over
one-step approach to the dual functionalisation of disulfide a range of concentrations (from 1 nM to 10 µM) to allow con-
bonds, which would complement the elegant double click struction of a dose–response curve. These experiments were
strategy described by Chudasama, Caddick and co-workers.13 repeated using native octreotide (at concentrations from
Thus we report herein the synthesis of a multi-modal NGM, 0.01 nM to 100 nM) to serve as a control (Fig. 1).
carrying a fluorophore to enable optical imaging, and radio-
From the dose–response analysis, an EC50 of 118 nM was
iodine for nuclear imaging techniques. We tested the ability of calculated for the modified octreotide 2. In comparison to the
this novel reagent to insert into the disulfide bond of a model native peptide (EC50 calculated as 0.66 nM), there is an attenu-
targeting peptide, octreotide.
ation of biological activity as a result of disulfide bridging
The chemistry of the NGM motif allows for the facile func- using the maleimide reagent 1. This is likely due to the small,
tionalisation of the ring nitrogen. The incorporation of an rigid nature of octreotide, leading a 2 carbon-bridge to be
alkyne at this position has been reported and creates a reactive sufficient to perturb the pharmacophore. This attenuation of
chemical handle available for further derivatisation.11e,14 We activity revealed that a MOMIA based on labelling the disulfide
envisaged exploiting this reactivity to install our desired func- bridge of octreotide would not be ideal for in vivo imaging.
tionalities, and thus N-alkyne dithiophenolmaleimide 1 was However this peptide still represented a suitable model system
synthesised.15 This reagent was employed to test the efficiency for testing the dual labelling strategy, in the knowledge that
of the bridging protocol for octreotide, and to investigate the future applications on larger, structured proteins would be
effect of the bridging on the biological activity.
unaffected by disulfide bridging.11a,c,e,f,13,18 Furthermore,
Octreotide was first reduced using 1.2 equivalents of tris- in vitro experiments could still be explored to reveal infor-
(2-carboxyethyl)phosphine (TCEP) for 1 h before addition of mation about the cellular distribution of the peptide.
1.2 equivalents of maleimide 1 (Scheme 2). Complete conver-
sion to the modified octreotide 2 was observed within 10 min cycloaddition reaction as a route to functionalising the malei-
(see ESI Fig. 1†). mide with the imaging agents of choice. Rhodamine B was
The free alkyne moiety of maleimide 1 lends itself to a click
To examine whether the bridging modification had an chosen as the fluorophore as it has good photostability,
effect on the biological activity of octreotide, a signalling assay quantum yield and water solubility,19 as well as a carboxylic
was established to test its activity. Somatostatin and its ana- acid group which can be readily functionalised with an azide.
logues such as octreotide are known to exert their pharmaco- The targeted rhodamine B-azide was synthesised in 4 steps
logical
effects
through
the
hyperpolarisation
of (Scheme 3). The carboxylic acid of rhodamine B was first con-
neuroendocrine and neuronal cell membranes.16 This hyper-
polarisation is facilitated by the activation of the G protein-
Fig. 1 Mean absolute current increase (pA/pF) with increasing concen-
tration of modified octreotide
2 (solid line) and native octreotide
Scheme 2 Octreotide disulfide bridging using maleimide 1.
(dashed line).
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Scheme 3 The synthetic route to rhodamine B-azide 3.
Fig. 2 (A) HPLC trace of the non-radioactive reference compound 4;
(B) radio-chromatogram of the dual optical and nuclear labelling
reagent [125I]4.
verted to the acid chloride and then reacted with 4-(N-Boc-
amino)piperidine to form the tertiary amide.20 Deprotection
with TFA led to quantitative formation of the TFA salt, which
was then coupled with 6-azidohexanoic acid21 to afford the
desired rhodamine B-azide 3 in good yield.
With the fluorescent azide and dithiophenolmaleimide
alkyne in hand, focus turned to the click cycloaddition reac-
tion. Yan et al. recently reported on a novel route to incorpor-
ation of the radiolabel 125I into the triazole product of a click
radiochemical yield was determined to be 58%, while the iso-
lated radiochemical yield was 47%.
To test whether this multi-modal maleimide was efficient in
the re-bridging of the disulfide of octreotide, it was trialled in
the method previously employed; octreotide was first reduced
with 1.2 equivalents of TCEP before addition of 1.2 equivalents
of maleimide 4. Efficient conversion to octreotide-MOMIA was
observed, with full conversion reached within 15 min (see ESI
Fig. 3†). With all components in place, the octreotide-MOMIA
was tested for its efficacy as an imaging agent for somatostatin
receptor-positive tumours. To examine whether the modified
octreotide still bound to the somatostatin receptor and whether
the attached rhodamine moiety maintained its fluorescence, con-
focal microscopy experiments were conducted. HEK293 cells were
transiently transfected with SSTR2 and enhanced green fluo-
rescent protein (eGFP) as a transfection marker. The cells were
incubated with the octreotide-MOMIA (1 µM) for 20 min before
its removal. The cells were washed and then visualised at two
wavelengths: 488 nm to visualise eGFP and confirm SSTR2
expression and 543 nm to visualise rhodamine fluorescence
(Fig. 3) and to confirm octreotide-MOMIA binding. Non-trans-
fected HEK293 cells were included as a negative control.
cycloaddition in
a one-pot, three-component, copper(II)-
mediated reaction.22 We envisaged employing this reaction in
a highly efficient synthesis affording a NGM bearing both a
fluorophore and a radiolabel. To enable structural characteris-
ation the non-radioactive iodinated compound was first syn-
thesised. Maleimide 1, azide 3, triethylamine, and
N-chlorosuccinimide were added to a solution of copper(I)
iodide and stirred in an overnight reaction to form the triazole
4 (Scheme 4).23 With this reference reagent in hand, work pro-
gressed on to the synthesis of the radioactive compound. In
this case, maleimide 1, copper(II) chloride and triethylamine
were combined prior to the addition of [125I]NaI and azide 3 to
form radioactive reagent [125I]4 (Scheme 4). The structural
identity of the radiolabelled product was confirmed by com-
parison of the radio-chromatogram with the UV trace from the
non-radioactive reference compound (Fig. 2). The analytical
Good fluorescence correlation was observed; octreotide-
MOMIA bound to cells expressing the SSTR2. Octreotide has
maintained its ability to bind to SSTR2 despite chemical modifi-
cation. Control cells with no SSTR2 expression revealed no non-
specific binding of octreotide-MOMIA. A high concentration of
rhodamine fluorescence was observed on the cell membrane
while intracellular fluorescence was less concentrated.
The confocal imaging experiments were repeated on a
second model system with endogenous expression of the
SSTR2. Human pancreatic BON-1 cells derived from a neuro-
endocrine tumour are reported to display considerable SSTR2
expression and served as an ideal cell line to test the efficacy of
octreotide-MOMIA.24 Confocal experiments were conducted as
previously described, with visualisation at 543 nm (Fig. 4).
Cell membrane localisation of octreotide-MOMIA was
observed, confirming SSTR2 binding of the peptide. This
Scheme 4 The synthesis of reagents 4 and [125I]4.
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accuracy of the staging and localisation of cancers. The
reagent described could also be readily applied to other recep-
tor-specific peptides and proteins, to access a range of dual
modality imaging agents. Furthermore, this strategy represents
a general approach to the efficient construction of dual-func-
tionalised protein conjugates. For example the use of a drug in
the place of the fluorophore would enable efficient access to
protein–drug conjugates detectable by nuclear imaging tech-
niques, with prospective application in theranostics.
Acknowledgements
The authors gratefully acknowledge the BBSRC, EPSRC, GE
Healthcare and UCL for supporting this research, Dr Abil Aliev
for NMR technical support and Dr Kersti Karu for assistance
with mass spectrometry. We would also like to acknowledge
Prof. Tim Meyer for the kind donation of the BON-1 cell line.
Part of the work was undertaken at UCLH/UCL who received a
proportion of funding from the Department of Health’s NIHR
Biomedical Research Centres funding scheme (E. A). This
work was supported by the British Heart Foundation and The
National Institute for Health Research Barts Cardiovascular
Biomedical Research Unit.
Fig. 3 Confocal images of HEK293 cells after incubation with octreo-
tide-MOMIA (1 µM) for 20 min at 37 °C prior to washing. (A) Cells trans-
fected with SSTR2 and eGFP and visualised at 488 nm. (B) Cells
transfected with SSTR2 and eGFP and visualised at 543 nm. (C) Non-
transfected cells visualised at 488 nm. (D) Non-transfected cells visual-
ised at 543 nm.
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