Enzymatic radiosynthesis of a18F-Glu-Ureido-Lys ligand for the prostate-specific membrane antigen (PSMA)

Article abstract of DOI:10.1039/c8ob03150a

Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour s

Full text of DOI:10.1039/c8ob03150a

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Enzymatic radiosynthesis of a 18F-Glu-Ureido-Lys
ligand for the prostate-specific membrane antigen
(PSMA)†
Cite this: DOI: 10.1039/c8ob03150a
b
Phillip T. Lowe, a Sergio Dall’Angelo, b Ian N. Fleming, c Monica Piras,
a
Matteo Zanda
*
b,d and David O’Hagan
*
Prostate cancer represents a major public health threat as it is one of the most common male cancers
worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer
cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been
identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the
focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high
affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours.
Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-mole-
cule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5’-fluo-
rodeoxy-adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhi-
bits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be pro-
duced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of
radiolabelled [18F]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advan-
tageous conditions (in water at neutral pH and at ambient temperature). Specific binding of
Received 19th December 2018,
Accepted 17th January 2019
[
18F]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA
DOI: 10.1039/c8ob03150a
diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and
demonstrates its first application towards targeting the PSMA.
rsc.li/obc
dependant integral membrane protein that is overexpressed in
almost all prostate cancers.4–6 PSMA catalyses the hydrolysis of
Introduction
Prostate cancer represents one of the most common malignan- N-acetylaspartylglutamate (NAAG) to generate glutamate and
cies in men and is diagnosed annually in over 1 million N-acetylaspartate (NAA),7,8 the excessive production of which
patients worldwide.1 With a substantial morbidity and mor- has been linked to a number of health issues including a
tality rate, it is a major public health issue.2 The selection of variety of neurological diseases.9,10 Primarily restricted to the
appropriate treatments using diagnosis derived from high prostate, PSMA expression levels in cancerous cells are approxi-
resolution imaging is therefore of the foremost importance.2,3 mately 1000 times higher than that of healthy tissue in other
The prostate-specific membrane antigen (PSMA), also known organs (such as those of the kidney and small intestine).11
as glutamate carboxypetidase II, N-acetyl-α-linked acidic dipep- Significantly, its expression correlates with both the stage and
tidase I (Naaladase I) or folate hydrolase, is a type II zinc grade of the tumour progression;12,13 due to each of these
factors, PSMA has become an increasingly important diagnos-
tic and therapeutic target.10,14–16
aSchool of Chemistry and Biomedical Sciences Research Centre, University of
Numerous low molecular weight PSMA inhibitors derived
St Andrews, North Haugh, St Andrews KY16 9ST, UK. E-mail: do1@st-andrews.ac.uk
from NAAG have been developed,17 with 2-(phosphono-
bJohn Mallard Scottish PET Centre School of Medicine, Medical Sciences and
methyl)-pentandioic acid (2-PMPA)18 being the first potent
Nutrition University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK.
inhibitor synthesised. Subsequently a number of thiol,19
E-mail: m.zanda@abdn.ac.uk
cAberdeen Biomedical Imaging Centre, Institute of Medical Sciences Foresterhill,
indole20 and hydroxamate21 derivatives were designed, see
Aberdeen, AB25 2ZD, UK
dC.N.R. – I.C.R.M., via Mancinelli 7, 20131 Milan, Italy
Fig. 1. Of particular interest in recent years has been a class of
urea-based inhibitors which possess the Glu-NH-CO-NH-Lys/
†Electronic supplementary information (ESI) available: Compound synthesis
Glu (GUL/GUG) pharmacophore.10,17,22 A number of com-
and characterisation, assay conditions and fluorinase overexpression and purifi-
pounds of this class have shown improved potency as PSMA
cation. See DOI: 10.1039/c8ob03150a
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Glu-NH-CO-NH-Lys/Glu pharmacophore, along with the devel-
opment of novel methodology to incorporate the [18F]fluoride
ion into these motifs, is of great interest.
The fluorinase enzyme, originally isolated from
Streptomyces cattleya,35 mediates the conversion of fluoride ion
and S-adenosyl-L-methionine (AdoMet) to 5′-fluoro-5′-deoxy-
adenosine (5′-FDA) and L-methionine (L-Met), Scheme 1.36
Furthermore, it can also utilise L-Met (or L-SeMet) in the
reverse direction and catalyse the displacement of chloride
from 5′-chloro-5′-deoxyadenosine (5′-ClDA) to generate AdoMet
(or AdoSeMet), which allows for an overall transhalogenation
reaction to occur if fluoride is present (Scheme 1).37 The fluori-
nase enzyme has been shown to possess a distinct localised
tolerance in its substrate promiscuity, allowing for decoration
of the C-2 position of the adenine ring of 5′-ClDA with exten-
Fig. 1 The PSMA substrate N-acetylaspartylglutamate (NAAG) and
examples of low-molecule-based PSMA inhibitors.
inhibitors. Their relative synthetic accessibility and inherent sively functionalised acetylene substituents.38 This promiscuity
modularity have permitted many of these inhibitors to be offers the opportunity to exploit its function to accommodate
modified for use in clinical imaging modalities via incorpor- the direct 18F-radiolabelling of biologically relevant molecules
ation of various radioisotopes.23–27
to generate chemically stable C–18F bonds under aqueous
Due to its high sensitivity, positron emission tomography ambient conditions at near-neutral pH. In this manner, the
(PET)28 has emerged as a valuable tool for imaging prostate fluorinase offers advantage when compared to many other
cancer using radiolabelled urea-based inhibitors of PSMA. radiolabelling methodologies, as its activity in aqueous
With its moderate half-life (t1/2 = 109.8 min, affording environments circumvents the requirement to secure anhy-
sufficient time for radioisotope incorporation), short positron drous [18F]fluoride via ion-exchange chromatography and as a
range (2.3 mm in water, allowing for high resolution images) Kryptofix® [2,2,2] formulation. The enzyme has the additional
and ease of production (with availability at any PET facility in benefits of not requiring the use of protecting groups,
possession of a cyclotron) [18F]fluoride is a preferred isotope increased temperatures or high/low pH levels. Furthermore,
for PET imaging.28 Thus, incorporation of [18F]fluoride into conventional HPLC methodology can be used to separate non-
urea-based inhibitors represents a potentially valuable tool for radiolabelled starting material from radiolabelled product,
imaging prostate tumours.29 To date, methods to integrate regardless of the targeting scaffold tethered to the fluorinase
[18F]fluoride into these compounds have been developed using binding motif. To date, this late-stage enzymatic 18F-radio-
indirect, multistep strategies involving the initial synthesis of a labelling has been accomplished with cancer targeting pegylated
18F-labelled prosthetic group such as [18F]fluorobenzaldehyde RGD peptides38–40 and A2A adenosine receptor agonists,41 as well
([18F]FBA) or 2,3,5,6-tetrafluorophenylester-6-[18F]-fluoronicoti- as tetrazine and biotin motifs,42 for use as radiotracers in PET.
nate ([18F]FPyl-TFP), which are then conjugated to the urea
For this study we have designed and synthesised a
based inhibitor.30,31 Such a strategy has been applied to the ClDA-PEG-GUL 15 construct to be used as a novel substrate for
synthesis of [18F]DCFPyl and [18F]PSMA-1007, both of which the fluorinase enzyme. It is designed as such to encompass
have shown promise during preclinical investigation.31 both
a 5′-chloro-5′-deoxyadenosine unit (responsible for
Alternatively, direct radiolabelling of urea-based inhibitors via binding to the fluorinase active site and undergoing an overall
[18F]AlF-complexation is also being investigated and has been fluorination), a PEGylated spacer unit (to project tethered
used for the synthesis of Al[18F]PSMA-HBED;32–34 once opti- cargo away from the enzyme) and a PSMA binding GUL teth-
mised, such a strategy may provide a potentially more efficient ered to the terminus of the PEG unit through amide coupling,
route to radio-fluorinated PSMA tracers for clinical application. see Scheme 2. ClDA-PEG-GUL 15 has been utilised in a fluori-
As such, new [18F]fluoride-radiotracers possessing the nase mediated transhalogenation reaction to generate its
Scheme 1 Fluorinase-catalysed transhalogenation reactions with C-2 modified 5’-chloro-5’-deoxyadenosine substrates.
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fluorinated analogue FDA-PEG-GUL 16, which was sub-
sequently assessed in vitro for its ability to inhibit the PSMA
protein.
Results and discussion
Synthesis of ClDA-PEG-GUL 15
The synthesis of ClDA-PEG-GUL 15 was accomplished using a
three-step approach (Scheme 3B) to allow for the individual
synthesis and then successive coupling of each component
required to assemble the substrate. These components are the
ClDA binding moiety, the functionalised PEG spacer and the
GUL PSMA binding motif. Firstly, the synthesis of the Glu-
NH-CO-NH-Lys 7 (Scheme 3A) was achieved in a three-step
Scheme 2 Design of the last step 18F-radiolabelling strategy of
ClDA-PEG-GUL.
Scheme 3 (A) Synthesis of (t-butyl protected)GUL 14. (B) Synthesis of ClDA-PEG-GUL 15 and FDA-PEG-GUL 16. Reagents and conditions: (a)
Triphosgene, DCM/NaHCO3 sat., 99%; (b) H-Lys(Cbz)-OtBu·HCl pyridine, DCM, 77%; (c) H2, Pd(OH)2, MeOH, 99%; (d) THF, NaH, propargyl bromide,
58%; (e) THF, NaH, tert-butyl bromoacetate, 60%; (f) Pd2(dba)3, Et3N, CuI, DMF, 59%; (g) TFA, DCM, 98%; (h) PyBOP, DIPEA, DMF, 42%; (i)TFA, DCM,
57%; ( j); fluorinase, L-SeMet, KF, phosphate buffer (pH 7.8), 70%.
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manner from H-Glu(OtBU)-OtBu 4. Briefly, 4 was treated
with triphosgene under basic conditions to afford isocyanate
intermediate 5, which was used directly in subsequent cou-
pling reactions, without the need for further purification.
Isocyanate 5 was then reacted with H-Lys(Cbz)-OtBu in the
presence of pyridine to afford the fully protected peptidomimetic 6
in good yield after flash chromatography. Finally, the Cbz pro-
tecting group was removed via hydrogenation to yield (t-butyl
protected) GUL 7, which was used in subsequent coupling
reactions without further purification. The synthesis of the
5′-chlorodeoxy-2-iodo-adenosine 11 was achieved via a five-
step protocol using previously established methods39 and the
alkyne/protected carboxylate functionalised linker 10 was
obtained via a two-step process.42
With 7, 10 and 11 in hand, their assembly was addressed.
Firstly 10 and 11 were combined via a Sonogashira cross-coup-
ling reaction, which, after successive flash chromatography
and C18 cartridge purification, afforded 12 in good yield. tert-
Butyl ester 12 was then deprotected with TFA to afford the
corresponding carboxylic acid 13. Carboxylic acid 13 was then
utilised in a peptide coupling reaction using an excess of 7,
PYBOP and DIPEA to generate ClDA-PEG-(t-butyl protected)
GUL 14 in good yield after purification by semi-prep HPLC
(Scheme 3B). Finally, the tert-butyl groups of ClDA-PEG-(pro-
tected)GUL 14 were removed by TFA. Particular care was taken
during this procedure and the reaction was monitored by
analytical HPLC to ensure unnecessarily long reaction times
were avoided (see ESI†). Semi-preparative HPLC was then used
to provide the candidate fluorinase substrate ClDA-PEG-GUL
15 in good yield (70%) and high purity (>95%.).
Fig. 2 HPLC time course (UV, 254 nm) of the incubation of
ClDA-PEG-GUL 15, green (tR = 9.5 min), with the fluorinase, L-SeMet,
KF, phosphate buffer (pH 7.8) at 37 °C. Traces show the formation of
FDA-PEG-GUL 16, yellow (tR = 8.8 min), and the consumption of
ClDA-PEG-GUL 15. For full conditions see the Experimental section and
ESI.†
Evaluation of 16 as an inhibitor of PSMA
The ability of FDA-PEG-GUL 16 to inhibit PSMA activity was
determined using a fluorescence-based assay, essentially as
previously reported.43 Briefly, purified recombinant human
PSMA protein was incubated with each test compound in the
presence of N-acetylaspartylglutamate as the substrate, at
37 °C for 60 min. The reaction was then stopped by heating at
95 °C for 5 min and the resulting solution incubated with a
solution of ortho-phthaldialdehyde (OPA, 15 mM) in OPA
buffer (0.2 M NaOH and 0.1% (v/v) β-mercaptoethanol) for
10 min at room temperature. Aliquots were then assessed for
fluorescence in a microplate reader using an excitation wave-
length of 330 nm and an absorption wavelength of 450 nm, to
determine the amount of free glutamate present. The binding
affinity of each test compound to purified recombinant human
PSMA was expressed as its 50% inhibitory concentration (IC50
value) in the assay (IC50 values were calculated using
GraphPad Prism 5, see ESI† for full experimental detail).
FDA-PEG-GUL 16 and 2-PMPA were both tested in the assay.
As expected,44 2-PMPA was a potent PSMA inhibitor, exhibiting
an IC50 value of 18 nM in this assay (Table 1). The fluorine
containing 16 was also a similarly good PSMA inhibitor, with
an IC50 value in the nanomolar range. These data establish
that 16 binds PSMA with high affinity.
Fluorinase mediated transhalogenation of ClDA-PEG-GUL 15
In order to establish whether ClDA-PEG-GUL 15 can undergo
biotransformation to its fluorinated analogue, small scale
analytical transhalogenation reactions were performed.
Accordingly, ClDA-PEG-GUL 15 was incubated in aqueous
buffered media (pH 7.8) with the fluorinase (1 mg mL−1),
L-SeMet and KF (see ESI† for full experimental detail) and reac-
tion progress was monitored by analytical HPLC over a period of
48 h. The time course profile generated from this assay revealed
that 15 was an efficient substrate for the fluorinase with a 40%
conversion between 0–1 h, at which point FDA-PEG-GUL 16 pro-
duction levels off (reaching 60% in 4 h and ∼90% in 10 h), see
Fig. 2. This data reveals that the GUL motif does not interfere
with the ability of the fluorinase to perform an overall trans-
halogenation from ClDA-PEG-GUL. Importantly, no degradation
of ClDA-PEG-GUL 15 or the fluorinated FDA-PEG-GUL 16
product was observed during these assays.
A larger scale transhalogenation reaction was then per-
formed to allow for the acquisition of FDA-PEG-GUL in order
to evaluate its ability to bind and inhibit PSMA. The reaction
was carried out on a ∼3 mg scale and monitored by HPLC.
Radiosynthesis of [18F]FDA-PEG-GUL ([18F]16)
After a 90% conversion, the protein was precipitated by A protocol was developed to enzymatically radiolabel the pro-
heating and the reaction centrifuged in an eppendorf. spective radiotracer [18F]FDA-PEG-GUL ([18F]16). Briefly, for
Purification of the lysate was accomplished using semi-prep hot labelling experiments using the fluorinase enzyme, [18F]
HPLC to afford 16 (∼2 mg) in high purity.
fluoride was generated in an [18OH2] aqueous solution at GBq
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Table 1 IC50 values of selected PSMA analogues were measured for
their ability to compete with a standard glutamate containing peptide
(Ac-Asp-Glu) as substrate for purified recombinant human PSMA
enzyme. Results are the average standard error (s.e.) from three inde-
pendent experiments, each performed in triplicate
Compound
IC50 s.e. (nM)
FDA-PEG-GUL 16
2-PMPA
98.6 22.5
18 5.0
levels. An aliquot of this (MBq of [18F]fluoride, picomolar
levels) was then added to a buffered solution (pH 7.8) of 15,
L-SeMet and a considerable excess of fluorinase enzyme
(micromolar, see Experimental section), and the reaction
mixture was incubated at 37 °C. For this protocol, the stoichio-
metry of the radiolabelling biotransformation reaction is dra-
matically reversed with respect to the non-radioactive trans-
halogenation reaction described above. Preliminary radiolabel-
ling experiments showed that 30–45 min was the optimal reac-
tion time for this biotransformation, at which point the
enzyme was cleanly heat-precipitated, diluted and removed by
centrifugation. The lysate was then subjected to semi-prep
HPLC and the peak corresponding to [18F]16 was collected
(free of 15, see Fig. S26–27†). The collected fraction was
diluted with water and loaded onto a C18 reverse phase car-
tridge and after a wash with water, [18F]16 was eluted from the
cartridge with EtOH. The purity of [18F]16 was determined by
analytical radio-HPLC (see Fig. 3), and its identity further con-
firmed by examination of a sample spiked with FDA-PEG-GUL
16 (see Fig. S27a†). A typical procedure from [18F]fluoride (572
MBq) to [18F]16 (19.2 MBq) elution in EtOH took 1.5 h, and
afforded a radiochemical yield of ∼3.4% (decay uncorrected)
and a radiochemical purity of >99%. This yield is in line with
that reported for other fluorinase catalysed radiolabelling. The
success of this protocol, along with the robust nature of the
fluorinase, allows for its further development and the potential
incorporation of immobilised enzyme. In such an instance,
Fig. 4 Bound [18F]16 [cpm mg−1] protein per MBq radiotracer.
the methodology could be then integrated into an automated
system which utilises greater amounts of [18F]fluoride to
increase the scale and yields of [18F]16 production.
Specific cell binding of [18F]FDA-PEG-GUL 16 to PSMA
expressing cancer cells
The binding of [18F]16 to PSMA on the surface of cancer cells
was evaluated in LNCaP (which are well known to express high
levels of PSMA) and PC3 (which do not express PSMA) cell
lines.45 Western blotting analysis of cell lysates confirmed that
the LNCaP cells used in this study expressed PSMA, whereas
the PC3 cells did not (data not shown). A high level of [18F]16
binding was observed in LNCaP cells (Fig. 4), but not in PC3
cells. 2-PMPA (10 µM) was included in binding assays to
permit identification of specific radiotracer binding to PSMA
from non-specific binding. Inclusion of 2-PMPA decreased
[18F]16 binding to LNCaP cells by approximately 95%, but had
no effect on radiotracer binding to PC3 cells (Fig. 4). These
data are consistent with the non-radiolabelled inhibition assay
and provide strong support that [18F]16 binds selectively to
PMSA in prostate cancer cells.
Conclusions
This study established FDA-PEG-GUL 16 as a new low mole-
cular weight PSMA-targeting ligand, which was generated
using the fluorinase enzyme by biotransformation from its
chlorinated precursor ClDA-PEG-GUL 15, further extending the
repertoire of substrates accepted by this enzyme.
FDA-PEG-GUL 16 was utilised for in vitro studies and found to
be a potent inhibitor of the PSMA protein. As part of this
study, a protocol was developed to allow for the enzymatic
production of [18F]FDA-PEG-GUL ([18F]16), making it a poten-
tial new PET radiotracer for imaging prostate cancer.
Candidate tracer [18F]16 was evaluated in a cell-based assays
and demonstrated to selectively bind to cells expressing PSMA.
Fig. 3 (A) Reaction scheme of the fluorinase catalysed transhalogena-
tion of 15 to [18F]FDA-PEG-GUL ([18F]16). (B) Analytical HPLC radio trace
of [18F]FDA-PEG-GUL ([18F]16) after purification. For additional infor-
mation on HPLC set up and conditions for radiolabelling experiments
see ESI.†
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Experimental
Acknowledgements
Non-radioactive transhalogenation assay
We thank the Engineering and Physical Sciences Research
In a total reaction volume of 1000 μL (in 50 mM phosphate Council, UK, for a research grant (EP/M01262X/1).
buffer, at pH 7.8), recombinant fluorinase (0.8 mg mL−1
)
was incubated with ClDA-PEG-GUL 15 (0.08 mM), L-SeMet
(0.075 mM) and KF (50 mM) at 37 °C. Samples (50 μL) were
periodically removed, the protein precipitated by heating at
95 °C for 5 min, before being clarified by centrifugation
(13 000 rpm, 10 min). Samples of the supernatant (40 μL)
were removed for analysis by HPLC, which was performed
on a Shimadzu Prominence system using a Kinetix 5 μm
XB-C18 100A (150 mm × 4.6 mm) column and a guard car-
tridge. Mobile phase: 0.05% TFA in water (solvent A) and
0.05% TFA in MeCN (solvent B); linear gradient: 15%
solvent B to 95% solvent B over 25 min, 95% for 5 min, and
back to 15% B for 10 min to re-equilibrate the column. Flow
rate: 1 mL min−1; detection: 254 nm; injection volume:
40 μL.
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A typical 18F-labelling experiment of 15 was performed as
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centrifugation (13 000 rpm, corresponding to 16 060g, 5 min).
The supernatant was injected into a Shimadzu Prominence
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There are no conflicts of interest to declare.
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