Spacer length effects on in vitro imaging and surface accessibility of fluorescent inhibitors of prostate specific membrane antigen

Article abstract of DOI:10.1016/j.bmcl.2011.09.115

Prostate-specific membrane antigen (PSMA), a type II transmembrane protein, has been becoming an active target for imaging and therapeutic applications for prostate cancer. Recently, the development of its various chemical inhibitor scaffolds has been exp

Full text of DOI:10.1016/j.bmcl.2011.09.115

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7013–7016
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spacer length effects on in vitro imaging and surface accessibility
of fluorescent inhibitors of prostate specific membrane antigen
Tiancheng Liu a, Jessie R. Nedrow-Byers a, Mark R. Hopkins a, Clifford E. Berkman a,b,
⇑
a Department of Chemistry, Washington State University, Pullman, WA 99164-4630, United States
b Cancer Targeted Technology, Woodinville, WA 98072, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Prostate-specific membrane antigen (PSMA), a type II transmembrane protein, has been becoming an
active target for imaging and therapeutic applications for prostate cancer. Recently, the development
of its various chemical inhibitor scaffolds has been explored to serve as carriers for therapeutic or
diagnostic payloads targeted to PSMA-positive tumor cells. However, there have been few efforts to
definitively determine the optimal length of linker between PSMA inhibitor cores and their payload mol-
ecules with regard to the affinity to PSMA and in vitro performance. In our present model study, three
spacer-length varied fluorescent inhibitors (FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG8-CTT-54) were
synthesized, and further enzymatic inhibition studies displayed linker length-dependent changes in:
inhibitory potency (IC50 = 0.41 nM, 0.35 nM, 1.93 nM), modes of binding (reversible, slowly reversible,
irreversible), respectively. Furthermore, cell-labeling imaging revealed the spacer length-related change
of fluorescence intensity (FAM-X-CTT-54 > FAM-PEG8-CTT-54 > FAM-CTT-54). These results suggest that
selection of linkers and their lengths will be important considerations in the development of next-
generation prostate tumor-targeted imaging probes and therapeutic agents that specifically home to
PSMA on tumor cells.
Received 22 August 2011
Accepted 27 September 2011
Available online 4 October 2011
Keywords:
Tumor-targeting imaging
PSMA
Confocal microscopy
Fluorescein conjugate
PEG linker
Ó 2011 Elsevier Ltd. All rights reserved.
The cell-surface enzyme prostate-specific membrane antigen
(PSMA) is perhaps the most important enzyme-biomarker and
target in prostate cancer research and is being aggressively pursued
as a target for the selective delivery of novel diagnostic and
therapeutic agents. PSMA is up-regulated and strongly expressed
on prostate cancer cells, including those that are metastatic.1
Endothelial-expression of PSMA in the neovasculature of a variety
of non-prostatic solid malignancies has also been detected.2,3 The
unique enzymatic activity of PSMA4–6 and the resolution of its crys-
tal structure7–9 have enabled the development of various high-affin-
ity chemical inhibitor scaffolds for this enzyme-biomarker.10–17
Recently, the successfully deployment of PSMA inhibitors as target-
ing motifs for imaging and therapeutic agents suggests that such
constructs can serve as pharmacokinetic alternatives to antibod-
ies.18–23
Although we previously reported that phosphoramidate pepti-
domimetic PSMA inhibitors were capable of both cell-surface label-
ing of prostate tumor cells for imaging18,19,24 and intracellular
delivery for targeted photodynamic therapy,20,25,26 there have been
few efforts to definitively determine the optimal spacer length
between a PSMA inhibitor core and its diagnostic or therapeutic
payload with respect to inhibitory potency and in vitro performance.
The polyethylene glycol (PEG) spacer has been widely applied in
nanotechnology to covalently couple small-molecule ligands or
antibodies onto the surfaces of nanoparticles for targeted imaging
or drug delivery.27–30 In general, PEG spacers can lead to improved
plasma circulation and biocompatibility of nanoparticles due to
their enhanced hydrophilicity, and provide sufficient flexibility for
a targeting molecule to overcome spatial limitations in order to
effectively interact with
a corresponding target protein or
receptor.28 In the presentstudy, we examinedthe effect of the spacer
length between a representative phosphoramidate PSMA inhibitor
core (CTT-54) and fluorescein-based dye (Fig. 1) upon both the
inhibitory potency against PSMA and the cell-labeling of PSMA+
cells. The preparation of both the phosphate PMSA inhibitor and
its fluorescein conjugates is provided in the Supplementary data.
In this study, a series of fluorescent PSMA inhibitor conjugates
(FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG8-CTT-54) were syn-
thesized according to a previously reported method.18 As shown
in the Supplementary data (Figs. S1 and S2), the absorption spectra
(400–800 nM) and fluorescence emission (at ꢀ520 nM) of free
fluorescein dye and the fluorescent PSMA inhibitor conjugates
displayed similar absorbance spectra and fluorescence intensity.
These data suggest that that conjugation of CTT-54 through various
spacer lengths had little impact on the spectral properties.
In Figure S3A–D, PSMA inhibition studies confirmed that
⇑
Corresponding author. Tel.: +1 509 335 7613; fax: +1 509 335 8867.
E-mail address: cberkman@wsu.edu (C.E. Berkman).
conjugation of CTT-5419,20,31 to fluorescein-based dyes through
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.115

7014
T. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7013–7016
Figure 1. Structures of PSMA inhibitor core CTT-54, and its fluorescein conjugates: FAM-CTT-54, FAM-X-CTT-54, and FAM-PEG8-CTT-54.
various spacer lengths (FAM-CTT-54, IC50 = 0.41 nM; FAM-X-CTT-
54, IC50 = 0.35 nM;19 FAM-PEG8-CTT-54, IC50 = 1.93 nM) had no
adverse effect upon the inhibitory potency of the parent inhibitor
core CTT-54 (IC50 = 14 nM).19 To understand the impact of spacer
length on the fluorescent inhibitor conjugates, we examined the
enzymatic activity recovery profiles for PSMA inhibition by CTT-
54, FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG8-CTT-54, according
to our previously reported method.16,18 Both CTT-54 and FAM-
PEG8-CTT-54 were shown to be irreversible inhibitors, while
FAM-CTT-54 was completely reversible and FAM-X-CTT-54 exhib-
ited characteristics of slowly reversible inhibitors (Fig. 2). These
data suggest that the placement of the fluorophore too close to
the PSMA active may prevent essential conformational changes
necessary for irreversible inhibition.
these cells were treated with each of the fluorescent inhibitor
conjugates in the presences of 0.2% NaN3 to block energy-depen-
dent PSMA internalization.19,32 Confocal microscopy revealed that
the surfaces of LNCaP cells treated with FAM-X-CTT-54 and FAM-
PEG8-CTT-54 were considerably more fluorescent than cells trea-
ted with FAM-CTT-54 (Fig. 3). This data suggested quenching of
the fluorophore when bound deeper into PSMA due to the absence
of a spacer to link the fluorophore and the inhibitor core.
An anti-fluorescein antibody-coupled to AlexaFluor 594 (red)
was used to probe the surface accessibility of the fluorophore on
the fluorescent inhibitor conjugates when bound to PSMA on
LNCaP cells (Fig. 4). Red fluorescence was only observed on the sur-
face of LNCaP cells treated first with FAM-PEG8-CTT-54. This data
suggested that unlike the shorter spacers, a spacer length such as
PEG8 would allow the fluorophore to be sufficiently remote from
the PSMA surface and accessible to its antibody binding. This data
were consistent with the finding above indicating that with the
shorter linker, the fluorophore was likely buried in the PSMA bind-
ing cavity resulting in fluorescence quenching.
To determine whether the spacer length would affect the
in vitro imaging of PSMA-positive prostate cancer cells (LNCaP),
In order to gain further insight on the mode of binding, fluores-
cence quenching, and decrease of the antigenicity for some of the
fluorescent inhibitor conjugates, molecular docking was performed
for CTT-54, FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG8-CTT-54 in
a crystal structure of the extracellular domain of PSMA (PDB:
3BI1).33 Due to limitations of the software, the molecular docking
of FAM-PEG8-CTT-54 failed. The surface images for the docking of
PSMA-CTT-54, PSMA-FAM-CTT-54 and FAM-X-CTT-54 (Fig. S4A–C)
revealed that CTT-54 was buried in the active site of PSMA and
the fluorescein moieties of FAM-CTT-54 and FAM-X-CTT-54 were
localized in a cleft in the spillway toward the active site of the
enzyme. This latter observation suggests that the fluorescein group
would be inaccessible to the anti-fluorescein antibody, which is
consistent with the decreased antigenicity observed in the
anti-fluorescein antibody labeling experiments above. In Figure
S4D–E, it was noted that in contrast to FAM-X-CTT-54, the fluores-
cein moiety of FAM-CTT-54 was in close proximity to the indole
Figure 2. The enzymatic activity recovery profiles for PSMA inhibited by FAM-CTT-
54, FAM-XCTT-54, and FAM-PEG8-CTT-54 and CTT-54. On the basis of recovery
profiles, CTT-54 and FAMPEG8-CTT-54 are irreversible; FAM-CTT-54 is completely
reversible and FAM-X-CTT-54 is slowly reversible. Uninhibited PSMA served as a
control.
group of PSMA0s Typ541, indicating a possible
p-interaction and
supporting the putative fluorescence quenching observed above
for FAM-CTT-54 bound in PSMA on LNCaP cells.

T. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7013–7016
7015
Figure 3. Direct fluorescent labeling of PSMA-positive cells with fluorescent inhibitors. Live LNCaP cells were labeled with 5
lM each of fluorescent inhibitors (green) for
30 min at 37 °C: (A) FAM-CTT-54, (B) FAM-X-CTT-54, and (C) FAM-PEG8-CTT-54. All cells were fixed and nuclei stained with Hoechst 33342 (blue). Distance scale is 20 lm.
Figure 4. Indirect immunofluorescence imaging of PSMA-positive cells with an anti-fluorescein antibody. LNCaP cells were incubated with (A) 5
FAM-X-CTT-54, (C) 5 M FAM-PEG8-CTT-54, and (D) no inhibitor all followed by incubation with anti-fluorescein antibody-coupled to AlexaFluor 594 (red). All cells were
fixed and nuclei stained with Hoechst 33342 (blue). Distance scale is 20 m.
lM FAM-CTT-54, (B) 5 lM
l
l
FAM-CTT-54, FAM-X-CTT-54 and FAM-PEG8-CTT-54 were con-
firmed to be potent inhibitors of PSMA with no loss of inhibitory
potency due to conjugation of the parent core scaffold, CTT-54. This
trend has been observed in our previous work19 and is consistent
with what we observed previously for other dye conjugates of
PSMA inhibitors.18,24 All these studies suggest that an additional
interaction between the conjugated fluorescent dye and PSMA
may contribute to the enhanced potency of these fluorescent
inhibitors.
Based on the relatively low IC50 value of FAM-CTT-54 (0.41 nM),
the poor performance in the in vitro cell imaging studies of FAM-
CTT-54 compared to either FAM-X-CTT-54 or FAM-PEG8-CTT-54
was unexpected. Previously reported studies on fluorescein-
quenching were mainly focused on biotin–streptavidin or anti-
fluorescein antibody-fluorescein binding pairs. When all four
biotin-binding sites of streptavidin are bound with biotin-4-fluores-
cein (B4F), approximately 88% of the B4F fluorescence is quenched.35
Both free and conjugated fluorescein has also been found to be
quenched by an anti-fluorescein antibody.36 The crystal structure
of the fluorescein-bound antibody 4-4-20 demonstrated that the
aromatic residues Tyr37 (light chain) and Trp33 (heavy chain) were
key to the observed fluorescence quenching.37 In contrast to FAM-X-
CTT-54, the molecular docking of FAM-CTT-54 in PSMA revealed
that the fluorescein group of FAM-CTT-54 could be positioned near
In addition to the S1 and S10 substrate-binding sites of PSMA
that have been shown to accommodate P1 and P10 residues of
various substrates or inhibitors,7–9 recent structural data support
the presence of an arene binding site remote from PSMA0s active
site.34 The S10 site specifically defines the critical structural fea-
tures (C-terminal glutamate or analogs) of substrates or inhibitors
required for the recognition and binding to PSMA.33 On the other
hand, S1 site is flexible and can accommodate both polar and
nonpolar molecules. The arrangement of both the S1 and recently
identified remote arene binding sites in addition to the ‘open’ or
‘closed’ conformation of the entrance lid (Trp541-Gly548) appear
to be dependent on a ligand’s structure.33 Molecular docking
results Fig. S4A–C) suggest that the inhibitor core CTT-54 should
allow for a ‘closed’ conformation of the entrance lid. In contrast,
docking FAM-CTT-54 or FAM-X-CTT-54 would be more conducive
to an ‘open’ conformation. The correlation of the entrance lid
conformation and the mode of binding is consistent with the
observation that CTT-54 exhibits irreversible inhibition of PSMA
while FAM-CTT-54 completely reversible inhibitor or FAM-X-
CTT-54 slowly reversible inhibitor (Fig. 2). Based on this trend, it
was expected that FAM-PEG8-CTT-54 (irreversible PSMA inhibitor)
should allow for a ‘closed’ entrance lid conformation by allowing
the fluorescein group to be positioned closer to the protein surface
and beyond the landscape of the entrance lid.
the indole group of Trp541 for a possible p-interaction (Fig. S4DE).
Therefore, we hypothesize that Trp541 is an important contributor
to the fluorescein-quenching of FAM-CTT-54 in the in vitrocellimag-
ing studies (Fig. 3).
Although S1 and arene binding sites in PSMA exhibit consider-
able flexibility in accommodating aromatic and polyaromatic
groups, we have concluded that there may be size limitations. For
example, in our previous work20,26 we observed that a pyropheo-
phorbide-a conjugated PSMA inhibitor (IC50 = 102 nM) exhibited
considerably weaker affinity for PSMA compared to its inhibitor
core CTT-54 (IC50 = 14 nM). It is expected that the installation of
as spacer of sufficient length could allow a diagnostic or therapeutic
payload conjugated to a PSMA inhibitor to bypass the arene binding
site, project beyond the protein surface, and enable a ‘closed’
conformation of the entrance lid. These are particularly important
considerations in developing drug conjugates to PSMA inhibitors
while preserving both affinity and mode of binding to PSMA. A
structural study of PSMA has revealed that an approximately 20 Å

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T. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7013–7016
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deep funnel-shaped cavity leads from the surface of the enzyme to
its active site.9 In the present study, fluorescein-based immunoflu-
orescence imaging provided the experimental proof that a PEG8
spacer was sufficiently long enough to escape the PSMA protein
surface and render the fluorescein accessible to its antibody bind-
ing. In addition, we have found that unlike biotin-PEG12-CTT-54,
biotin-PEG4-CTT-54 labeled cells cannot be detected by extravi-
din-conjugated fluorescein (unpublished data). Furthermore,
crystal structure studies have revealed that PEG linkers are highly
dynamic and absent of specific interactions with PSMA.34 Therefore,
the current study together with these previous findings suggest
that a PEG8–12 linker between an irreversible PSMA inhibitor core
and a therapeutic payload should be sufficiently long enough to
retain both the binding affinity of inhibitor core and mode of bind-
ing in subsequent tumor-targeted therapeutic applications.
In conclusion, the present study revealed that fluorescent PSMA
inhibitor conjugates exhibited spacer length-dependent effects on
inhibitory potency, mode of inhibition, and fluorescence-quenching.
Therefore, the results presented herein suggest that length-optimal
spacers will be critical in the development of novel drug conjugates
of high-affinity, small-molecule inhibitors for both PSMA-based
targeted prostate tumor and tumor neovasulature chemotherapy.
22. Hillier, S. M.; Maresca, K. P.; Femia, F. J.; Marquis, J. C.; Foss, C. A.; Nguyen, N.;
Zimmerman, C. N.; Barrett, J. A.; Eckelman, W. C.; Pomper, M. G.; Joyal, J. L.;
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Acknowledgments
23. Kularatne, S. A.; Wang, K.; Santhapuram, H. K.; Low, P. S. Mol. Pharmacol. 2009,
6, 780.
24. Liu, T.; Jabbes, M.; Nedrow-Byers, J. R.; Wu, L. Y.; Bryan, J. N.; Berkman, C. E. Int.
J. Oncol. 2011.
25. Liu, T.; Wu, L. Y.; Choi, J. K.; Berkman, C. E. Int. J. Oncol. 2010, 36, 777.
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28. Wang, M.; Thanou, M. Pharmacol Res. 2010, 62, 90.
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The authors extend their gratitude for technical assistance to
G. Helms and W. Hiscox at the WSU Center for NMR Spectroscopy,
as well as C. Davitt and V. Lynch-Holm at the WSU Franceschi
Microscopy and Imaging Center. This work was supported in part
by the Washington State Life Sciences Discovery Fund (LSDF 08-01
2374880) and the National Institutes of Health (5R01CA140617-02).
Supplementary data
30. Lee, J.; Choi, Y.; Kim, K.; Hong, S.; Park, H. Y.; Lee, T.; Cheon, G. J.; Song, R.
Bioconjugate Chem. 2010, 21, 940.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.115.
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J. Oncol. 2011, 38, 1349.
32. Liu, H.; Rajasekaran, A. K.; Moy, P.; Xia, Y.; Kim, S.; Navarro, V.; Rahmati, R.;
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