Structure-Activity Relationship of 18F-Labeled Phosphoramidate Peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate Cancer

Article abstract of DOI:10.1021/acs.jmedchem.5b01850

A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC₅₀ values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [¹⁸F]4, [¹⁸F]5, and [¹⁸F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [¹⁸F]5 and [¹⁸F]6 as favorable candidates for future prostate cancer imaging clinical trials.

Full text of DOI:10.1021/acs.jmedchem.5b01850

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Article
Structure-Activity Relationship of 18F-labeled phosphoramidate
peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-
targeted inhibitor analogues for PET imaging of prostate cancer
Shorouk Dannoon, Tanushree Ganguly, Hendry Cahaya, Jonathan J Geruntho, Matthew S Galliher,
Sophia K Beyer, Cindy Jan Choy, Mark R Hopkins, Melanie Regan, Joseph E Blecha, Lubica Skultetyova,
Christopher R Drake, Salma Jivan, Cyril Barinka, Ella F Jones, Clifford E. Berkman, and Henry F. VanBrocklin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01850 • Publication Date (Web): 26 May 2016
Downloaded from http://pubs.acs.org on May 31, 2016
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Structure-Activity
Relationship
of
18F-labeled
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phosphoramidate peptidomimetic Prostate-Specific
Membrane Antigen (PSMA)-targeted inhibitor analogues
for PET imaging of prostate cancer
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1#
Shorouk Dannoon , Tanushree Ganguly 2#, Hendry Cahaya1, Jonathan J. Geruntho2,
Matthew S. Galliher2, Sophia K. Beyer2, Cindy J. Choy2, Mark R. Hopkins2, Melanie
Regan1, Joseph E. Blecha1, Lubica Skultetyova3, Christopher R. Drake1, Salma Jivan1,
Cyril Barinka3, Ella F. Jones1§, Clifford E. Berkman2,4§*, Henry F. VanBrocklin1§*
1Department of Radiology and Biomedical Imaging, University of California San
Francisco, USA; 2Department of Chemistry, Washington State University, USA;
3Institute of Biotechnology, CR, Prague; 4Cancer Targeted Technology, USA
#
Contributed equally
§ Contributed equally as senior authors
*Correspondence to:
Prof. Henry F. VanBrocklin
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Department of Radiology and Biomedical Imaging
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University of California San Francisco
185 Berry Street, Suite 350
San Francisco, CA 94107
tel: (415) 353ꢀ4569
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fax: (415) 514ꢀ8242
henry.vanbrocklin@ucsf.edu
* Prof. Clifford E. Berkman
Department of Chemistry
Washington State University
Pullman, WA 99164–4630.
tel: (509) 335ꢀ7613
fax: (509) 335ꢀ8389.
cberkman@wsu.edu
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ABSTRACT
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A series of phosphoramidateꢀbased prostate specific membrane antigen (PSMA)
inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorineꢀ18
analogs were evaluated for use as positron emission tomography (PET) imaging agents
for prostate cancer. To gain insight into their modes of binding, they were also coꢀ
crystallized with the extracellular domain of PSMA. All analogs exhibited irreversible
binding to PSMA with IC50’s ranging from 0.4ꢀ1.3 nM. In vitro assays showed binding
and rapid internalization (80ꢀ95%, 2h) of the radiolabeled ligands in PSMA(+) cells. In
vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with
tumor to blood ratios of 25.6:1, 63.6:1 and 69.6:1 for [18F]4, [18F]5 and [18F]6,
respectively, at 2h post injection . Installation of aminohexanoic acid (AH) linkers in the
phosphoramidate scaffold improved their PSMA binding and inhibition, and was critical
for achieving suitable in vivo imaging properties, positioning [18F]5 and [18F]6 as
favorable candidates for future prostate cancer imaging clinical trials.
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INTRODUCTION
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Prostate Specific Membrane Antigen (PSMA) is an ideal cell surface biomarker and
has been extensively pursued as a target in the development of imaging and therapeutic
agents for prostate cancer (PCa).1ꢀ9 In the past several years, radiolabeled PSMAꢀ
targeted small molecules have successfully detected prostate tumor xenografts in mouse
models and some have advanced to clinical studies.10 These small molecular scaffolds
exhibit high affinity and specificity for PSMA, some of which rapidly internalize into the
PSMA(+) tumor cells, overcoming the in vivo pharmacokinetic drawbacks presented by
antibodies.1, 11 Several ureaꢀbased inhibitors, pioneered by Kozikowski and Pomper have
been labeled with various isotopes such as 111In, 99mTc, 123I for SPECT12, 13 and 68Ga, 18F,
124I for PET11, 14, 15 imaging. Some of these ureaꢀbased inhibitors (e.g. [18F]DCFBC and
[18F]DCFPyL) have been translated to human imaging in clinical trials.16, 17 While the
pharmacokinetic profile and imaging with these ureaꢀbased agents appears superior to
antibodyꢀbased diagnostics, the reversible mode of binding to PSMA may explain the
observed tumor washout over several hours.18
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In parallel, we developed a class of phosphoramidate compounds that exhibit
irreversible or slowꢀreversible binding to PSMA, depending upon the structure, that have
shown similar promise for detection of PSMA(+) cells and tumors in mouse models.19ꢀ22
Based on their mode of binding to PSMA, we envisioned that this would result in reduced
tumor washout. In PSMA(+) cells, we found that the mode of binding exhibited a
significant effect upon cell uptake and internalization.23 Furthermore, when conjugated
to either 18Fꢀlabeled pendant groups22 or chelators bearing 99mTc21, these compounds
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displayed consistent uptake and retention in PSMA(+) tumors in mice with minimal
washout over several hours.
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More recently, we reported a unique phosphoramidateꢀbased PSMA inhibitor as a
promising candidate for PET imaging of PCa (Figure 1).24 The 18Fꢀlabeled analogue
[18F]5 displayed rapid uptake (2.35% ID/g at 1 h) and retention (2.35% ID/g at 4 h) in
PSMA(+) CW22Rv1 tumor xenografts in mice with an exceptional tumorꢀtoꢀblood ratio
of 265:1 at 4 h postꢀinjection. An important feature of 5, revealed by Xꢀray
crystallography, was the πꢀstacking and πꢀcationic interaction of its fluorobenzamido
(FB) ring with the Arg511/Try541 and Arg 463 residues, collectively known as the arene
binding site (ABS) located at the entrance PSMA’s active site. This additional unique
interaction was thought to be responsible for enhanced PSMA affinity and favorable in
vivo characteristics of this radiotracer compared to our previous reported analogues.
In the current study, we modified the phosphoramidate scaffold of 5, by either
omitting (4), or installing an extra (6) aminohexanoate (AH) linker, to further understand
the structure activity relationship (SAR) of phosphoramidates with respect to the
interactions with the PSMA arene binding site and their corresponding in vivo
pharmacokinetics and biodistribution (Figure 1). Herein, we report the synthesis,
radiolabeling, and in vivo performance of phosphoramidates [18F]4 and [18F]6 in
comparison to previously reported [18F]5. The in vitro cell uptake and internalization in
PSMA(+) CWR22Rv1and PSMA(–) PC3 cells was determined at 1 and 2 h postꢀ
incubation. Additionally, in vivo bioꢀdistribution data at 1 and 2 h time points as well as
microPET/CT imaging at 2 h postꢀinjection were obtained in mice implanted with
PSMA(+) CWR22Rv1 and PSMA(–) PC3 tumors.
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RESULTS
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Synthesis of cold standards and radiolabeling precursors. The
phosphoramidateꢀbased PSMA inhibitors and synthetic intermediates were prepared
using the same general methodology as previously reported.24 Compound 1 was prepared
in a final step by global deprotection of benzyl esters in precursor 7 (Scheme 1).
Radiolabling precursor 1 represents a common structural core of the phosphoramidate
analogues examined in the present study. The Bocꢀprotected precursor 8 was N-
deprotected and coupled to CbzꢀAHꢀOH or CbzꢀAH2ꢀOH to generate 9 and 10,
respectively. Subsequent global deprotection of 9 and 10, respectively yielded 2 and 3
(Scheme 2).
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All the fluorobenzamide (FB and [18F]) derivatives were synthesized in
quantitative yields via coupling of the phosphoramidate radiolabeling precursors 1, 2 and
3 with succinamidyl 4ꢀfluorobenzoate (SFB) and [18F]SFB to generate 4 and [18F]4
(Scheme 1) 5 and[18F]5, 6 and [18F]6 (Scheme 2), respectively. The nonꢀradioactive
analogues served as cold standards to determine IC50 values, mode of inhibition and as
reference compounds to optimize the HPLC conditions for purification of their [18F]ꢀ
labeled analogues.
IC50 and mode of inhibition. IC50 values and mode of inhibition (reversible,
slowly reversible, or irreversible) were determined as described previously.25ꢀ28 As is
common for smallꢀmolecule inhibitors of PSMA,19, 22 derivatization of the N-terminal
amine of the parent compounds 1 (IC50 = 27 nM), 2 (IC50 = 19 nM), and 3 (IC50 = 7.8
nM) with SFB led to enhanced inhibitory potency against PMSA (4, IC50 = 1.3 nM; 5,
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IC50 = 0.4 nM; 6, IC50 = 0.9 nM) (Figure 1). As observed previously for a number of
phosphoramidateꢀbased PSMA inhibitors, compounds 4, 5 and 6 exhibited an irreversible
mode of binding to PSMA.
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Co-crystallization of phosphoramidate-based PSMA inhibitors with
Glutamate carboxypeptidase II and structural determination. The extracellular
domain of PSMA, also known as human glutamate carboxypeptidase II (hGCPII; amino
acids 44 ꢀ750), was expressed in an S2 cell and purified using described protocols.29 To
evaluate the effect of the AH chain length on PSMA (or hGCPII) binding, the
phosphoramidate PSMA inhibitors 4 and 6 (the crystal structure of hGCPII/5 was
recently reported24) were coꢀcrystallized with hGCPII and the crystal structures of
complexes hGCPII/4 and hGCPII/6 were determined at a resolution of 1.77 Å and 1.71
Å, respectively. The structural characteristics of hGCPII/4 (PDB code: 4LQG, reported
as hGCPII/CTT1056) and hGCPII/6 were compared to the previously reported
hGCPII/524 (PDB code: 4JYW, reported as hGCPII/CTT1057).
Structures were determined using difference Fourier methods, and the final
models had wellꢀdefined crystallographic parameters (Sectionꢀ3, Table ST1). The overall
fold of all three structures is nearly identical with a maximum root mean square deviation
of 0.33 Å for the 681 equivalent Cα pairs between hGCPII/5 and hGCPII/6 complex.
The most pronounced difference in the loop arrangement is observed for the amino acids
stretch spanning Ser501 – Pro510.
Inhibitors were fitted into the Fo-Fc positive density peaks in the final stages of
the refinement. In the case of 4, there is the strong Fo – Fc electron density for the
inhibitor parts including the Cꢀterminal glutamate, phosphoramidate group, and the linker
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up to the P1 carboxylate, and weaker, yet interpretable density, for the remaining part of
the molecule. As for 6, we built the model with the full confidence encompassing the Cꢀ
terminal glutamate, phosphoramidate group, and the linker up to the P1 carboxylate and
the adjacent peptide bond. However, the distal part of the inhibitor including the P2
residue, the lipophilic linker, and the fluorobenzoyl group, is not seen in the Fo – Fc
electron density; they are flexible and do not contribute to the interactions with GCPII
(Figure 2A). In contrast to poorly defined distal parts of 4 and 6, positioning of 5 within
the hGCPII binding pocket can be unambiguously assigned for whole inhibitor (Figure
2B).
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The core structural elements found to be common in all three inhibitors are the C-
terminal glutamate, the phosphoramidate group, the P1 hydroxypropylglycine residue,
and the P2 glutamate linker. There is an overlapping of the C-terminal glutamate and the
phosphoramidate moieties between the three inhibitors. The C-terminal glutamate
occupies the S1´pocket of the enzyme in the same mode as previously reported. This
includes direct interactions between αꢀ and γꢀcarboxylates of inhibitors and the side
chains Arg210, Asn257, Tyr552, Lys699, and Tyr700.30, 31 Free oxygen atoms from the
phosphoramidate group chelate Zn2+ ions as well as interact with activeꢀsite residues
including sideꢀchains of Tyr552, His553, Asp387, His377, Asp453, and Glu425.
Additionally, the phosphoramidate amidoꢀgroup is involved in the hydrogen bonding
with the Gly518 mainꢀchain carbonyl and the Glu424 ꢀcarboxylate, whereas the oxygen
atoms from the PꢀO bond and P1 hydroxypropylglycine form waterꢀmediated contacts
with the side chains of Asp453 and Arg536.
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It is interesting to note that there are slight differences in the positioning between
the AH linker and P1 carboxylate in 5 compared to that in 4 and 6. While the P1
carboxylate of 4 and 6 interact directly with both Arg534 and Arg536, this motif in 5 is
shifted by approximately 1.1 Å (for the carbon atom of the P1 carboxylate), engaging
NH1 of Arg536 only (3.1 Å).
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The most important and prominent differences in the positioning of the inhibitor
distalꢀcomponents are found in the lipophilic aminohexanoic linker and the fluorobenzoyl
ring. For 4 (Figure 2C), the distance between the linker to the distal ring is
approximately 13 Å. The distal fluorobenzoyl ring of 4 is positioned parallel to the
guanidinium group of the Arg463 at a distance of approximately 4.0 Å with weak πꢀ
cation interactions in the arene binding site. The terminal fluorobenzoyl functionality of 5
is wedged into the areneꢀbinding cleft located at the “entrance lid” of the enzyme that is
shaped by the side chains of Trp541 and Arg511 on sides and by the Arg463 sideꢀchain at
the bottom. The plane of 5’s fluorobenzoyl ring is virtually parallel to both the indole and
guanidinium groups of Trp541 and Arg511, respectively, and both these residues
contribute to inhibitor binding (Figure 2D). In contrast to 4 and 5, the distal part of 6,
possessing the longest linker (approximately 28 Å), is not observed in the electron
density.
In vitro uptake and internalization study. Compounds [18F]4, [18F]5 and [18F]6
demonstrated specificity for PSMA showing uptake in PSMA(+) CWR22Rv1 cells but
not in PSMA(–) PC3 cells. As early as 1 h postꢀincubation, [18F]6 exhibited statistically
significant higher uptake compared to that of [18F]4 and [18F]5 with P values of <0.0001
as determined by student tꢀtest; [18F]5 uptake was also statistically higher than [18F]4
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with P value of 0.0012 (Table 1). The same trend was observed at 2 h. The activity
measured within CWR22Rv1 cells, representing internalization for [18F]4, [18F]5 and
[18F]6, at 1 h was 80.7%, 81.4% and 84.9% respectively, which increased to 94.2%,
84.2% and 91.3% respectively, at 2h postꢀincubation (Table 1).
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In vivo imaging and biodistribution studies. As seen in the microPET/CT
images in Figure 3, uptake of the tracers [18F]4, [18F]5 and [18F]6 was clearly observed in
the PSMA(+) CWR22Rv1 tumors at 2 h postꢀinjection but not in the PSMA(–) PC3
tumors. While all compounds showed uptake in the kidneys and clearance through the
bladder, minimal uptake was seen in all other organs, including bone (Figure 3).
Biodistribution data are provided for all three compounds in Table 2. Uptake of
[18F]4, [18F]5 and [18F]6 at 1 h postꢀinjection in the PSMA(+) CWR22Rv1 tumor were
1.54 ± 0.40, 3.16 ± 0.39, and 2.92 ± 0.30 %ID/g with a tumorꢀtoꢀblood ratio of 10, 20,
and 24 respectively. At 2 h postꢀinjection, the tumor accumulations were 1.57 ± 0.50,
1.65 ± 0.32 and 1.86 ± 0.14 %ID/g with rapid clearance from blood, providing a tumorꢀ
toꢀblood ratio of 26, 64 and 70, respectively. Kidneys showed the expected high uptake
and retention of all compounds. At 1 h postꢀinjection, kidney uptake of [18F]4, [18F]5 and
[18F]6 was 8.94 ± 2.93, 24.38 ± 3.72 and 5.87 ± 0.67 %ID/g respectively, and at 2 h was
9.97 ± 2.81, 21.54 ± 6.12 and 7.13 ± 1.45 %ID/g respectively. For the PSMA(–) PC3
xenografts, tumor uptake was similar to that in the background and nonꢀtarget organs
while the uptake in kidneys for [18F]4, [18F]5 and [18F]6 at 2 h post injection was 5.64 ±
2.41, 18.98 ± 4.75 and 4.44 ± 1.03 %ID/g, respectively.
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DISCUSSION
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A series of FB ringꢀcontaining compounds was designed with or without a linker to
separate it from the common phosphoramidate core 1. Compound 4, in which the FB
group was directly attached to the inhibitor 1 via its terminal amine, was expected to
behave like our first generation analogue II. The other new compound in the series was
compound 6 with the FB ring separated from the core inhibitor by two AH units.
Compound 5, where the inhibitor core was separated from the FB ring by one AH unit,
has previously shown desirable PSMA targeting properties.24 [18F]5 retained high affinity
for PSMA and showed consistent uptake and rapid internalization into CW22Rv1
(PSMA+) cells. The in vitro properties translated well to mouse models implanted with
CW22Rv1 tumors xenografts. High uptake and retention of [18F]5 over 4 h was observed
with exceptional bloodꢀtoꢀtumor ratios of 265:1 at 4 h postꢀinjection, suggesting rapid
clearance of the radiotracer from blood and all nonꢀtarget organs. Coꢀcrystallization of
PSMA with 5 revealed the binding of the fluorobenzoyl (FB) ring in the arene binding
site (ABS), initially identified by Zhang et al.32
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It was previously revealed that an optimal distance between the targeting core and
the FB group is necessary to achieve efficient binding of the FB ring with the ABS.
Based on the results obtained from modeling studies, and literature precedent, it was
anticipated that the FB ring of the [18F]4 would be too short to reach the ABS, the linker
distance of >20Å in [18F]6 was expected to allow for ABS binding as a contribution to a
bimodal binding interaction. In addition to the pharmacodynamic consequences of linker
length, we sought to asses the effect of varying the AH linker units on the in vivo
pharmacokinetics of the targeting agents.
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The synthesis of compounds 1, 2, 5 and [18F]5 were reported previously,22, 24 while
the synthesis of 4/[18F]4 and 6/[18F]6 are reported herein. Globally protected 3 was
generated by acid deprotection of the Bocꢀgroup of 8 followed by installation of Cbzꢀ
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(AH)2ꢀOH linker. Global deprotection afforded
3
in quantitative yield.
Fluorobenzoylation of the N-terminal amines of 1 and 3 yielded fluorobenzamide (FB)
compounds 4 and 6, respectively. As observed with 5 and other phosphoramidate
analogues, these compounds maintained high affinity and irreversible modes of binding
to PSMA upon derivatization of the N-terminal amine. Owing to the high affinity and the
irreversible nature of the common scaffold, it was expected that compounds [18F]4 and
[18F]6 would show uptake and retention in PSMA(+) tumors in vivo, while rapidly
clearing from all nonꢀtarget organs, as was observed in the case of [18F]5.24
To gather insight into the molecular interactions of these analogues with PSMA
and draw a comparison with the previous analogue 5, compounds 4 and 6 were also coꢀ
crystallized with the extracellular domain of PSMA. While the P1' carboxylate, the
phosphoramidate binding group and the P1 residue interact almost identically to PSMA,
the distinct differences were the aminohexanoic acid linker(s) and the fluorobenzoyl ring.
As anticipated, the linkage between the distal FB ring and the phosphoramidate core in 4
(approximately 13 Å) was insufficient to access the recently identified ABS.32 Rather,
the distal FB group was found parallel to the guanidinium group of the Arg463 at a
distance of approximately 4.0 Å (Figure 2C). Clearly, πꢀ‘ interactions play a prominent
role in the stabilization of this inhibitor’s positioning although the strength of this
interaction is likely much weaker compared to interactions in the ABS as observed for 5.
The terminal fluorobenzoyl moiety of 5 was found to be parallel to both indole and
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guanidinium groups of Trp541 and Arg511 in the ABS residues contributing to enhanced
inhibitor binding (Figure 2D). In the case of 6, possessing the longest linker
(approximately 28 Å), the portion of the inhibitor distal to the active site was not
observed in the electron density map suggesting its positional flexibility and the absence
of significant PSMAꢀinhibitor interactions (Figure 2A and 2B).
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The structural data are in a good agreement with the inhibitory properties toward
PSMA contributed from the terminal FB group and the AH linker on the tested
compounds. The coupling of the terminal FB group to the core structures 1, 2, and 3 led
to an improvement of the IC50 values for 4, 5, and 6 by approximately 20ꢀ, 50ꢀ and 9ꢀ
fold, respectively. A mechanistic explanation for the increase in inhibition potency may
be the result of additional binding interactions between the distal motifs of the inhibitors
and the protein in addition to entropic contributions. The most prominent effect (50ꢀfold
improvement) was observed for 5, for which the terminal FB group is fully engaged with
the ABS. Considerably less (20ꢀfold) enhancement of inhibition was observed for 4,
where there was less pronounced interactions between the SFB and the ABS. Interactions
with the ABS were absent for 6, which exhibited the least enhancement of inhibitory
potency compared to its inhibitor core 3. However, it should be noted that more complex
effects (including entropic factors and solvation effects) may also play a role in defining
the absolute values of inhibition constants for individual molecules. The simple bimodal
binding as in 5 or unimodal binding in 6 cannot be the sole determining factor for the
inhibition potencies.
The PSMA(+) LNCaP cell lines, have been used most widely in the PSMA
research by various research groups, including us.1, 21, 22 Although LNCaPs have a high
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PSMA concentration, their growth rate and proliferation as tumors in mice are
unpredictable, and often leads to tumor necrosis. In contrast, the CWR22Rv1 cells have
a moderate PSMA expression (8ꢀfold lower than LNCaPs), and a predictable growth
pattern as tumor xenografts.24, 33 In addition, the cellular PSMA concentration in the
CRW22Rv1 cells is more akin to the expression levels expected in human prostate cancer
metastases. Therefore, for the in vitro and in vivo studies in the present research,
CWR22Rv1 has been the PSMA(+) cell line of choice.
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The in vitro studies indicated uptake of all the radiotracers in PSMA(+)
CW22Rv1 cells, where an increase in uptake was observed as the lipophilic linker length
in these compounds increased from [18F]4 to [18F]6. While the increased uptake of [18F]5
can be attributed to its unique bimodal interactions with PSMA, no such binding
interaction was observed with 6. Therefore, the enhanced uptake in PSMA(+) cells in
vitro cannot be explained solely on the basis of PSMA affinity. This trend has also been
reported previously in the literature, where high uptake in PSMA(+) cells in vitro and
tumors in vivo, has often been correlated with increased lipophilicity.12 As expected, all
three compounds showed negligible uptake in PSMA(–) PC3 cell lines at all time points,
due to lack of PSMA expression in this cell line. In addition, all compounds showed rapid
internalization into PSMA(+) CW22Rv1 cells with more than 80% internalization at 1 h.
We have previously reported that the mode of binding of PSMA inhibitors has a direct
impact on the degree of internalization.28, 33, 34 The irreversible nature of the scaffolds
and findings from in vitro uptake and internalization suggest that the radiotracers will
likely lead to both uptake and retention in the PSMA(+) tumors in vivo over time,
allowing clearance from the nonꢀtarget organs.
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Tumor uptake was observed for [18F]4, [18F]5 and [18F]6 at 1 and 2 h postꢀ
injection in mice bearing PSMA(+) CWR22Rv1 tumor cells. At 1 h, the uptake of [18F]5
(3.16%) and [18F]6 (2.92%) was about twoꢀfold higher than [18F]4 (1.54%) (p < 0.05).
However, there was no significant difference in tumor uptake between the three
analogues at 2 h post injection. At 2 h postꢀinjection, all three tracers exhibited rapid
clearance from blood and nonꢀPSMA expressing organs, resulting in high tumorꢀtoꢀblood
ratios for [18F]5 and [18F]6, 64 and 70, respectively, that were significantly (p < 0.05)
greater than [18F]4. The irreversible mode of binding of these compounds, possibly
contributes to its tumor retention, while rapidly clearing from other nonꢀtarget organs, in
contrast to the washout that is reported over time for most ureaꢀbased agents 35. As
expected, kidney uptake was observed with all three analogues, due to the high
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expression of PSMA in mouse kidneys , with the highest uptake shown in the case of
[18F]5, which can be considered as a secondary positive indicator of PSMA specificity.
The kidney uptake of [18F]5 was significantly higher (p < 0.05) at both time points and in
both tumor bearing animal models. It is unknown why the kidney uptake of [18F]5 was 2ꢀ
2.5 fold higher as the magnitude of the difference does not correlate to a significant
chemical or in vitro characteristic. For compounds [18F]4 and [18F]6 there was no
significant difference in kidney uptake at either time point, nor was there any difference
for all three tracers between the kidney uptake in the PC3 versus the CWR22Rv1 animal
model at 2 hrs.
Given the structural similarities, it was expected that [18F]4 would exhibit similar
in vivo properties to our firstꢀgeneration PET agent. In our previous work, [18F]II was
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evaluated in PSMA(+) LNCaP tumor xenografts in mice with imaging and
biodistribution data collected at 2 h postꢀinjection. PSMA expression in LNCaP cells is
approximately 5ꢀ10 fold greater than that in CW22Rv1 cells.24 In comparison to the
current work, [18F]4 had a modest uptake at 1.57% with a tumorꢀtoꢀblood ratio of 64:1 in
the lower PSMA expressing CW22Rv1 tumors vs. 1.24% and tumorꢀtoꢀblood ratio of 9:1
for [18F]II in LNCaP tumors. Though this may not be a headꢀtoꢀhead comparison
because different tumor types were used, the in vivo uptake and clearance of [18F]4
suggests an overall trend toward higher affinity and more superior properties compared to
[18F]II.
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The most promising compounds of this study were [18F]5 and [18F]6, in which the
installation of lipophilic AH linker units between the phosphoramidate targeting core and
the FB ring improved the in vivo tumor uptake and clearance compared to that of [18F]4.
While the excellent in vivo properties of [18F]5 can be attributed to its unique interactions
with the ABS which resulted in higher PSMA affinity, the added lipophilicity from the
additional AH spacer in [18F]6 may have contributed to reduced renal clearance and
consequent tumor uptake. Although we observed no direct crystallographic evidence for
the FB group of [18F]6 interacting with the ABS, the high degree of rotational freedom
afforded by the two AH units may allow for the secondary binding at the ABS in solution.
The PET/CT images confirmed the observations of the biodistribution studies.
All three tracers demonstrated significant uptake in the PSMA(+) CWR22Rv1 tumors but
not in PSMA(–) PC3 tumors, confirming their specificity for PSMA. All these analogues
exhibited high tumorꢀtoꢀbackground ratios at 2 h postꢀinjection with rapid blood
clearance and minimal uptake and retention in nonꢀtarget organs. No evidence of
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metabolic defluorination and subsequent bone accumulation by any of the analogues was
observed.
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The tumor uptake and biodistribution patterns are in good agreement with the
other known Fꢀ18 PSMA urea agents, that have been translated into the clinical setting.
Although it is hard to make a direct headꢀtoꢀhead comparison between these compounds
due to the different models used between the different studies and the variable expression
levels of PSMA in the PSMA(+) cell lines used. However, the observed
pharmacokinetics are indicative of an overall trend of the uptake and clearance of these
agents. [18F]DCFBC, the first generation urea compound to be tested in human clinical
trials, showed an uptake of 4.7% in PSMA(+) PC3ꢀPIP cells and a tumor:blood ratio of
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13:1 . In comparison, compounds [18F]5 and [18F]6 show an uptake of 1.67 and 1.8%,
with tumor:blood ratios of 64:1 and 69:1 respectively, at the 2 h time point. It has been
shown that the concentration of PSMA in the PC3ꢀPIP cells is significantly higher than
37
that in CWR22Rv1 cells . Although the tumor uptake values are comparatively higher
in the second generation urea agent, [18F]DCFPyL (39.4% at 2h in PC3ꢀPIP cells), the
renal and liver uptake for this compound is also significantly higher than our
phorphoramidates.17 The renal and liver uptake values for [18F]DCFPyL at 2h are 15.7%
and 2.14% respectively, as compared to 7.13% and 0.25% for [18F]6.
As both these reported urea compounds have been able to successfully detect
primary and metastatic lesions in clinical trials,15, 18 the comparable trend in tumor uptake
and pharmacokinetics, and excellent clearance from nonꢀtarget organs, observed for
[18F]5 and [18F]6 are positive indicators of their success in future clinical trials. In fact,
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after successful completion of preclinical, dosage and toxicology studies, the firstꢀinꢀ
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human clinical trials for compound [18F]5 is currently underway.
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CONCLUSION
We have successfully synthesized and evaluated a bracketed series of PSMAꢀ
targeted phosphoramidate analogues, differing in the lipophilicity compared to compound
5, and evaluated them as PET imaging agents for prostate cancer. While capitalizing the
unique bimodal interaction with the “arene binding site” in PSMA can be a key element
for future designs of inhibitors with improved affinity, a balance between hydrophilic and
lipophilic properties in the core structure may also be critical for favorable in vivo
properties of the radiotracer. With their exceptional binding, tumor uptake and retention,
and remarkable tumorꢀtoꢀblood ratios, [18F]5 and [18F]6 are wellꢀpositioned as favorable
candidates for translation to future prostate cancer imaging studies in human.
EXPERIMENTAL SECTION
Cell Lines, Reagents and General Methods. CWR22Rv1 and PCꢀ3 cells were
obtained from the American Type Culture Collection (Manassas, VA). NCrꢀnu/nu mice
(strain code 088) were purchased from Charles River (Hollister, CA).
Zꢀ6ꢀ
Aminohexanoic acid (CbzꢀAHꢀOH) was purchased from Sigma Aldrich (St. Louis, MO).
All chemicals and cellꢀculture reagents were purchased from Fisher Scientific
(Sommerville, NJ) or SigmaꢀAldrich. All solvents used in chemical reactions were
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anhydrous and obtained as such from commercial sources or distilled prior to use. All
5
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other reagents were used as supplied unless otherwise stated.
Liquid flash
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chomatography (silica or C18) was carried out using a Flash Plus chromatography system
(Biotage, Charlotte, NC). Highꢀresolution mass spectrometry was performed using an
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1
ABS 4800 MALDI TOF/TOF Analyzer (Applied Biosystems, Framingham, MA). H
NMR chemical shifts were referenced to tetramethylsilane (
= 0.00 ppm), CDCl3 (δ =
7.26 ppm) or D2O (δ = 4.87 ppm). 13C NMR chemical shifts were referenced to CDCl3 (δ
31
=77.23 ppm). P NMR chemical shifts in CDCl3 or D2O were externally referenced to
85% H3PO4 (δ = 0.00 ppm) in CDCl3 or D2O. Aqueous buffered solutions for in vitro
experiments and HPLC chromatography were prepared with deionized distilled water
(MilliꢀQ water system, Millipore, Billerica, MA).
The HPLC analysis and purification system for radioactive compounds were
performed on a Waters model 600 Multisolvent System pump equipped with a Shimudzu
model SPDꢀ10A UV detector and an inꢀline radioactivity detector (model 105s, Carroll
and Ramsey Associates, Berkeley, CA) that was coupled to a data collection system
(PeakSimple model 304, SRI, Torrance, CA).
Purity of compounds 4, 5 and 6 was verified to be >95% via 31P (formation of
single product) and 1H NMR.
18
1. Syntheses of phosphoramidate compounds and their respective F analogues.
The synthetic sequence of compounds 4 and [18F]4 is shown in Scheme 1. Synthesis and
characterization of 7 and its precursors is provided as supporting information (Sectionꢀ1).
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The general synthetic scheme for compounds 2, 3, 5, 6, [18F]5 and [18F]6 is shown in
Scheme 2. Synthesis of compounds 2, 5, 18F-5 and 8 have been reported previously.24
1.1. Synthesis of 6-(6-(((benzyloxy)carbonyl)amino)hexanamido)hexanoic acid,
Cbz-AH2-OH.
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Synthesis of CBZ-AH-OH: 6ꢀamino caproic acid (8 g, 61 mmol, 1 eq) and sodium
carbonate (61 mmol, 1 eq) were dissolved in water (18 mL) in a round bottom flask
(100mL) and placed in an ice bath. Benzoyl chloroformate (67.1 mmol, 1.1 eq) was
added to the flask dropwise. Sodium hydroxide (2N, 15mL) was added in 5 mL portions
over a 15 min period. The reaction was monitored by TLC and stirred until completion.
The crude product was washed with diethyl ether (2 x 20mL) and the aqueous phase was
acidified to pH= 2 and placed in 5°C. The white precipitate was filtered and washed with
10% HCl .TLC (silica, 4:1 DCM:EtOAc, 1% AcOH) showed the presence of CBZꢀAHꢀ
OH (Rf= 0.27). CBZꢀAHꢀOH was isolated as a white solid by column chromatography
(silica, 4:1 DCM:EtOAc, 1% AcOH), yield 25.5%. M.P. = 57ꢀ59°C. 1H NMR (300 MHz,
CDCl3): δ 1.36 (m, 2H), 1.50 (m, 2H), 1.65 (m, 2H), 2.35 (t, 2H), 2.35 (t, 2H), 3.19 (m,
4H), 4.78 (s, 1H), 5.09 (s, 2H), 5.64 (s, 1H), 7.35 (m, 5H). 13C NMR (100 MHz, CDCl3):
δ 24.23, 26.08, 29.57, 33.82, 40.79, 66.64, 128.08, 128.48, 136.53, 156.44, 179.11. HR
mass spectroscopy: calculated 288.1212, found 288.12106 (M Na+) for C14H19NO4.
Synthesis of CBZ-AH2-OH: CBZꢀAHꢀOH (0.20g, 0.754 mmol, 1 eq) and HBTU (0.83
mmol, 1.0 eq) was dissolved in distilled DCM (10 mL) and allowed to stir for 1h under
Ar(g) in a flame dried flask (25 mL). A solution of NHS (0.83 mmol, 1.1 eq) and TEA
(0.75 mmol, 1 eq) was prepared in distilled DCM (4 mL) and added to the reaction
dropwise. This was allowed to stir overnight. The DCM was dried under vacuum. The
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crude product was extracted with ethyl acetate (25 mL) and washed with 10% HCl (3x 25
mL), 10% NaHCO3 (3x 25 mL), and brine (3x 25 mL) and dried with Na2SO4. The
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presence of CBZꢀAHꢀNHS was determined by H NMR (300 MHz, CDCl3): δ 1.43 (m,
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2H), 1.51 (m, 2H), 1.73 (m, 2H), 2.59 (t, 2H), 2.35 (t, 2H), 2.76 (s, 4H), 3.19 (m, 4H),
4.98 (s, 1H), 5.07 (s, 2H), 5.64 (s, 1H), 7.35 (m, 5H). CBZꢀAHꢀNHS was used as a crude
mixture without further purification.
6ꢀamino caproic acid (0.12g, 0.90 mmol, 1.2 eq) and sodium bicarbonate (1.35 mmol,
1.8 eq) were dissolved in water (8 mL). CBZꢀAHꢀNHS (0.752 mmol, 1 eq) was dissolved
in acetone (5.2 mL) and was added dropwise to the round bottom flask and stirred
overnight. The reaction mixture was acidified to pH 2 and the white precipitate was
filtered and washed with 10% HCl. The product was isolated as a white solid without any
further purification (68% yield, 0.61 mmol). TLC (Silica, 49:1 EtOAc:AcOH) showed
the presence of CBZꢀAH2ꢀOH (Rf= 0.17). M.P= 104ꢀ106°C .1H NMR (300 MHz,
CDCl3): δ 1.36 (m, 4H), 1.50 (m, 4H), 1.61 (m, 4H), 2.16 (t, 2H), 2.35 (t, 2H), 3.16ꢀ3.34
(m, 4H), 4.94 (s, 1H), 5.09 (s, 2H), 5.69 (s, 1H), 7.34 (m, 5H). 13C NMR (300 MHz,
CDCl3): δ 24.28, 25.28, 25.93, 26.06, 28.65, 29.15, 33.36, 35.57, 35.63, 38.73, 40.19,
65.85, 127.30, 127.48, 128.00, 137.05, 157.47, 174.60, 176.02. HR mass
spectroscopy:calculated 378.22, found 379.22906 (M H+) for C20H30N2O5.
1.2.
2-(((S)-((2R)-2-(4-amino-4-carboxybutanamido)-2-
carboxyethoxy)(hydroxy)phosphoryl) amino)pentanedioic acid, [1]. To a solution of a
benzyl ester protected phosphoramidate (7) (0.100 g, 0.095 mmol) in THF (1 ml), was
added 10% Pd/C (10mg), K2CO3 (0.033 mg, 0.241 mmol) and H2O (1 ml). The mixture
was stirred vigorously, purged with Ar(g) and then charged with H2(g) under balloon
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pressure overnight at room temperature. The solution was filtered through a 0.2 mm
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PTFE micropore filtration disk (Whatman). The solvent was removed in vacuo to yield a
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white solid, 1 in 96% yield. H NMR (300 MHz, D2O): δ 1.44ꢀ 1.54 (m, 3H), 1.62ꢀ 1.67
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(m, 3H), 1.86ꢀ 1.93 (m, 2H), 2.02ꢀ 2.05 (m, 2H), 2.21 (m, 1h), 2.25 (m, 1h), 3.31 (m, 1H),
3.47 (m, 1h), 3.56 (t, 2H), 3.92 (m, 1H). 31P NMR (300 MHz, D2O): δ 8.46. HR mass
spectroscopy:calculated 471.3, found 470.20 (MꢀH) for C15H26N3O12P+.
1.3.
2-(((S)-((2R)-2-carboxy-2-(4-carboxy-4-(4-
phosphoryl)amino)pentanedioic
fluorobenzamido)butanamido)ethoxy)(hydroxy)
acid, [4]. A solution of SFB (20.14 ꢁmol, 1 eq) in 400 ꢁL THF was added to a stirred
solution of 1 (30.22 ꢁmol, 1.5 eq) in 600 ꢁL of 0.1M KHCO3. The reaction mixture was
stirred for 6 h in the dark at room temperature. The unreacted 1 was scavenged by stirring
with 25 mg Siꢀisocyanate resin (SiliCycle, Inc., Quebec, Canada) overnight at room
temperature. The solution was subsequently centrifuged (7800 rcf, 10 min) and the
supernatant was lyophilized in a 2 mL microꢀcentrifuge tube. The unreacted materials
and/or hydrolyzed SFB were removed by successively triturating the lyophilized solid
with 1 mL portions of DMSO and centrifuging the mixture (16,200 rcf, 1 min) after each
wash; this process was repeated 10 times. The resulting solid was dried in vacuo
1
providing the desired 4ꢀfluorobenzamidoꢀphosphoramidate 4 in quantitative yield. H
NMR (300 MHz, D2O): δ 1.44ꢀ 1.54 (m, 2H), 1.56ꢀ1.68 (m, 2H), 1.86ꢀ2.14 (m, 4H),
2.27ꢀ2.35 (m, 2H), 3.27ꢀ3.34 (m, 1H), 3.50 (t, 2H), 3.73ꢀ3.87 (m, 2H), 4.05ꢀ4.08 (t, 1H,
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3.9 Hz), 4.15ꢀ4.19 (dd, 1H, 4.4 Hz, 9.15 Hz), 7.04ꢀ7.10 (t, 2H), 7.66ꢀ7.70 (dd, 2H). P
NMR (300 MHz, D2O): δ 8.42. HR mass spectroscopy: calculated 720.03, found 719.96
(M+4Na+K) for C22H29FN3O13P+.
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1.4.
Synthesis
of
dibenzyl
2-(((R)-(benzyloxy)(((24R)-19,24-
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bis((benzyloxy)carbonyl)-3,10,17,22-tetraoxo-1-phenyl-2-oxa-4,11,18,23-
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tetraazaheptacosan-27-yl)oxy)phosphoryl)amino) pentanedioate, [10]. CbzꢀAH2ꢀacid
(AH = aminohexanoic acid) (0.1g, 0.264mmol) was preꢀactivated with HBTU
(0.29mmol, 1.1 eq) and TEA (0.29mmol, 1.1 eq). 8 was treated with a mixture of dry
TFA/DCM for deprotection of N-terminal Bocꢀ group and then added to the flask above
with activated CbzꢀAH2ꢀacid. Purification was carried out using reversed phase C18
chromatography with 80% MeOHꢀwater as the mobile phase. Pure 10 was isolated in
49% yield.1H NMR (300 MHz, CDCl3): δ 1.28ꢀ1.30 (m, 4H), 1.41ꢀ1.46 (m, 4H), 1.56ꢀ
1.61 (m, 6H), 1.86ꢀ1.89 (m, 2H), 2.09ꢀ2.27 (m, 10H), 2.37–2.39 (m, 2H), 3.12ꢀ3.18 (m,
4H), 3.74 (m, 1H), 3.89 (m, 2H), 4.51 (m, 2H), 4.91ꢀ4.96 (m, 2H), 5.05ꢀ5.11 (m, 10H) ,
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5.95 (d, 1H, ꢀNH), 6.98 (d, 1H, ꢀNH), 7.03 (d, 1H, ꢀNH), 7.27ꢀ7.31 (m, 27H). P NMR
(300 MHz, CDCl3): δ 8.47. ESI mass spectroscopy: calculated 1281.4, found 1282.4
(M+H), 1305.6 (M+Na) for C70H84N5O16P+.
1.5. Synthesis of 2-(((S)-((4R)-4-(4-(6-(6-aminohexanamido)hexanamido)-4-
carboxy butanami -do) -4-carboxybutoxy)(hydroxy)phosphoryl)amino)pentanedioic
acid, [3]. To a solution of benzyl ester protected phosphoramidate (10) (0.160 g, 0.124
mmol) in THF (1 ml), was added 10% Pd/C (16mg), K2CO3 (0.044 mg, 0.318 mmol) and
H2O (1 ml). The mixture was stirred vigorously, purged with Ar(g) and then charged with
H2(g) under balloon pressure overnight at room temperature. The solution was filtered
through a 0.2 mm PTFE micropore filtration disk (Whatman). The solvent was removed
1
in vacuo to yield a white solid, 3 in 94% yield. H NMR (300 MHz, D2O): δ 1.14ꢀ1.19
(m, 2H), 1.36 (m, 4H), 1.38ꢀ1.50 (m, 10H), 1.59ꢀ1.68 (m, 2H), 1.89 (m, 2H), 1.99ꢀ2.19
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(m, 8H), 2.86 (t, 2H), 3.34 (m, 1H), 3.56 (dd, 1H), 3.94 (m, 3H). P NMR (300 MHz,
D2O): δ 8.43. HR mass spectroscopy: calculated 698.30, found 698.35 (M+H) for
C27H49N5O14P+.
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1.6. Synthesis of 2-(((S)-(((22R)-17,22-dicarboxy-1-(4-fluorophenyl)-1,8,15,20-
tetraoxo-2,9,16,21
-tetraazapentacosan-25-
yl)oxy)(hydroxy)phosphoryl)amino)pentanedioic acid, [6]. A solution of 3 (0.028g,
0.003mmol, 1.5 eq) was made in 500 µL of 100 mmol KHCO3 and SFB (0.005g, 1 eq) in
400 µL THF was added and stirred for 5 h. The unreacted 3 was scavenged by stirring
with 5 mg Siꢀisocyanate resin (SiliCycle, Inc., Quebec, Canada) overnight at room
temperature. The solution was subsequently centrifuged (7800 rcf, 10 min) and the
supernatant was lyophilized in a 2 mL microꢀcentrifuge tube. The unreacted materials
and/or hydrolyzed SFB were removed by successively triturating the lyophilized solid
with 1 mL portions of DMSO and centrifuging the mixture (16,200 rcf, 1 min) after each
wash; this process was repeated 10 times. The resulting solid was dried in vacuo
1
providing the desired 4ꢀfluorobenzamidoꢀphosphoramidate 6 in quantitative yield. H
NMR (300 MHz, D2O): δ 1.09ꢀ1.19 (m, 2H), 1.16ꢀ1.24 (m, 6H), 1.30ꢀ1.35 (m, 2H), 1.41ꢀ
1.45 (m, 5H), 1.61ꢀ1.68 (m, 5H),1.99ꢀ2.07 (m, 6H), 2.14ꢀ2.21 (m, 2H), 2.91ꢀ2.95 (m,
2H), 3.16ꢀ3.21(m, 2H), 3.25ꢀ3.33 (m, 2H), 3.54ꢀ3.56 (m, 2H), 3.87ꢀ3.96 (m, 2H), 7.02ꢀ
7.08 (m, 2H) , 7.56ꢀ7.61 (m, 2H). 31P NMR (300 MHz, D2O): δ 8.43. HR mass
spectroscopy: calculated 820.38, found 820.43 (M+H) and 858.40 (M+K) for
C34H51N5FO15P+.
1.7. Synthesis of [18F]4, [18F]5 or [18F]6- Succinimidylꢀ[18F]fluorobenzoate
([18F]SFB) was synthesized in a Neptis® Synthesizer (ORA – Optimized Radiochemical
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Applications, Belgium)) equipped with commercially available kits and cassettes (ABX
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GmbH, Germany) that was then coupled to 1, 2 and 3 as was previously described with
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modifications 22. The PSMA analogues (2 mg) were dissolved in 100 L of H2O, 20
L
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of 0.1 M K2CO3 and 100 L of [18F]SFB in acetonitrile were added to a 1ꢀdram vial
charged with a stir bar (pH 9.5ꢀ10). The coupling reaction took place at 40 ºC for 15 min.
Prior to in vitro and in vivo studies, the radioconjugate was purified on a semiꢀpreparative
RPꢀHPLC using a Phenomenex C18(2) 100Å, 250x10 mm, 5 µm column. Linear
gradient (20% to 90% over 22 min) of solvent B in solvent A (A: 0.1% formic acid in
water; B: 0.1% formic acid in acetonitirile) at a flow rate of 5 mL/min and UV detection
at 254 nm. [18F]4, [18F]5 or [18F]6 peak was collected and concentrated via QMA light
SPE method using 0.5% NaCl for elution and diluted with 1X PBS for in vitro and in vivo
studies. Radiochemical yields ranged between 50ꢀ60% decayꢀcorrected from [18F]SFB.
Analytical RPꢀHPLC of the purified peak confirmed >95% purity of all three compounds
via coinjection with the respective nonꢀradioactive analogs.
2.1. General method of determining IC50 values. Inhibition studies were performed
as previously described with minor modifications.25, 34 Description is provided in
supporting information (Sectionꢀ2).
2.2. Mode of inhibition Study. The mode of inhibition studies followed the
procedure described in our previous work.19 A description is provided in supporting
information (Sectionꢀ2).
3.1. GCPII expression, purification, crystallization and data collection. The
extracellular part of human GCPII (hGCPII; amino acids 44 ꢀ750) was expressed in S2
cell and purified according to procedures described previously.29 The final protein
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preparation in 20 mM TrisꢀHCl, 150 mM NaCl, pH 8.0, was concentrated to 9 mg/mL
and stored at ꢀ80°C until further use.
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Complexes of hGCPII/4 and hGCPII/6 were prepared by mixing stock solutions of
hGCPII (9 mg/mL) and a given inhibitor (20 mM in water, pH adjusted to 8.0 by the
addition of NaOH) at the 9:1 ratio (v/v). Crystals were grown from 2 µL droplets made
by mixing equal volumes of GCPII/inhibitor and reservoir solutions (33% pentaerythritol
propoxylate, (Sigma), 1.5% polyethylene glycol 3350 (Sigma), and 100 mM Tris–HCl,
pH 8.0) using the hangingꢀdrop vapor diffusion setup at 293 K. Diffraction quality
crystals were flashꢀfrozen in liquid nitrogen directly from the crystallization droplet. The
diffraction data for both complexes were collected at 100 K using synchrotron radiation
at the MX 14.2 beamline (BESSYII, Helmholtz-Zentrum Berlin, Germany; 0.918 Å). The
complete dataset was collected from a single crystal and data were processed XDSAPP
package.38
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3.2. Structure determination and refinement. Structures were determined by the
difference Fourier methods using the structure of the hGCPII/NAAG complex (PDB
entry 3BXM;39) as the template model. Model building was accomplished using Coot40
and calculation steps were performed using Refmac 5.1.41 The restrains library and the
coordinate files for individual inhibitors were prepared using the PRODRG server42 and
the inhibitors/substrates were fitted into the positive electron density map in the final
stages of the refinement.
The stereochemical quality of final models was evaluated using MolProbity43 and
the final model, together with experimental amplitudes, was deposited in the RCSB
Protein Data Bank under the entry code 4LQG (hGCPII/4; deposited as
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hGCPII/CTT1056). Data collection and structure refinement statistics are provided in the
supporting information, Sectionꢀ3, table ST1.
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4. Cell lines and cell culture. CWR22Rv1 and PCꢀ3 cells were incubated in Tꢀ75
flasks with complete growth medium (RPMI 1640 containing 10% heatꢀinactivated fetal
calf serum (FBS), 100 units of penicillin and 100 ꢁg/mL streptomycin) in a humidified
incubator at 37 °C and 5% CO2.
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4.1. In vitro cell uptake studies. Confluent CWR22Rv1 and PCꢀ3 cells were
detached with 0.25% trypsinꢀ0.53 mM EDTA solution. The cells were washed three
times with 1% FBS 1XꢀRPMI 1640 phosphateꢀfree medium. Cells were subdivided into
microꢀcentrifuge tubes to contain approximately 500,000 cells each in 250 µL of 1% FBS
1XꢀRPMI 1640 phosphateꢀfree medium and 6.25 µl ethanol. A solution of [18F]4, [18F]5
or [18F]6 (2 µCi in 3 µL) was added to microꢀcentrifuge tubes containing one of the
following: CWR22Rv1 cells (n=5) in 1% FBS 1XꢀRPMI 1640 phosphateꢀfree medium;
PCꢀ3 cells (n=5) in 1% FBS 1XꢀRPMI 1640 phosphateꢀfree medium; 1 mL of 1% FBS
1XꢀRPMI 1640 phosphateꢀfree medium (n=3) which served as reference for the total
activity incubated with the cells. All samples above were incubated at 37 °C and 5% CO2
for 1 and 2 h. At each time point, the cell pellets were repeatedly washed with PBS (1
mL), centrifuged (2 min at 14700g) and the media was removed. The radioactivity of the
cell pellets were counted and compared to the total activity references.
4.2. Internalization studies. Internalization studies followed the method describe for
the in vitro cell uptake studies with a minor modification. Following the incubation
periods (1 and 2 h) the cells were washed as described in the uptake studies with PBS
cooled to 4 °C. The resulting cells were then treated with a solution of 50 mM glycine
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and 100 mM NaCl at pH 3 for 2 min at 37 °C and 5% CO2. Cells were then centrifuged
(2 min at 12000g) and the supernatants were collected. This treatment was repeated two
additional times and the combined supernatants were counted for external binding while
the cell pellet was counted for internalization.44, 45
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5. In vivo PET imaging studies. All animal experiments were conducted in
accordance with the UCSF IACUC approved protocol. Approximately 106 CWR22Rv1
PSMA(+) or PC3 PSMA(ꢀ) cells in 50:50 mixture of complete medium and matrigel
matrix were implanted in the right shoulder of athymic NCrꢀnu/nu male mice
(approximately 8 weeks old) from Charles River (Hollister, CA). Approximately four
weeks after implantation, animals with tumors reaching 150 to 300 mm3 were
anesthetized by isoflurane inhalation and were administered with [18F]4, [18F]5 or [18F]6
at 200ꢀ250 µCi in 250 µL of saline through tail vein injection. The resulting animals
were imaged with 10 min acquisition by a microPET/CT imaging system (Inveon,
Siemens, Germany) at 2 h postꢀinjection. PET imaging data were acquired in list mode
and reconstructed with the iterative OSEM 2ꢀD reconstruction algorithm provided by the
manufacturer.
6. Biodistribution studies. Four to five weeks after the implantation of CWR22Rv1
PSMA(+) cells or three to four weeks after the implantation of PC3 PSMA(ꢀ) cells, tumor
bearing mice were anesthetized by isoflurane inhalation and administered with 50 µCi of
[18F]4, [18F]5 or [18F]6 in 150 µL of saline through tail vein injection. These mice
(CWR22Rv1 at 1 h and 2 h postꢀinjection; PC3 at 2 h post injection; n = 4 for each time
point) were euthanized for biodistribution analysis. Blood was collected by cardiac
puncture. Major organs (heart, lung, liver, spleen, kidney, muscle, bone and tumor
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xenografts) were harvested, weighed, and counted in an automated gamma counter
(Wizard 2, PerkinElmer, Waltham, MA). The percent injected dose per gram (%ID/g) of
tissue was calculated by comparison with standards of known radioactivity. Statistical
analysis was performed using twoꢀtailed student tꢀtest (Microsoft Excel Prism software).
A p value of < 0.05 was considered statistically significant.
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