Increasing time on target: utilization of inhibitors of cysteine cathepsins to enhance the tumor retention of receptor-targeted agents

Article abstract of DOI:10.1039/c8cc05982a

We report a strategy of utilizing irreversible cysteine cathepsin inhibitor as trapping agent to increase the tumor residence time of receptor-targeted agents. The targeted constructs incorporating these cysteine cathepsin trapping agents were able to for

Full text of DOI:10.1039/c8cc05982a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: W. Fan, W. Zhang,
S. Alshehri and J. C. Garrison, Chem. Commun., 2018, DOI: 10.1039/C8CC05982A.
This is an Accepted Manuscript, which has been through the
Volume 52 Number
1 4 January 2016 Pages 1–216
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Accepted Manuscripts are published online shortly after
Chemical Communications
www.rsc.org/chemcomm
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
=
3
rsc.li/chemcomm

Please do not adjust margins
ChemComm
Page 1 of 5
View Article Online
DOI: 10.1039/C8CC05982A
Journal Name
COMMUNICATION
Increasing Time on Target: Utilization of Inhibitors of Cysteine
Cathepsins to Enhance the Tumor Retention of Receptor-Targeted
Agents
Received 00th January 20xx,
Accepted 00th January 20xx
Wei Fan, †ab, Wenting Zhang,†ab Sameer Alshehri, ab and Jered C. Garrison *abcd
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report a strategy of utilizing irreversible cysteine cathepsin however, upregulation of both the expression and activity of
inhibitor as trapping agent to increase the tumor residence time of CCs has been observed5 and has garnered interest in the
receptor-targeted agents. The targeted constructs incorporating development of reversible and irreversible inhibitors of these
these cysteine cathepsin trapping agents were able to form high proteases for diagnostic and therapeutic purposes.6
molecular weight adducts with intracellular cysteine cathepsins, Dipeptidyl acyloxymethyl ketones (AOMKs) are one example of
a class of irreversible inhibitors for CCs.7 These inhibitors have
high selectivity for the active site of CCs. They can also form
irreversible thioether linkages with the cysteine responsible for
the catalytic function of the protease.8a To date, a variety of
AOMK inhibitors has been reported for diagnostic and
therapeutic purposes related to cysteine cathepsins known role
in cancer.7a, 8
thus achieving superior retention in tumor tissues.
The development of receptor-targeted radiopharmaceuticals
that selectively bind to overexpressed receptor populations in
cancerous tissues has been, and continues to be, extensively
investigated.1 The targeting constructs for these agents can be
broadly divided into carriers of low-molecular weight (e.g.,
small molecules and peptides) and high-molecular weight (e.g.,
proteins and antibodies). Low-molecular weight carriers offer
several advantages relative to macromolecules, such as rapid
accumulation in the target and clearance from non-target sites.2
Unfortunately, compared to high-molecular weight carriers,
smaller molecules generally have inherently higher metabolism
and diffusion characteristics, leading to decreased tumor
residence times that often diminish translational potential,
particularly for therapeutic applications.
Herein, we propose a synergistic concept that utilizes CC
inhibitors, such as AOMKs, as novel and powerful CC-trapping
agents (CCTAs) to improve the retention of low-molecular
weight, receptor-targeted radiopharmaceuticals (Figure 1A).
Upon binding of the agonistic-targeting vector to its
corresponding cellularreceptor and intracellular trafficking to
the endolysosomal compartments, we hypothesized that
targeting vectors incorporating these CCTAs can irreversibly
bind to the highly expressed and active CCs within these
compartments. As a result, high-molecular weight, intracellular
CC-adducts, which would limit cellular efflux and diffusion of the
radiopharmaceutical were expected, thereby enhancing its
long-term retention in target tissues. It was anticipated that by
using this strategy, significant increases in the target/non-target
(T/NT) ratios could be achieved, thereby increasing the
likelihood of clinical translation. To examine the utility of this
concept, the neurotensin (NT) peptide / NTSR1 was utilized as
the model platform, a system for which our laboratory is well
versed.9
Cysteine cathepsins (CCs) are a family of 11 endolysosomal
proteases with
a variety of functions, but are primarily
attributed to protein catabolism.3 These proteases are highly
expressed (i.e., mM) in endolysosomal compartments, but are
also known to exist extracellularly. The extracellular activity of
CCs is generally very low and tightly regulated in normal tissue
through a number of biological mechanisms.4 In cancers,
a. Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
985830 Nebraska Medical Center, Omaha, NE 68198, United States.
b. Center for Drug Delivery and Nanomedicine, University of Nebraska Medical
Center, 985830 Nebraska Medical Center, Omaha, NE 68198, United States.
c. Department of Biochemistry and Molecular Biology, University of Nebraska
Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198, United
States
d. Eppley Cancer Center, University of Nebraska Medical Center, 985950 Nebraska
Medical Center, Omaha, NE 68198, United States.
† These authors contributed equally to this work.
NTSR1 is a receptor known to be overexpressed in a number of
cancers, including pancreatic, prostate, and colon2a, 10. In this
study, the synthesized NTSR1-targeted agents utilizes an NT
fragment (i.e., NT(6-13)) as the targeting vector. This peptide
has a low-molecular weight with nanomolar binding affinity to
the NTSR1. Figure
1 provides a schematic outlining the
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
synthesized NTSR1-targeted conjugates. Briefly, the AOMK
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 5
View Article Online
DOI: 10.1039/C8CC05982A
COMMUNICATION
Journal Name
Table 1 The Log D7.4, CatB inhibition constant, and competitive binding (IC50) to NTR1 of
the compounds
Compound
LogD7.4
CatB Ki (nM)
NTSR1 IC50 (nM)
1a
1b
2a
2b
2c
2d
2e
2f
2.08 ± 0.08a
2.51 ± 0.02a
-1.46 ± 0.02b
-1.49 ± 0.02b
-0.98 ± 0.02b
-1.79 ± 0.06b
-1.71 ± 0.05b
-1.95 ± 0.05b
-1.56 ± 0.07b
-2.01 ± 0.06b
23 ± 1
nic
о
о
25 ± 4
26 ± 5
69 ± 13
73 ± 8
72 ± 2
50 ± 13
ni
20 ± 2
22 ± 2
19 ± 1
20 ± 3
20 ± 3
18 ± 2
49 ± 5
52 ± 8
3a
3b
ni
a
b
The Log D7.4 was determined by HPLC analysis. The Log D7.4 was determined by
radiometric analysis. c No inhibition observed.
(Table 1). These results suggest minimal impact of the CCTA on
the conjugate affinity for the NTSR1 and vice versa (i.e., the
impact of the peptide on the CCTA efficacy).
Fig. 1 (A) Scheme outlining the structure of the CCTA-incorporated, NTSR1-targeted
analogs. (B) Table detailing the structural components of the synthesized analogs. 177Lu-
DOTA
–
lutetium-177-labeled-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
Efflux studies were performed to examine the HT-29 cellular
retention profile of the radioconjugates over a 24 h period, as
shown in Figure 2A. Increased retention was observed as the
CCTA linker (X) increased in length from null (2a) to acetyl (2b)
to PEG3 (2c), suggesting the length impacts the cellular activity.
Unexpectedly, the longest linker Gly-(D)Ser3 (2d), did not follow
this trend and had a cellular retention profile similar to 2a.
However, introduction of a (D)Ser3 in the peptide linker (Y)
resulted in a substantial increase in the cellular retention of the
analogous 2f. This data implies that introducing a PEG linker
between the CCTA and the peptide or inserting a three-D-Serine
linker in the peptide sequence could benefit the intracellular
binding of the conjugates.
acid; Cy5 – Cyanine 5; PEG3 - 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid.
inhibitor 1a was utilized as the model CCTA and the non-
reactive 1b (no inhibition) was used as a structurally analogous
control. The lutetium-177
(
177Lu)-labeled-conjugates 2a-2d
were used to access the impact of the CCTA linker (X) on the
activity of the radioconjugate. For radioconjugates 2e-2f,
modifications were made to the peptide (Y) and the CCTA linker
to examine how increases in hydrophilicity impacted biological
performance. Radioconjugates 3a-3b utilized the inactive CCTA
(1b) and served as matching controls to the experimental
analogs. Lastly,
a fluorescent dye (Cyanine 5, Cy5) was
conjugated to the CCTA-incorporated peptide to yield the
experimental compound 4a, with 4b serving as the matched
control. Synthetic schemes and details regarding the synthesis
of these compounds can be found in the supporting
information. The 177Lu radiolabeling efficiencies of the
conjugates were determined to be from 54.5 % to 88.8 % (Figure
S1 and S2). No radiolysis was detected under the radiolabeling
condition for all the conjugates.
Compound 2c exhibited substantially reduced efflux (36%)
compared to the structurally analogous inactive CCTA control
3a (66%) at 24 h. Similarly, 2f yielded reduced efflux results
To assess the in vitro CC-trapping potency of these conjugates,
the inhibition constant of the conjugates relative to the
unmodified inhibitor 1a were determined. Cathepsin B (CatB)
was chosen as the model CC due to its ubiquitous expression in
mammalian cells and the selectivity of 1a for this protease. By
monitoring the initial hydrolysis rates of the substrate by CatB
at different concentrations in the presence of the conjugates
(Figure S4), the observed rate constant (Kobs) was calculated and
converted to the inhibition constants (Ki) (Figure S4, S5 and S6)
according to the determined Michaelis-Menten constant (Km) of
the CatB (Figure S3). The results showed that only the
hydrophilic CCTA conjugates (2a-2f) demonstrated low
nanomolar Ki and inhibition IC50 to CatB, similar to the 1a
control (Table 1 and Table S4 and S5). As expected, the
unlabeled control analogs of 3a and 3b did not demonstrate any
inhibition over the concentrations investigated. In addition, the
NTSR1-binding affinity of the conjugates was investigated using
a competitive binding assay with HT-29 human colon cancer
Fig. 2. (A) Efflux of the internalized 2a-f and 3a-b in HT-29 cells, values are means ± SD
(n = 3). (B) Representative confocal microscopy images of the efflux of Cy5 labeled 4a
and 4b (red) formHT-29 cells. Cell endolysosomal compartments were stained with
Lysotracker (green). Scale bar = 50 Pm. (C) Time-dependent fluorescence intensity of
Cy5 per cell as quantified from the confocal images. (D) Co-localization efficiency of Cy5
(red) overlapping with Lysotracker (green). All the analysis was performed in 6 random
images and were presented as mean ± SD. **p < 0.01, ***p < 0.001, NS = not significant.
cells,
a . All of the
well-known NTSR1-positive cell line9a
conjugates exhibited comparable nanomolar binding affinities
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 3 of 5
View Article Online
DOI: 10.1039/C8CC05982A
Journal Name
COMMUNICATION
(39%) relative to 3b (69%). The percentage surface bound vs Indeed, co-incubation with CA-074 (Figure 3A), a CatB selective
internalization of 2c and 2f (Figure S7) was also investigated. inhibitor,11 eliminated observable CatB adducts. No
The surface bond for both of the conjugates was observed to be interference on CatB binding of the conjugates was observed
significantly (4 - 5 fold) higher than that of our previous NTSR1 when co-incubated with the competitive NTSR1 ligand
ligand at 2h,9b which is likely due to the increased confirmed the minimal impact of the NTSR1 peptide on affinity
hydrophobicity after the incorporation of the CCTAs. Overall, of the conjugates to CatB (Figure S10A). These CCTA-
the inclusion of an active CCTA into the NTR1-targeted peptide incorporated conjugates have been shown to bind to the Cys-
construct led to a clear increase in cellular retention.
29 residue12 in the active site of the protease. Autoradiographic
The cell trafficking study of the Cy5-labeled conjugate 4a and its SDS-PAGE demonstrated adduct formation of 2f (Figure 3B) and
CCTA-inactive counterpart 4b was carried out utilizing confocal 2c (Figures S10B) with CatB (~24 kDa heavy chain). Incubation
microscopy. The conjugates were efficiently internalized by the of 2f and 2c in live HT-29 cells resulted in multiple
cells within 2 h, providing strong fluorescence intensity (red) in macromolecule adducts formed, including CatB and possibly
the cytoplasm (Figure S8 and S9). This internalization could be other cysteine cathepsin adducts. In addition, the intracellular
effectively blocked by the addition of an unlabeled NTSR1- adduct formation of the conjugates was found to be
targeted agent, demonstrating that the cellular uptake is substantially inhibited by the co-incubation with a competitive
NTSR1-mediated. The co-localization of the Cy5 signal of NTSR1 ligand (Figure 3C), indicating that adduct formation is
conjugates 4a or 4b with the LysoTracker™ (green) signal dependent on receptor-mediated endocytosis.
indicated intracellular trafficking by the endolysosomal The intracellular adduct formation was also confirmed by the
pathway. The intracellular retention of the conjugates was observation of multiple adducts in the GPC analysis (Figure 3D
further investigated over time (Figure 2B). The incorporation of and Figure S11). Based on the GPC profile, the percentages of
CCTA significantly prolonged the residence time of 4a in the macromolecular adducts, with respect to total intracellular
cells, imparting a 5.5 fold increase in florescence compared with activity, were calculated as 61% for 2c and 66% for 2f, indicating
its inactive counterpart, conjugate 4b at 24 h (Figure 2C). that the radioconjugates efficiently bind to CCs after
Remarkably, in contrast to 4b, the co-localization of 4a within internalization. Furthermore, the radiolabeled adducts of 2f
the endolysosomal compartments (LysoTracker™ signal) could still be detected by SDS-PAGE after 24 h (Figure 3E),
persisted throughout the 24 h time period (Figure 2D). These suggesting a significant portion of these adducts are able to
observations strongly suggest the CCTA in 4a enabled the CC- remain intact over this timespan. Quantification of the
mediated trapping of this conjugate in the endolysosomal radioactive signal from the SDS-PAGE experiments revealed
compartments.
that signal at 24 h was 51%, relative to 2 h, suggesting that the
To examine the ability of conjugates 2f and 2c to form half-life of these cellular adducts is approximately 24 h. As
macromolecular adducts with CCs, gel permeation expected, the inactive controls (3a and 3b) demonstrated no
chromatography (GPC) and sodium dodecyl sulfate adduct formation by either technique.
polyacrylamide gel electrophoresis (SDS-PAGE) were utilized. The excellent serum stability (Figure S12 and S13) and more
hydrophilic profile (Table 1) of 2f prompted the examination of
the in vivo targeting and retention profiles of this agent. Using
an HT-29 xenograft mouse model, the biodistribution profile of
2f and the inactive control 3b, were determined (Table S5). Both
conjugates demonstrated good muscle and blood clearance.
Tumor uptake (Figure 4A) of the two radioconjugates was
statistically identical at 4 hours. However, by 24 hours, 2f had a
25% increase in tumor uptake, while the tumor retention of 3b
decreased significantly by 40%. Percent decreases of about 33%
were seen for both conjugates at 72 h. Overall, 2f demonstrated
a nearly two-fold increase in retention time in the tumors,
compared to the control 3b after 24 h. Uptake in the liver and
kidney were substantial for both conjugates; however,
conjugate 2f demonstrated significantly increased retention
compared to the CCTA-inactive 3b. The uptake in the liver is
likely due to hepatic clearance and non-specific internalization
of these rather lipophilic conjugates.9a, 13 We have found (data
not shown) that the liver and spleen uptake of similar CCTA-
Fig.3. (A) The autoradiography of the SDS-PAGE showing the cathepsin B binding of the
conjugates can be completely inhibited by cysteine proteases inhibitor CA-074. (B) The
incorporated NTR1-targeted agents can be eliminated by simply
increasing the hydrophilicity of the utilized agent. Renal uptake
is most likely due to the well-known renal reuptake/non-specific
internalization mechanism of charged peptides by the proximal
tubule cells during renal excretion.14 Given this, it is probable
autoradiography of the SDS-PAGE gel from the CatB and live HT-29 cell samples after
incubation with 2f and 3b. (C) The autoradiography of the SDS-PAGE demonstrated that
co-incubation with competitive NTSR1 ligand N1 could result in the significant inhibition
of the intracellular adduct formation of the conjugates. (D) The GPC profiles of 2f and
HT-29 cells samples after incubation with 2f and 3b. (E) Autoradiographic image of a SDS-
PAGE gel examining the time-dependent retention of CatB-conjugate adducts in HT-29
cells after pre-incubation with 2f for 4 h.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 5
View Article Online
DOI: 10.1039/C8CC05982A
COMMUNICATION
Journal Name
We thank Janice A. Taylor and James R. Talaska from the Advanced
Microscopy Core Facility at the UNMC for providing assistance with
confocal microscopy. In addition, we gratefully acknowledge Ed Ezell
at the Nuclear Magnetic Resonance (NMR) Core Facility at UNMC for
assistance in collecting NMR data. We thank Melody A. Montgomery
for the professional editing of this manuscript. Lastly, we
acknowledge Dr. Shaheen Ahmed for providing technical support
with western blot studies. This study was supported by grants from
the Nebraska Department of Health and Human Services (2017-21)
and the National Institutes of Health (1R01CA17905901A1).
Fig.4. (A) The %ID/g in HT-29 xenograft tumors at 4, 24 and 72 h postinjection of 2f and
3b in mice (n = 5). (B) The autoradiography of SDS-PAGE of the HT-29 xenograft tumors
at 24 h postinjection of 2f and 3b in mice. (C) Percentage of the macromolecule
associated radioactivity (Mw > 10 kDa) in tumor tissues after administration of 2f and 3b
(n=3). *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant. (D) and (E) The western
blot of cathepsin B (human Liver) and the tumor homogenates after injected with 4a and
4b. The transferring membrane was stained by cathepsin B antibody and visualized by
fluorescence with excitation at 635 nm (D) for Cy 5 and at 532 nm (E) for Alexa Fluor 488
of the secondary antibody.
Conflicts of interest
There are no conflicts to declare
Notes and references
that 2f forms adducts in the liver and kidney in a manner similar
to the internalization mechanism for NTSR1-positive tumors.
To confirm this hypothesis, SDS-PAGE analysis was performed
on tumor (Figure 4B) as well as liver and kidney (Figure S14)
samples ex vivo for conjugates 2f and 3b. For 2f, identical
adduct profiles were observed in the tumor and non-target
tissue samples. These results suggest that these agents form
macromolecular adducts in these tissues most likely due to the
same CC-trapping mechanism. Control conjugate 3b
demonstrated no signs of adduct formation. In addition, using
centrifugal filtration (10 kDa MWCO) to separate
macromolecules from low-molecular weight compounds,
greater than 68% of the radioactivity resident in the HT-29
tumor tissues was found to be associated with macromolecules
(Figure 4C) at 24 and 72 h for 2f, indicating that the increased
retention in these tumors is indeed due to the CC binding.
Lastly, Cy5-labeled conjugates 4a and 4b were injected into
mice to further evaluate in vivo adduct formation. Similar to the
biodistribution data for the radioconjugates, the ex vivo imaging
results indicated that the tumor retention of 4a was greater
than its counterpart 4b at 24 hours (Figure S15). Analysis of the
fluorescently labeled proteins by western blot at 532 nm (Figure
4D) showed that 4a, based on corresponding CatB antibody
staining (Figure 4E), was mainly bound to CatB in tumor tissues.
This is thought to be due to the high CatB expression/activity
profiles in cells as well as the CatB selectivity of the CCTA
trapping agent.7, 15
1
M. J. Welch and W. C. Eckelman, Targeted Molecular Imaging,
Taylor & Francis, Oxfordshire, 2012
(a) M. Fani, H. R. Maecke and S. M. Okarvi, Theranostics, 2012,
2, 481; (b) A. J. Fischman, J. W. Babich and H. W. Strauss, J.
Nucl. Med., 1993, 34, 2253; (c) S. M. Okarvi, Cancer Treat. Rev.
2008, 34, 13.
(a) J. Reiser, B. Adair and T. Reinheckel, J. Clin. Investig., 2010,
120, 3421; (b) C. Palermo and J. A. Joyce, Trends Pharmacol.
Sci., 2008, 29, 22.
2
3
4
5
6
V. Turk, V. Stoka, O. Vasiljeva, M. Renko, T. Sun, B. Turk and
D. Turk, Biochim. Biophys. Acta, 2012, 1824, 68.
(a) J. A. Joyce and D. Hanahan, Cell Cycle, 2004, 3, 1516; (b) N.
Aggarwal and B. F. Sloane, Proteomics Clin. Appl., 2014, 8, 427.
(a) J. Kos, A. Mitroviđ and B. Mirkoviđ, Future Med. Chem.,
2014, 6, 1355; (b) D. R. Sudhan and D. W. Siemann,
Pharmacol. Ther., 2015, 155, 105; (c) S. J. Salpeter, Y. Pozniak,
E. Merquiol, Y. Ben-Nun, T. Geiger and G. Blum, Oncogene,
2015, 34, 6066.
7
8
(a) A. Krantz, L. J. Copp, P. J. Coles, R. A. Smith and S. B. Heard,
Biochemistry, 1991, 30, 4678; (b) B. M. Wagner, R. A. Smith,
P. J. Coles, L. J. Copp, M. J. Ernest and A. Krantz, J. Med. Chem.,
1994, 37, 1833.
(a) J. C. Powers, J. L. Asgian, Ö. D. Ekici and K. E. James, Chem.
Rev., 2002, 102, 4639; (b) H. H. Otto and T. Schirmeister,
Chem. Rev., 1997, 97, 133; (c) L. O. Ofori, N. P. Withana, T. R.
Prestwood, M. Verdoes, J. J. Brady, M. M. Winslow, J. Sorger
and M. Bogyo, ACS Chem Biol, 2015, 10, 1977.
(a) Y. Jia, W. Zhang, W. Fan, S. Brusnahan and J. Garrison,
Bioconjugate Chem., 2016, 27, 2658; (b) Y. Jia, W. Shi, Z. Zhou,
N. K. Wagh, W. Fan, S. K. Brusnahan and J. C. Garrison, Nucl.
Med. Biol., 2015, 42, 816.
9
10 (a) Z. Wu, D. Martinez-Fong, J. Trédaniel and P. Forgez, Front.
Endocrinol., 2012, 3, 184; (b) R. M. Myers, J. W. Shearman, M.
O. Kitching, A. Ramos-Montoya, D. E. Neal and S. V. Ley, ACS
Chem Biol, 2009, 4, 503.
11 T. Towatari, T. Nikawa, M. Murata, C. Yokoo, M. Tamai, K.
Hanada and N. Katunuma, FEBS Lett., 1991, 280, 311.
12 G. Blum, G. von Degenfeld, M. J. Merchant, H. M. Blau and M.
Bogyo, Nat Chem Biol, 2007, 3, 668.
13 K. L. Audus and T. J. Raub, Biological Barriers to Protein
Delivery, Springer, Boston, 1993
14 E. Vegt, M. Melis, A. Eek, M. de Visser, M. Brom, W. J. Oyen,
M. Gotthardt, M. de Jong and O. C. Boerman, Eur. J. Nucl.
Med. Mol. Imaging, 2011, 38, 623
15 D. Kato, K. M. Boatright, A. B. Berger, T. Nazif, G. Blum, C.
Ryan, K. A. Chehade, G. S. Salvesen and M. Bogyo, Nat. Chem.
Biol., 2005, 1, 33.
In summary, this study reinforces the concept that synergistic
incorporation of CC inhibitors (i.e., CCTAs) into a NTSR1-
targeted peptide can lead to the ability to efficiently form
adducts in the endolysosomal compartments of NTSR1-positive
cells. Furthermore, the formation of these macromolecular
adducts substantially prolonged the in vivo retention of the
radioconjugates in NTSR1-positive tumors. This strategy has the
potential to provide an unprecedented means to enhance the
efficacy of NTR1-targeted agents for an array of diagnostic and
therapeutic applications. Also, this technology is expected to be
easily adaptable to a range of receptor-avid small molecules,
peptides, and other targeted agents to improve the selective
retention of these agents, thereby leading to substantial
improvements in translational potential.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C8CC05982A
An efficient strategy of utilizing cysteine cathepsin inhibitor for enhanced tumor residence of the
receptor-targeted agents was presented.