A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy

Article abstract of DOI:10.1016/j.bmcl.2008.01.060

Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathepsin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells.

Full text of DOI:10.1016/j.bmcl.2008.01.060

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1632–1636
A cell-penetrating peptidic GRP78 ligand for tumor
cell-specific prodrug therapy
a
a
a
a
ˇ
´
Yoshiyuki Yoneda, Sebastian C. J. Steiniger, Katerˇina Capkova, Jenny M. Mee,
Ying Liu,a Gunnar F. Kaufmanna and Kim D. Jandaa,b,
*
aDepartments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bThe Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 22 December 2007; revised 12 January 2008; accepted 15 January 2008
Available online 19 January 2008
Abstract—Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that
specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for
tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathep-
sin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Chemotherapy remains an important treatment option
for cancer patients. However, its success is limited by
such drawbacks as insufficient intratumor drug concen-
trations and systemic toxicity due to lack of target spec-
ificity. Restricting the action of a cytotoxic drug to the
tumor site would greatly alleviate systemic toxicity and
improve therapeutic efficacy. Tumor targeting peptides
are promising vehicles for site-directed cancer therapy.
Recently, we have reported the selection and character-
demonstrated in a variety of tumors, such as prostate,
colon, skin, and breast cancers.3 In human cancers, ele-
vated GRP78 level generally correlates with higher path-
ologic grade, recurrence, and poor patient survival in
breast, liver, prostate, and colon cancers.4,5
For a drug deliverable release strategy, we focused on a
proteolytically cleavable linker as we have shown that
Pep42 will be internalized through the GRP78 receptor,
endocytosed and trafficked to the lysosomes that con-
tain proteases, such as cathepsin B.6 In terms of prote-
ases, cathepsin B is a ubiquitously expressed cysteine
protease located in the lysosomes.7 It is confined strictly
to the lysosomes and not found extracellularly, except in
pathological conditions such as cancer or rheumatoid
arthritis.8 Therefore Pep42-drug conjugates containing
cathepsin B-cleavable linkers are likely to be stable in
circulation and selectively release their drug specifically
in the targeted tissue.
ization of Pep42,
a cyclic 13-mer oligopeptide,
CTVALPGGYVRVC, that specifically binds to glu-
cose-regulated protein 78 (GRP78).1 Furthermore, we
have also demonstrated the internalization of Pep42 into
a number of cancer cell lines.2 In cancer cells, the over-
expression of GRP78, an intracellular chaperone and
member of the heat shock protein 70 (HSP70) family,
provides a protective cellular response against stress
conditions. Indeed, GRP78 overexpression also repre-
sents an attractive target as it results in the specific pres-
ence of GRP78 molecules on the cancer cell surface.
Normal GRP78 expression is maintained at low levels
but is upregulated in stress environments and induced
in tumor environments. GPR78 overexpression has been
For our linker release strategy, we decided to employ the
Val-Cit motif that is rapidly cleaved by cathepsin B, but is
very stable in plasma.9 The cytotoxic drugs of choice were
the two well characterized and widely used anti-tumor
agents, paclitaxel (Taxol) and doxorubicin (Fig. 1).1,10
Keywords: Cell-penetrating peptide; Prodrug; Chemotherapy;
Doxorubicin.
Thus, Pep42 was linked through an amidic bond to p-
aminobenzylalcohol, a commonly used self-immolative
spacer, attached to paclitaxel through a carbonate and
*
Corresponding author. Tel.: +1 858 784 2515; fax: +1 858 784
2595; e-mail: kdjanda@scripps.edu
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.060

Y. Yoneda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1632–1636
1633
Figure 1. Potent anticancer drugs Taxol and Doxorubicin.
to doxorubicin through a carbamate functionality
(Fig. 2).11,12 The enzymatic cleavage triggers a 1,6-elim-
ination reaction, resulting in the unmodified drug, car-
bon dioxide and the remnants of the spacer being
released (Fig. 3).13
Subsequent protection of the amino group (3) and treat-
ment with bis-PNPC afforded the carbonate (4), which
was converted to the corresponding paclitaxel (5a) and
doxorubicin (5b) derivatives. Deprotection with tetra-
zole in TFE (6a–b), followed by coupling with Pep 42-
Val-Cit (7), synthesized by standard Fmoc/DIC/HOBt
protocols, gave the desired conjugates (1a) and (1b) in
good yields (Scheme 1).
The general synthetic strategy involved coupling of the
Pep42-Val-Cit (7) fragment with an appropriately func-
tionalized derivative of paclitaxel or doxorubicin (6a–b)
(Scheme 1). The starting Val-Cit-PABOH (2) was read-
ily obtained using a previously described procedure.14
The cytotoxicity of the Pep42-prodrug conjugates on
SJSA-1 osteosarcoma cells, a GRP78-expressing cell
Figure 2. Conjugates of Pep 42 with taxol (1a) and doxorubicin (1b). Amino acids are described with the appropriate one-letter codes.

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Y. Yoneda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1632–1636
Figure 3. Release cascade of the drug.
Scheme 1. Synthesis of the Pep 42 conjugates.
line, was studied in vitro by using an MTT assay. SJSA-
1 cells (5 · 103) were plated in each well of a 96-well tis-
sue culture plate. Medium supplemented with 10% FBS
was added, and cells were allowed to adhere for 24 h.
Cells were then incubated with serial dilutions of Tax-
ol-Pep42 and doxorubicin-Pep42 for 6 h in triplicate,
and a MTT assay was performed (Figs. 4 and 5).18 As
shown in Figure 4, Taxol-Pep42 showed greater cytotox-
icity than free Taxol alone against SJSA-1 cells. At the
Taxol equivalent concentration of 10À3 lM, cell viabil-

Y. Yoneda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1632–1636
1635
this resulted in an increase in cytotoxicity as Pep42 facil-
itates the uptake of Taxol and doxorubicin into cells and
thus, delivers its toxic payload directly into the cancer
cell.
Acknowledgment
This work was supported by The Skaggs Institute for
Chemical Biology and the National Institutes of Health
(CA 094193).
Supplementary data
Figure 4. Viability of cells incubated with serial dilutions of Taxol and
Taxol-Pep42 by MTT assays.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.01.060.
References and notes
1. Kim, Y.; Lillo, A. M.; Steiniger, S. C.; Liu, Y.; Ballatore,
C.; Anichini, A.; Mortarini, R.; Kaufmann, G. F.; Zhou,
B.; Felding-Habermann, B.; Janda, K. D. Biochemistry
2006, 45, 9434.
2. Liu, Y.; Steiniger, S. C.; Kim, Y.; Kaufmann, G. F.;
Felding-Habermann, B.; Janda, K. D. Mol. Pharm. 2007,
4, 435.
3. Shin, B. K.; Wang, H.; Yim, A. M.; Le Naour, F.;
Brichory, F.; Jang, J. H.; Zhao, R.; Puravs, E.; Tra, J.;
Michael, C. W.; Misek, D. E.; Hanash, S. M. J. Biol.
Chem. 2003, 278, 7607.
4. Fu, Y.; Lee, A. S. Cancer Bio.l Ther. 2006, 5, 741.
5. Zhang, J.; Jiang, Y.; Jia, Z.; Li, Q.; Gong, W.; Wang, L.;
Wei, D.; Yao, J.; Fang, S.; Xie, K. Clin. Exp. Metastasis
2006, 23, 401.
Figure 5. Viability of cells incubated with serial dilutions of Doxoru-
bicin and Doxorubicin-Pep42 by MTT assay.
6. Schmid, B.; Chung, D. E.; Warnecke, A.; Fichtner, I.;
Kratz, F. Bioconjug. Chem. 2007, 18, 702.
7. Yan, S.; Sloane, B. F. Biol. Chem. 2003, 384, 845.
8. Bettio, F.; Canevari, M.; Marzano, C.; Bordin, F.;
Guiotto, A.; Greco, F.; Duncan, R.; Veronese, F. M.
Biomacromolecules 2006, 7, 3534.
ity was about 40% for Taxol-Pep42 treated cells, while
the viability of Taxol treated cells decreased to only
80%. The IC50 values for Taxol were 3.2 and 1.1 nM
for Taxol-Pep42 (Fig. 4).
9. Dubowchik, G. M.; Firestone, R. A. Bioorg. Med. Chem.
Lett. 1998, 8, 3341.
10. Liu, C.; Sun, C. Z.; Huang, H. N.; Janda, K. D.;
Edgington, T. Cancer Res. 2003, 63, 2957.
11. Niculescu-Duvaz, I.; Niculescu-Duvaz, D.; Friedlos, F.;
Spooner, R.; Martin, J.; Marais, R.; Springer, C. J.
J. Med. Chem. 1999, 42, 2485.
12. Toki, B. E.; Cerveny, C. G.; Wahl, A. F.; Senter, P. D.
J. Org. Chem. 2002, 67, 1866.
13. Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone,
R. A. Bioorg. Med. Chem. Lett. 1998, 8, 3347.
14. Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner,
D.; Hofstead, S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J.;
Trail, P. A. Bioconjug. Chem. 2002, 13, 855.
The doxorubicin-Pep42 conjugate also demonstrated en-
hanced cytotoxicity against SJSA-1 cells (Fig. 4). For
unconjugated doxorubicin, the IC50 was 6.2 nM, while
cells treated with doxorubicin-Pep42 showed a viability
of 50% at 1.7 nM (Fig. 5). These results are consistent
with or superior to previously reported analogous stud-
ies.15,16 When employing the same activation-release
cascade, antibody conjugates have demonstrated a high-
er increase in cytotoxic activity.17 However, here one
must also consider that the payload exceeds more than
one toxin molecule per antibody molecule, while the
coupling ratio of prodrug to Pep42 was 1:1.
15. Safavy, A.; Raisch, K. P.; Khazaeli, M. B.; Buchsbaum,
D. J.; Bonner, J. A. J. Med. Chem. 1999, 42, 4919.
16. Chen, X.; Plasencia, C.; Hou, Y.; Neamati, N. J. Med.
Chem. 2005, 48, 1098.
17. Jeffrey, S. C.; Nguyen, M. T.; Andreyka, J. B.; Meyer, D.
L.; Doronina, S. O.; Senter, P. D. Bioorg. Med. Chem.
Lett. 2006, 16, 358.
18. Cells were plated in 96-well plates (5000 cells per well)
and incubated overnight at 37 ꢁC to allow for attach-
ment and spreading of the cells. Serial dilutions of the
In summary, we have demonstrated that the peptidic
GRP78 ligand Pep42 can be utilized to efficiently deliver
two well characterized cytotoxic drugs frequently used
in current cancer therapy treatment regimes into cancer
cells. Based on our previously reported data we
exploited Pep42’s translocation into the lysosomal com-
partment to design prodrug conjugates that specifically
release the drugs upon entering the cells. Gratifyingly,

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Y. Yoneda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1632–1636
Pep42, Pep42-prodrug conjugate as well as doxorubicin
reagent was then added to individual wells and allowed
to further incubate for 1 h at 37 ꢁC. Samples were
assayed in triplicate and the absorbance was measured
at 490 nm. Cells incubated with PBS were used as
untreated control cells and cell culture medium was a
background control.
and paclitaxel were prepared in cell culture medium in a
separate plate. The medium of the plate containing the
cells were taken out and replaced with medium con-
taining the Pep42-prodrug conjugate dilutions, followed
by an incubation period of 6 h at 37 ꢁC. The MTT