Synthesis and biological evaluation of a peptide-paclitaxel conjugate which targets the integrin αvβ6

Article abstract of DOI:10.1016/j.bmc.2011.07.046

The integrin α_vβ₆ is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other carcinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds α_vβ₆ and displays

Full text of DOI:10.1016/j.bmc.2011.07.046

Bioorganic & Medicinal Chemistry 19 (2011) 5480–5489
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a peptide–paclitaxel conjugate
which targets the integrin
avb6
⇑
Shunzi Li, Bethany Powell Gray, Michael J. McGuire, Kathlynn C. Brown
Department of Internal Medicine and The Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,
Dallas, TX 75390-8852, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The integrin
cinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds
displays minimal affinity for other RGD-binding integrins. Here we report the use of this peptide to
actively deliver paclitaxel to vb6-positive cells. We synthesized a water soluble paclitaxel–H2009.1 pep-
tide conjugate in which the 20-position of paclitaxel is attached to the tetrameric peptide via an ester link-
age. The conjugate maintains its specificity for vb6-expressing NSCLC cells, resulting in selective
cytotoxicity. Treatment of vb6-positive cells with the conjugate results in cell cycle arrest followed by
induction of apoptosis in the same manner as free paclitaxel. However, initiation of apoptosis and the
resultant cell death is delayed compared to free drug. The conjugate demonstrates anti-tumor activity
in a H2009 xenograft model of NSCLC with efficacy comparable to treatment with free paclitaxel.
Ó 2011 Elsevier Ltd. All rights reserved.
a
vb6 is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other car-
Received 28 June 2011
Revised 19 July 2011
Accepted 22 July 2011
Available online 4 August 2011
a
vb6 and
a
a
Keywords:
Peptide
Paclitaxel
Lung cancer
Drug delivery
a
1. Introduction
biomarker. Active targeting is anticipated to further improve drug
accumulation and/or increase cellular uptake of a therapeutic while
Non-small cell lung cancer (NSCLC) is a notoriously deadly cancer
with a 5-year survival rate of less than 15%.1 Paclitaxel (TAXOLÒ) is a
widely used cytotoxic agent for the treatment of NSCLC as well as
various other carcinomas. However, its clinical utility is hampered
by poor aqueous solubility and non-selective toxicity. Delivery sys-
tems that specifically target paclitaxel to the tumor site are antici-
pated to (a) reduce systemic toxicity, (b) improve efficiency of
paclitaxel delivery to tumors and increase its dwell time within
the tumor, (c) lower the paclitaxel dosage required to achieve effec-
tive tumor growth reduction, and (d) improve paclitaxel’s solubility.
The nanoparticle formulation of paclitaxel, AbraxaneÒ (Nab-paclit-
axel), improves solubility and negates the need for formulation in
polyethoxylated castor oil (Cremophor EL).2,3 Additionally, in-
creased accumulation of paclitaxel is observed in tumors due to pas-
sive targeting of the nanoparticle that occurs as a result of the
enhanced permeability and retention effect (EPR). However, pa-
tients treated with Nab-paclitaxel still suffer from neutropenia
and neuropathy due to non-specific uptake in non-diseased tissues.
As such, there is a continuing need to develop active targeting drug
delivery systems that utilize homing ligands which transport thera-
peutics specifically to tumors that express a specific cell surface
decreasing non-specific uptake in other organs.
Most efforts in active tumor targeting have focused on conju-
gating paclitaxel to monoclonal antibodies (MAbs) specific for cell
surface cancer biomarkers in order to deliver paclitaxel preferen-
tially to the tumor.4–9 Yet, chemically modifying antibodies is chal-
lenging, production costs are substantial, and, post-translational
modifications on MAbs can trigger severe hypersensitivity reac-
tions.10 Synthetic peptides have attracted attention as targeting
molecules.11,12 Peptides are smaller than antibody-based targeting
agents, can be synthesized in large quantities and are amenable to
regiospecific derivatization.13 The toxicity profile of peptides is
low. Peptides can be chemically modified to alter affinity, charge,
hydrophobicity, stability, and solubility.
Several peptide–paclitaxel conjugates have been reported. Cell
penetrating peptides have been employed as carriers of paclit-
axel.14,15 In addition, peptides that bind cell surface biomarkers
have served as delivery vehicles for paclitaxel. Most of these are
based on naturally occurring peptides such as bombesin and
somatostatin or on the well-characterized
avb3 binding peptide
RGD.4,16–20 More recently, paclitaxel–peptide conjugates that tar-
get HER-221 and GRP7815,22 have met with success in vitro. How-
ever, the number of targeting ligands needs to expand in order to
address the diversity of cancer phenotypes found in the patient
population. Moreover, most of these ligands are unsuitable for
the treatment of NSCLC.
Abbreviations: NSCLC, non-small cell lung cancer; MAb, monoclonal antibody;
H2009.1, peptide sequence: RGDLATLRQL; scH2009.1, peptide sequence:
DALRLQGTLR.
We previously identified a peptide from a phage displayed pep-
⇑
Corresponding author.
E-mail address: Kathlynn.Brown@UTSouthwestern.edu (K.C. Brown).
tide library via biopanning on the NSCLC cell line H2009.23 This
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.046

S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
5481
peptide, referred to as H2009.1, binds the integrin
a
vb6 and dis-
obtained as white powder, which were soluble in cold water as
well as in most polar organic solvents such as chloroform and
methyl alcohol. The increased solubility of paclitaxel allows for
use of the drug in buffered saline solutions and avoids the need
for the use of CremophorÒ EL.
plays minimal affinity for other RGD-binding integrins.24 Expres-
sion of
avb6 is widespread in early stage human NSCLC, and it is
associated with poor patient survival.24,25 Additionally, this inte-
grin is over-expressed in many epithelial derived carcinomas but
is not found in normal primate tissues.26–36 In sum, the integrin
a
vb6 is emerging as an important target for anticancer therapies.
2.2. Cell-specific binding of H2009.1 tetrameric peptide–
paclitaxel conjugate
As such, our lab is focusing on developing the H2009.1 peptide
for clinical use.
Tetramerization of the H2009.1 peptide dramatically increases
its affinity for its cellular target resulting in a ligand with a
half-maximal binding affinity of 50 pM.37 In addition, the
H2009.1 tetrameric peptide mediates cellular uptake allowing for
the chemotherapeutics to be delivered intracellularly. Finally, we
have demonstrated that the peptide accumulates specifically in
Although paclitaxel is incorporated in the trilysine core away
from the peptide binding domain, it is important to verify that
the addition of this hydrophobic drug does not destroy H2009.1’s
binding nor affect its unique cell specificity. To address this issue,
the peptide–paclitaxel conjugates were labeled with fluorescein
(Fig. 1, compound 4) and binding was quantified by flow cytomet-
ric analysis. When H2009 cells were treated with scrambled
peptide–paclitaxel conjugate, no binding was observed (Fig. 2).
However, H2009 cells treated with labeled H2009.1 peptide–
paclitaxel had a mean fluorescence intensity (MFI) 3.3-fold greater
than that observed for the scrambled peptide conjugate, corrobo-
rating that binding is peptide-mediated and not affected by attach-
avb6-positive tumors but not in avb6-negative tumors nor in nor-
mal healthy tissues. Thus, this peptide has the properties necessary
to be an effective homing ligand for drug delivery. In the present
study, we report the synthesis and characterization of a water sol-
uble, cancer cell specific delivery drug system, in which H2009.1
tetrameric peptide is conjugated to paclitaxel (Schemes 1 and 2).
ment of paclitaxel. H460 cells express the integrins
vb5 but do not express
vb6.24 As such, these cells are a good
control with which to address specificity of the conjugate for
vb6. H460 cells exhibit minimal binding to the H2009.1 pep-
tide–paclitaxel conjugate, indicating that H2009.1 tetrameric
peptide–paclitaxel selectively targets vb6 positive cells and addi-
avb3, a5b1, and
a
a
2. Results and discussion
a
2.1. Synthesis and characterization of H2009.1 peptide–
paclitaxel conjugate
a
The 20-hydroxyl and 70-hydroxyl groups of paclitaxel are suit-
able sites for conjugation to a targeting ligand, with preferential
modification occurring at the 20-hydroxyl group due to steric
hindrance at the 70-hydroxyl group. 20-Maleimido-paclitaxel was
prepared in 68% yield through coupling paclitaxel with 3-maleim-
idopropionic acid (Scheme 1, compound 1). 1NMR analysis
revealed a shift of the C-20 proton to 5.47 ppm compared to
4.71 ppm in free paclitaxel, and the resonance of the 20-OH proton
at 3.55 ppm in paclitaxel disappeared. The resonance of the C-70
proton was unaffected. These data confirm the formation of an
ester bond at the 20 hydroxyl group of paclitaxel.
The H2009.1 10mer tetrameric peptide (Scheme 2, compound
2) was synthesized separately by a convergent strategy published
previously by our lab.37 Chemoselective reaction between the thiol
of the trilysine peptide core with the 20-maleimido-paclitaxel re-
sults in formation of the peptide–drug conjugate in ꢀ90% yield
(Fig. 1, compound 3). The same reaction conditions were employed
tion of the hydrophobic drug does not increase binding to non-
avb6
expressing cells.
2.3. Drug release study
Paclitaxel must be released from the peptide carrier to be ac-
tive; yet, the conjugate must remain stable until it reaches its tu-
mor target. Our linker design is based on the hypothesis that the
ester linkage will be stable until internalized into cells where in-
creased concentration of esterases and proteases will lead to
hydrolysis thus liberating paclitaxel. As the 20-hydroxyl group is
required for tubulin binding, the peptide–paclitaxel conjugate is
anticipated to be inactive unless internalized.38–40 To assure that
the drug-conjugate is stable at neutral pH but hydrolyzed by ester-
ases, release of paclitaxel from the H2009.1 peptide–paclitaxel
conjugate was determined in both PBS buffer (pH 7.4) and esterase
containing PBS buffer. In PBS, minimal paclitaxel was released even
after 96 h (Fig. 3). Similar results were observed under acidic con-
dition (pH 5.6 buffer, data not shown). The hydrolysis rate was in-
creased in the presence of porcine esterase; 34.1% of paclitaxel was
released by 48 h. This 7-fold increase of paclitaxel release indicates
that hydrolysis is enhanced in the presence of esterase; however,
using
a scrambled sequence tetrameric peptide (noted as
scH2009.1) in order to provide a control peptide–paclitaxel conju-
gate. These two constructs have the same chemical composition
but a different linear sequence. The H2009.1 peptide–paclitaxel
conjugate and scrambled peptide–paclitaxel conjugate were
Scheme 1. Synthesis of 20-maleimido-paclitaxel was prepared via coupling paclitaxel with 3-maleimidopropionic acid (compound 1).

5482
S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
O
O
N
NH
O
N
N
O
H
N
O
O
O
CONH2
N
H
O
NH
H2009.1 Peptide
HN
10
+
O
O
HS
O
O
O
O
O
O
H
N
OH
NH
N
H
O
O
O
S
1. PBS, 2 h, RT
2.TFA/anisole (99:1), AgOAc
3. Dithiothreitol in AcOH (1M)
HN
NH
O
O
O
N
O
O
H
N
O
O
CONH2
O
N
O
H2009.1 Peptide
10
H
O
S
O
N
O
NH
O
H
O
O
N
CONH2
N
O
H2009.1 Peptide
10
H
O
O
S
NH
HN
N
O
O
O
O
O
O
O
H
N
OH
N
NH
N
H
O
O
S
O
HS
O
H
N
H2009.1 Peptide
O
O
O
N
CONH2
10
H
NH
O
O
O
N
O
S
O
H
N
H2009.1 Peptide
O
O
O
N
H
CONH2
O
Compound 2
Scheme 2. Synthesis of tetrameric H2009.1 peptide by a convergent coupling reaction (compound 2). Michael addition of cysteine containing monomeric peptides with the
maleimide trilysine core results in formation of the tetrameric peptide. A PEG11 linker is placed between the peptide and the core to improve solubility and prevent peptide
aggregation. Removal of the acetamidomethyl protein group from the trilysine core by treatment with the Ag(OAc) reveals a unique thiol for coupling to compound 1.
drug release remains inefficient. By comparison, 70% of paclitaxel
is released from the peptide–paclitaxel conjugate after 48 h incu-
bation in rat serum, with a half-life of approximately 20 h. The
half-life of the conjugate in serum is similar to that observed for
other paclitaxel ester linkers.41 The increased release is likely due
to participation of other proteases or esterases present in rat ser-
um. This also raises some concern about the stability of the conju-
gate while in circulation; however, 86% of the conjugate remains
intact in rat serum at 1 h. As the overall size of the complex is be-
low the renal filtration cutoff, we believe most of the conjugate
that does not reach the tumor will be cleared by the kidney within
this time frame. Of note, the tetrameric peptide itself has a half-life
of 12 h indicating that peptide stability will not be an issue within
this context.
2.4. Evaluation of in vitro cytotoxicity
To evaluate the ability of the peptide conjugates to deliver ac-
tive paclitaxel in a peptide-specific manner, the cytotoxicity of free
paclitaxel, H2009.1 tetrameric peptide–paclitaxel conjugate and
scrambled peptide–paclitaxel conjugate was determined (Table
1). The IC50 value for H2009.1 peptide–paclitaxel conjugate is
460 nM on H2009 cells, whereas the scrambled peptide conjugate
does not reach a 50% reduction in cell viability, even at 1 lM. Thus,
H2009.1 peptide is mediating cellular delivery of paclitaxel in a se-
quence dependent manner. There is a reduction in the potency of
the paclitaxel when it is conjugated to the peptide (IC50 of free pac-
litaxel: 15 nM). This could be due to reduced uptake of the drug,
slow release of paclitaxel from its carrier, and/or drug delivery to

S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
5483
O
H
N
O
O
CONH2
H2009.1 Peptide
N
H
O
10
O
S
O
N
O
NH
O
O
H
N
O
O
CONH2
N
O
H2009.1 Peptide
10
H
O
O
S
NH
HN
N
O
O
OH
O
O
O
O
S
O
O
H
N
N
H
O
N
NH
N
H
NH
O
O
S
O
O
H
N
O
H2009.1 Peptide
O
O
N
O
N
H
O
O
CONH2
10
O
NH
O
O
O
O
O
O
O
HO
N
O
OH
H
O
O
O
S
O
O
H
N
O
H2009.1 Peptide
O
O
O
N
H
CONH2
10
O
Compound 3
O
H
O
O
N
CONH2
O
N
H
O
H2009.1 Peptide
10
O
S
O
N
O
OH
NH
O
H
N
O
O
CONH2
S
N
O
H2009.1 Peptide
H
10
O
O
HN
NH
O
S
NH
HN
N
N
O
O
OH
OH
O
O
O
O
H
N
O
O
H
N
HN
NH
N
N
H
O
O
H
O
S
O
O
O
S
O
O
H
N
H2009.1 Peptide
O
O
O
N
H
CONH2
N
10
NH
O
O
O
O
O
O
N
O
H
O
N
O
O
S
HO
O
O
O
H
N
O
O
H2009.1 Peptide
O
O
O
O
O
N
H
CONH2
H
10
O
O
O
OH
Compound 4
Figure 1. Structure of H2009.1 peptide–paclitaxel conjugate (compound 3) and FITC-labeled H2009.1 peptide–paclitaxel conjugate (compound 4). The H2009.1 peptide
displays the sequence RGDLATLRQL and the scrambled H2009.1 peptide is DALRLQGTLR.
and retention in an inappropriate cellular compartment. Drug con-
jugates often have reduced effectiveness compared to their non-
conjugated partners. Other peptide–paclitaxel conjugates have
shown reduced efficacy under short incubation times but the rea-
son for the observation has not been determined.16,41 However, im-
proved biodistribution of the targeted drug conjugates in vivo can
often overcome the loss of activity.
10 min followed by different time points of recovery. In the pres-
ence of free paclitaxel, a rapid reduction of cell viability was ob-
served within the first 48 h. Neither H2009.1 10mer tetrameric
peptide–paclitaxel conjugate nor scrambled peptide–paclitaxel
conjugate exhibited any effect within the first 48 h. However,
H2009.1–paclitaxel conjugate began to reduce cell viability at
72 h and inhibition of 40% is observed at 120 h. By contrast, the
scrambled peptide–paclitaxel conjugate exhibited less than 15%
cell growth inhibition at 120 h. As the cells are only exposed to
To understand the time frame in which cell death is initiated,
H2009 cells were incubated with 1 lM of each compound for

5484
S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
Figure 2. The paclitaxel–H2009.1 conjugate binds specifically to avb6-expressing cells. H2009 cells (avb6-positive) and H460 cells (avb6-negative) were incubated for 1 h with
1 M paclitaxel–H2009.1 conjugate labeled with FITC (compound 4) or scrambled peptide–paclitaxel–dye conjugate. Binding of the conjugate was measured by flow
cytometry. A total of 10,000 events were evaluated for fluorescence in channel 1 (excitation at 488 nm, emission at 500–550 nm).
To determine whether the H2009.1 conjugate is specifically
cytotoxic toward
a
vb6 positive cells, H2009 and
avb6-negative
H460 cells were incubated in the presence of 1
l
M of unconjugated
paclitaxel or of H2009.1 peptide–paclitaxel conjugate (Fig. 4). Both
cell types are sensitive to free paclitaxel; the IC50 of paclitaxel on
H460 cells is 24 nM, similar to that observed on H2009. However,
after treatment with H2009.1 peptide–paclitaxel conjugate, the
avb6 negative H460 cells remained almost unaffected while the
H2009 cells were sensitive to the conjugate. At 120 h, the differ-
ence in viability between the two cell types is 3.9-fold. In sum, con-
jugation of paclitaxel to H2009.1 converts paclitaxel from a general
cytotoxic agent to one that only affects
avb6-positive cells. Thus,
while decreased in vitro efficacy is observed with the H2009.1–
paclitaxel conjugate compared to free paclitaxel, the conjugate
has a wider therapeutic window.
Figure 3. Paclitaxel is released from the conjugate in the presence of esterase and
serum. Compound 3 was dissolved in phosphate-buffered solutions (PBS, 0.01 M) at
pH 4 (N), or esterase (18 u/mL) in PBS (j), or rat serum (d), individually. The
solutions were incubated at 37 °C. At time points 3, 8, 24, 48, 72, and 96 h during
incubation, the release of paclitaxel was analyzed by HPLC.
2.5. Competition assay
To further assure that the H2009 peptide delivers paclitaxel in a
receptor dependent fashion, we sought to determine if free
H2009.1 peptide could block the toxicity of the H2009.1–paclitaxel
conjugate. H2009 cells were treated with a 10-fold excess of free
H2009.1 peptide, followed by treatment with the H2009.1 pep-
tide–paclitaxel conjugate. At 120 h after exposure to the conjugate,
cells treated first with free peptide were significantly protected
from the cytotoxic effects of the H2009.1–paclitaxel conjugate; cell
viability was 73 0.08% for samples pre-treated with free H2009.1
versus 36 0.07% for cells that were incubated with the drug con-
jugate alone (Fig. 5), restoring viability to that observed with the
scrambled peptide–paclitaxel conjugate (79 0.06%). Additionally,
free H2009.1 tetrameric peptide showed only slight inhibition of
cell proliferation on H2009 cells (0.92 0.10% cell viability). These
data support that conjugate internalization is a receptor-mediated
process. The need for a 10-fold excess of free peptide to block the
peptide–paclitaxel conjugate’s cytotoxicity can be attributed to the
Table 1
IC50 values of paclitaxel conjugatesa
H2009 cells
H460.1
Paclitaxel
H2009.1–paclitaxel
Scramble H2009.1–paclitaxel
14.8 3.3 nM
460 75 nM
Not reachedb
23.6 7.4 nM
Not reachedb
Not reachedb
a
Cells were exposed to drug for 10 min followed by a 120 h recovery in media.
The short incubation time assures that cytotoxicity is due to the peptide conjugate
and not free paclitaxel released prematurely from the conjugate.
b
Not reached at highest concentration used, 1 lM.
the drug conjugates for 10 min, the delayed response is not due to
a slow accumulation of conjugate within the cells. Instead, this
data suggests that drug release is a limiting factor and that suffi-
cient exposure time is essential for the drug to be released from
the conjugate. This is consistent with the in vitro drug release as-
says which show release of paclitaxel from the peptide to be inef-
ficient. However, we cannot rule out the possibility that drug
release is rapid yet transportation to the cytoplasm is rate limiting.
high avidity of the conjugate and/or a rapid recycling of avb6.
2.6. Cell cycle analysis
Paclitaxel induces G2/M cell cycle arrest resulting in apoptosis.
Flow cytometry analysis was performed to analyze the cell cycle

S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
5485
Figure 4. The H2009.1–paclitaxel conjugate displays
a
vb6-selective cytotoxicity that is time dependent. Left panel: H2009 cells were exposed to 1
lM Taxol, H2009.1
peptide–paclitaxel (compound 3) and scH2009.1 peptide–paclitaxel for 10 min, washed, and incubated further in fresh medium for different time point. The cell viability is
normalized to untreated control cells. Right panel: H2009 cells and H460 cells were exposed to 1 M Taxol and compound 3 for 10 min, washed, and incubated further in
l
fresh medium for indicated times. The short exposure time to the conjugates is necessary to assure that the affects observed are due to the conjugate and not paclitaxel
released from the peptide before internalization.
H2009.1 Peptide
1.2
1
0.8
0.6
0.4
0.2
0
Figure 5. The affect of H2009.1–paclitaxel conjugate on cell viability is blocked by pretreatment with H2009.1 peptide. H2009 cells were treated with or without 10 lM
H2009.1 10-mer tetrameric peptide for 1 h, washed, and then treated with 1
lM H2009.1–paclitaxel conjugate (compound 3) or scH2009.1 peptide–paclitaxel. Viability was
determined at 96 h.
perturbations of H2009 cells treated with the H2009.1 peptide–
paclitaxel conjugate to assure that the conjugate is functioning in
a similar manner as free paclitaxel (Table 2). At 24 h, G2/M cell
by ꢀ2-fold and a 3-fold increase in polyploid cells is observed.
Increasing the peptide–paclitaxel conjugate concentration also in-
creases the percentage of cells in cycle arrest. Approximately 50%
more G2/M arrest occurred with a decrease of cells in G1 phase
at 24 h. The effect of paclitaxel on the cell-cycle progression of
H2009 cells thus occurs in a dose-dependent manner. These data
support the conclusion that the H2009.1–paclitaxel conjugate
cycle arrest was observed for H2009 cells treated with 1
lM free
paclitaxel. When H2009 cells were treated with 1 M H2009.1
l
peptide–paclitaxel conjugate, the cell cycle remains unperturbed
at 24 h. However, at 48 h, the number of cells in G2/M increases
Table 2
Cell cycle analysis of H2009 cells treated with paclitaxel–peptide conjugatesa
0
l
M
1
l
M
5 lM
Control cells
Paclitaxel
Conjugate 24 h
Conjugate 48 h
Conjugate 24 h
Conjugate 48 h
G1
S
G2/M
Polyploid
50.9
11.2
31.1
6.8
8.5
4.1
73.5
14
44.9
8.1
39.0
8.0
11.4
4.5
57.1
26.9
35.8
6.7
45.8
11.7
23.9
8.5
53.9
23.6
a
Represents the average of two experiments.

5486
S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
induces cell cycle arrest as observed for the free drug. However, the
conjugate shows a delayed effect as time is required to accumulate
sufficient concentrations of liberated paclitaxel.
2.7. Characterization of drug-induced apoptosis
Initiation of apoptosis is characterized by activation of a system
of caspases. To determine the time frame in which this process be-
gins upon treatment with H2009.1 peptide–paclitaxel conjugate,
caspase activation was followed by flow cytometry using VybrantÒ
FAM Poly Caspases Assay. Propidium iodine was employed to detect
non-viable cells. At 24 h, 37% of cells treated with H2009.1 peptide–
paclitaxel conjugate were positive for caspase activation. Of this
subpopulation, 70% are PI negative, suggestive of cells in early apop-
tosis. As time progressed, the number of caspase and PI double posi-
tive cells increased (Fig. 6). At day 4, 43% of the cell population is
positive for caspase activation and PI staining, indicating the cells
are in late apoptosisand have lost integrity ofthe plasma membrane.
By comparison, cells treated with the control peptide conjugate ex-
hibit minimal caspase activation with a maximum of 14% caspase
positive cells at 96 h. These data again confirm that the H2009.1 pep-
tide specifically delivers paclitaxel. The time course of H2009.1 pep-
tide-conjugate induced apoptosis is similar to that observed for both
the cell cycle arrest and cytotoxicity studies, again indicating that
the H2009.1–paclitaxel conjugate shows delayed action. However,
it should be noted that at 96-h, 40% of the cells treated with the
H2009.1–paclitaxel conjugate still remain caspase negative and PI
negative. At 144 h, this population drops to 27%. Thus there is a sig-
nificant subpopulation of viable cells that are not affected. It is pos-
sible that there is a subpopulation of cells that do not internalize the
peptide or do not accumulate enough paclitaxel during the 10 min
exposure to be effective. This is consistent with the flow cytometry
data which reveals a small subpopulation of cells that do not bind
significant amounts of the conjugate. This highlights a concern in
using targeted therapies; due to heterogeneity of the tumor, small
subsets of cells may remain refractory to treatment. Expanding the
suite of tumor targeting ligands is anticipated to minimize this prob-
lem by allowing a greater percentage of the tumor to be targeted.
Figure 7. Tumor growth rate is slowed by free paclitaxel and H2009.1–paclitaxel
conjugate by similar amounts. Subcutaneous tumors were established on the flank of
NOD/SCID mice (n = 5). At day 18, saline, free paclitaxel and compound 3 were
injected intravenously via the tail vein with 5 mg/kg, based on paclitaxel weight.
Animals were treated at days 18, 21, 24, 27, and 30. Measurements were made with
calipers by an independent scientist. Tumor volumes are calculated as V = (l ꢁ w2)/2.
Saline (d), paclitaxel (j), H2009.1–paclitaxel (N).
can overcome the loss of activity. We determined the efficacy of the
H2009.1–paclitaxel conjugate in a H2009 xenograph model. H2009
cells were implanted subcutaneously on the flank of immunocom-
promised mice. At day 18 when palpable tumors had formed, the
animals were treated with saline, 5 mg/kg paclitaxel or 5 mg/kg
H2009.1–paclitaxel (based on the total amount of paclitaxel). A total
of 5 injections per mouse were given every 3 days. While the
H2009.1–paclitaxel conjugate reduced the rate of tumor growth
compared to saline, there is no statistical difference between free pac-
litaxel and the conjugate in terms of tumor growth rate or survival
(Fig. 7). Similarly, the animal weights remained steady for both groups
suggesting that no gross toxicity was observed (data not shown). Thus,
conjugation to the targeting peptide did not provide any obvious ben-
efit over the free drug in vivo. Interestingly, the effect of the H2009.1–
paclitaxel is delayed; the growth rates for the control animals and
those treated with H2009.1–paclitaxel conjugate do not begin to di-
verge until day 46. This is 7 days after the free drug begins to show
an effect on tumor growth. Also of note, the conjugate does not de-
crease the performance of paclitaxel despite its higher IC50 found
in vitro. Complete biodistribution and pharmacokinetic experiments
are needed to determine if the observed results are due to a lack of drug
targeting, inefficient drug release, premature drug release, or poor dis-
tribution throughout the tumor. As the conjugate is below the renal fil-
tration limit, rapid clearance by the kidneys may contribute to the lack
of efficacy; however free paclitaxel is also cleared by the kidneys and
previous studies have demonstrated accumulation of the peptide in
positive tumors despite the rapid renal filtration. Additionally, maxi-
mally tolerated dosage needs to be examined; it is possible that
higher doses of paclitaxel can be administered as a conjugate be-
cause of reduced off target effects.
2.8. Tumor growth studies
While drug conjugates often have reduced efficacy in vitro com-
pared to their non-conjugated partners, the opposite is often true
in vivo as improved biodistribution of the targeted drug conjugates
100
90
80
70
60
50
40
30
20
10
0
3. Conclusions
In sum, the H2009.1 peptide is able to selectively deliver a che-
24 h
Caspase Negative/PI Negative Caspase Negative/PI Positive
Caspase Positive/PI Negative Caspase Positive/PI Positive
48 h
72 h
96 h
120 h
144 h
motherapeutic agent to avb6 positive cells, thus opening the ther-
apeutic window. Our data indicate that this conjugate effects cell
death via cell cycle arrest followed by the induction of apoptosis
in the same manner as paclitaxel. However, the time frame of
activity is delayed compared to free drug. We believe this is due
to inefficient release of paclitaxel or poor intracellular trafficking
to the cytoplasm where paclitaxel exerts its effects. Ester based
conjugates to paclitaxel have met with mixed results in vitro and
in vivo. Future optimization of drug linkers will be needed for pep-
tide–paclitaxel conjugates to be viable. Recently, self-immolative
Figure 6. H2009.1–paclitaxel activates caspases and initiates apoptosis. H2009
cells were exposed to 1 M H2009.1–paclitaxel conjugate for 10 min, washed, and
l
incubated in fresh media for indicated times. Floating and attached cells were
collected and subjected to caspases/PI staining using VybrantÒ FAM Poly Caspases
Assay and analyzed by flow cytometry. 10,000 events were measured using channel
1 (excitation 488 nm, emission 500 nm) and channel 3 (excitation 488 nm, emission
650 nm). The data represents the average of two experiments.

S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
5487
linkers based on para-aminobenzylalcohol and ethylenediamine
have shown promising results in vitro and in vivo.14,15,42,43 These
linkers undergo spontaneous drug release and may be more appro-
priate for paclitaxel delivery. However, drug release will depend on
the subcellular localization of the conjugate. As such, effective link-
ers for one targeting ligand may not be suitable for another. In
addition, further studies need to be performed to determine abso-
lute uptake of paclitaxel in order to assure that this is not limiting
the conjugate’s effectiveness.
4.4. Synthesis of H2009.1 10-mer tetrameric peptide (compound
2)
H2009.1 10-mer tetrameric peptide with a free thiol group was
obtained by convergent synthesis followed by deprotection of the
acetamidomethyl-cysteine to reveal a free thiol as previously de-
scribed.37 The peptide was purified by reverse phase HPLC using
a Vydac PR-C18 column (250 mm ꢁ 22 mm, 10
lm) on a Breeze™
HPLC (Water Inc.) with eluents of H2O/0.1% TFA (eluent A) and ace-
tonitrile/0.1% TFA (eluent B). The following elution profile (referred
to as Method B) was utilized: 0–1 min, 90% A, 10% B; 1–61 min,
eluent B was increased from 10% to 40% at a flow rate of
10 mL/min. Elution of the peptides was monitored by UV absor-
bance at 220 nM. The peptide mass was confirmed by matrix
assisted laser desorption ionization time of flight mass spectrome-
try (MALDI MS) in linear mode using sinapinic acid as matrix on a
Voyager DE™ Pro instrument (Applied Biosystems Inc., Foster City,
CA). Compound 1 (C364H652N92O130S5): MALDI MH+ (average mass
calculated/found: 8557.94/8558.38).
4. Experimental
4.1. Materials
All reagents, unless specified, were of analytical grade and pur-
chased commercially. Paclitaxel was obtained from LC Laboratories
(Woburn, MA). N,N0-Dicyclohexyl-carbodiimide (DCC) and N-
Methylmorpholine (NMM) were purchased from ACROS Organics
(Geel, Belgium). All Fmoc-protected amino acids and 2-(1H-benzo-
4.5. Conjugation of H2009.1 10mer tetrameric peptide to 20-
maleimido-paclitaxel (compound 3)
triazole-l-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HBTU) were purchased from EMD Biosciences Inc. (San Diego,
CA). Anhydrous Hydroxybenzotriazole (HOBt) was purchased from
SynBioSci (Livermore, CA). 3-Maleimidopropionic, Piperidine,
Porcine liver esterase, and rat serum were purchased from
Sigma–Aldrich Inc. (St. Louis, MO).
A solution of compound 1 (17.1 mg, 2
0.8 mL Ar-purged 1ꢁ PBS/0.01 M EDTA. A solution of compound
2 (3.0 mg, 3 mol) in 0.2 mL DMF was added to the peptide solu-
lmol) was prepared in
l
tion. The reaction mixture was stirred at RT for 30 min. The prod-
uct, H2009.1 10-mer tetrameric peptide–paclitaxel conjugate
(compound 3) was then purified with RP-HPLC using Method A
4.2. Cell lines
and characterized by MALDI Mass Spectra. Compound
(C418H708N94O147S5): MALDI MH+ (average mass calculated/found:
9562.96/9561.75).
3
Human lung cancer cell lines were obtained from the Hamon
Center for Therapeutic Oncology Research (UTSW) and were main-
tained according to standard protocols.44 RPMI1640 was purchased
from Mediatech (Herndon, VA). Fetal Bovine Serum (FBS) was pur-
chased from Gemini Bio-products (Woodland, CA). Enzyme-free Cell
Dissociation buffer was from Invitrogen (Grand Island, NY). R5
media consists of RPMI supplemented with 5% FBS.
4.6. Synthesis of H2009.1 peptide–paclitaxel–FITC (compound 4)
The maleimido tetrameric core for fluorescein isothiocyanate
(FITC) labeling was synthesized on Fmoc-b-Ala-CLEAR™ Acid Resin
(substitution level 0.52 mmol/g) using standard protocols. Fmoc-
Lys(Boc)-OH and Fmoc-Cys(Acm)-OH was coupled at a 5-fold ex-
cess using HBTU, HOBt and NMM coupling (45 min). Piperidine
in DMF (20%) was employed to remove N-terminal Fmoc protect-
ing groups. Fmoc-Lys(Fmoc)-OH and 3-maleimidopropionic acid
was coupled in the same fashion. Upon completion of the synthe-
sis, the maleimido tetrameric core was cleaved from the resin
using a TFA:triisopropylsilane:H2O cocktail (95%:2.5%:2.5%) and
precipitated in cold diethyl ether. The crude tetrameric core was
purified by reverse phase HPLC using Method B.
4.3. Synthesis of 20-maleimido-paclitaxel (compound 1)
Paclitaxel (8.6 mg, 10
propionic acid (1.0 mg, 6
(4.1 mg, 20 mol) in 500 lL
l
mol) was incubated with 3-Maleimido-
mol) and dicyclohexylcarbodiimide
anhydrous dichloromethane for
l
l
12 h at RT (Scheme 1). Dicyclohexylurea was removed by filtra-
tion. The organic solvent was removed under vacuum and the so-
lid was resuspended in 1 mL CH3OH:H2O (50:50 V/V) containing
0.1% trifluoroacetic acid. The reaction mixture was purified by
reverse phase HPLC with eluents of H2O/0.1% TFA (eluent A)
and acetonitrile/0.1% TFA (eluent B). The following elution profile
(referred to as Method A) was utilized: 0–1 min, 70% A, 30% B;
1–71 min, eluent B was increased from 30% to 100% at a flow rate
of 10 mL/min. Elution of the conjugate was monitored by UV
absorbance at 220 nM. The purified conjugate was charac-
terized by MALDI Mass Spectra and 1H NMR. Compound 2
(C54H56N2O17): MALDI MNa+ (monoisotopic mass calculated/
found: 1027.36/1027.19). 1H NMR reported in ppm (CDCl3):
1.82 (s, 1H, 1-OH); 5.68 (d, 1H, H-2); 3.80 (d, 1H, H-3); 2.45 (s,
3H, 4-OAc); 4.97 (d, 1H, H-5); 2.54 (m, 1H, Ha-6); 1.98 (m, 1H,
Hb-6); 4.43 (m, 1H, H-7); 2.40 (d, 1H, 7-OH); 6.28 (s, 1H, H-10);
2.23 (s, 3H, 10-OAc); 6.19 (t, 1H, H13); 2.20–2.30 (band, 2H,
H14); 1.24 (s, 3H, Me-16); 1.14 (s, 3H, Me-17); 1.68 (s, 3H, Me-
18); 1.93 (s, 3H, Me-19); 4.32 (d, 1H, Ha-20); 4.21 (d, 1H, Hb-
20); 5.47 (d, 1H, H-20); 6.06 (dd, 1H, H-30); 6.98 (d, 1H, 30-NH);
7.62–8.13 (m, 5H, C2-OBz); 7.32–7.52 (m, 5H, C30-Ph); 7.49–
7.77 (m, 5H, C30-NBz) and 2.75 (t, 2H); 3.84 (t, 2H); 6.51 (s, 2H)
contributed to 3-maleimidopropionic acid.
The free
was labeled with FITC. A solution of maleimido tetrameric core
with Lys ( -NH2) (2.9 mg, 2 mol) was prepared in 0.5 mL 0.1 M
borate buffer (pH 9.3). A solution of FITC (1.2 mg, 3 mol) in
e-amino group of Lys on the maleimido tetrameric core
e
l
l
0.5 mL DMF was added to the tetrameric core solution. The reac-
tion mixture was stirred at RT in the dark for 3 h and purified by
reverse phase HPLC using Method B. The FITC labeled maleimido
tetrameric core was applied for convergent synthesis of H2009.1
10-mer tetrameric peptide and paclitaxel conjugation as described
above.
4.7. In vitro release of paclitaxel from H2009.1 10-mer
tetrameric peptide–paclitaxel conjugate
Compound 3 was dissolved in phosphate-buffered solutions
(PBS, 0.01 M) at pH 7.4, or esterase (18 u/mL) in PBS, or rat serum,
individually. The solutions were incubated at 37 °C. At time points
3, 8, 24, 48, 72, 96 h during incubation, aliquots were removed and
mixed with 100 lL of ethanol to precipitate the serum proteins.

5488
S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
The mixture was centrifuged at 3000 rpm for 5 min. The pellets
were resuspended with 80% ethanol, followed by centrifugation
at 3000 rpm again for 5 min. The pellets were rinsed and washed
one more time. Then the pooled supernatants were analyzed by
HPLC to determine the amounts of paclitaxel liberated from the
conjugate. Paclitaxel was quantified by HPLC using a standard
curve prepared with authentic paclitaxel. UV–Vis detection at
220 nm was used for data collection and analysis.
peptide–paclitaxel conjugate at different concentrations for
10 min. Drugs were removed and the cells washed with R5 three
times followed by recovering for 24 or 48 h. At the end of each
treatment, cells were collected, resuspended in 0.5 mL PBS and
fixed in 70% ethanol for 12 h at 4 °C. Ethanol-suspended cells
were centrifuged at 3000 rpm for 5 min and washed twice in
PBS to remove residual ethanol. Cell pellets were suspended in
1 mL of PBS containing 0.02 mg/mL of PI, 0.5 mg/mL of DNase-
free RNase A and incubated at 37 °C for 1 h. Cell cycle profiles
were studied using CellQuanta™ flow cytometer and data were
analyzed by WinMDI 2.9 software.
4.8. Cell viability assay
H2009 and H460 cells were seeded in 96 well culture plates at
1000 cells/well in a volume of 50
lL R5 medium and allowed to ad-
4.12. Apoptosis assay
here overnight at 37 °C in humidified atmosphere of 5% CO2. Paclit-
axel or H2009.1 peptide–paclitaxel conjugate or scrambled
peptide–paclitaxel conjugate were added to quadruplicate wells
Drug induced apoptosis was analyzed by VybrantÒ FAM Poly
Caspases Assay which detects activated caspases 1, 3, 4, 5, 6, 7, 8,
and 9 (Invitrogen, Carlsbad, CA). Briefly, H2009 cells were plated
in 50
(ranging from 1 nM to 1
tration). Untreated control cells received 50
l
L R5 media to produce the final concentrations indicated
M, all based on total paclitaxel concen-
L R5 only and were
l
and exposed to 1 lM drug for 10 min followed by different recov-
l
ery times. Both suspended and adherent cells were collected and
subjected to caspases/PI staining using VybrantÒ FAM Poly Caspas-
es Assay Kit according to the protocol provided by the manufac-
cultured as a set of eight replicate wells per plate. After 10 min
of exposure to the drug, media was aspirated from all wells, and
the wells were washed four times with 200
lL R5 before contin-
turer. Stained cells were analyzed on a CellQuanta™ flow
uing the culture in 100 L R5 for varying periods of time (from
l
cytometer. Data were analyzed by WinMDI 2.9 software.
24 to 120 h). At the end of the post-treatment incubation cell
viability was assessed using CellTiter Glo™ reagent (Promega,
Madison, WI). Luminescence was detected using a plate reader
30–60 min after the addition of the CellTiter Glo™ reagent.
4.13. In vivo tumor growth
Female NOD/SCID mice were injected with 1 million H2009
cells in the right flank. At day 18, after palpable tumors had
formed, mice (5 per group) were injected via tail vein with PBS,
5 mg/kg of free paclitaxel, or 5 mg/kg of H2009.1 tetrameric pep-
tide–paclitaxel. The mice were treated every 3 days for a total of
5 injections. Tumor growth was determined by measuring tumor
length and width with calipers, and tumor volume was calculated
using the formula v = (l ꢁ w2)/2, where l is length and w is width.
4.9. Binding of H2009.1 tetrameric peptide–paclitaxel–FITC by
flow cytometry
Approximately 100,000 cells were seeded per well in 12 well
culture plates and allowed to adhere overnight. Cells were
incubated for 1 h with 1 lM H2009.1 10mer tetrameric peptide–
paclitaxel–FITC or scrambled peptide–paclitaxel–FITC. Cells were
washed four times with PBS+ containing 0.1% BSA, followed by 2
brief rinses with 20 mM HCl–glycine, pH 2.2, 150 mM NaCl, and a
final one with PBS. Cells were removed from the wells by incuba-
tion on ice for 30 min in 1 mL/well of Enzyme-free Cell Dissociation
Buffer. Cells were scraped from the plate and prepared as a single
cell suspension by passage through a 27 gauge needle. FITC–
peptide–paclitaxel binding was assessed for 10,000 cells per
treatment group by flow cytometry using a CellQuanta™ flow
cytometer. Cells were gated by size and side scatter properties,
Acknowledgments
This work was supported by the Welch Foundation (I1622 to
K.C.B.) and a Career Development Award to K.C.B. from the Special-
ized Program of Research Excellence in Lung Cancer (grant
P50CA70907). B.P.G. is supported by a fellowship from the Cancer
Research and Prevention Institute of Texas (RP 101496).
Supplementary data
and FITC assayed by fluorescence in channel
395 nm, emission 488 nm).
1 (excitation
Supplementary data (the 1HNMR of compound 1 and analytical
HPLC traces for compounds 3 and 4) associated with this article
can be found, in the online version, at doi:10.1016/j.bmc.
2011.07.046.
4.10. Drug competition assay
H2009 cells were plated at 1000 cell/well in 96-well plates
1 day prior to the treatment to allow the cells to adhere to the
References and notes
plate. The next day, the cells were pre-incubated with 10
H2009.1 10-mer tetrameric peptide for 1 h then 1 M H2009.1
10mer tetrameric peptide–paclitaxel conjugate was added. After
10 min, cells were washed three times with 200 L R5 before
continuing culture in 100 L R5 for 120 H. Cell viability was ana-
lM
l
1. Jemal, A.; Siegel, R.; Xu, J.; Ward, E. CA Cancer J. Clin. 2010, 60, 277.
2. Ibrahim, N. K.; Desai, N.; Legha, S.; Soon-Shiong, P.; Theriault, R. L.; Rivera, E.;
Esmaeli, B.; Ring, S. E.; Bedikian, A.; Hortobagyi, G. N.; Ellerhorst, J. A. Clin.
Cancer Res. 2002, 8, 1038.
3. Henderson, I. C.; Bhatia, V. Expert Rev. Anticancer Ther. 2007, 7, 919.
4. Safavy, A. Curr. Drug Deliv. 2008, 5, 42.
l
l
lyzed as described in Section 4.8.
5. Quiles, S.; Raisch, K. P.; Sanford, L. L.; Bonner, J. A.; Safavy, A. J. Med. Chem. 2009,
53, 586.
6. Safavy, A.; Bonner, J. A.; Waksal, H. W.; Buchsbaum, D. J.; Gillespie, G. Y.;
Khazaeli, M. B.; Arani, R.; Chen, D.; Carpenter, M.; Raisch, K. P. Bioconjug. Chem.
2003, 14, 302.
7. Safavy, A.; Georg, G. I.; Velde, D. V.; Raisch, K. P.; Safavy, K.; Carpenter, M.;
Wang, W.; Bonner, J. A.; Khazaeli, M. B.; Buchsbaum, D. J. Bioconjug. Chem.
2004, 15, 1264.
8. Wu, A. M.; Senter, P. D. Nat. Biotech. 2005, 23, 1137.
9. Gilbert, C. W.; McGowan, E. B.; Seery, G. B.; Black, K. S.; Pegram, M. D. J. Exp.
Ther. Oncol. 2003, 3, 27.
4.11. Cell cycle analysis
Cell cycle perturbations induced by H2009.1 10mer tetra-
meric peptide–paclitaxel conjugate were analyzed by propidium
iodide (PI) DNA staining with flow cytometric analysis. H2009
cells were seeded in 12 well culture plates at approximately
100,000 cells per well and allowed to adhere overnight. Cells
were treated with paclitaxel or H2009.1 10mer tetrameric

S. Li et al. / Bioorg. Med. Chem. 19 (2011) 5480–5489
5489
10. Chung, C. H.; Mirakhur, B.; Chan, E.; Le, Q.-T.; Berlin, J.; Morse, M.; Murphy, B.
A.; Satinover, S. M.; Hosen, J.; Mauro, D.; Slebos, R. J.; Zhou, Q.; Gold, D.; Hatley,
T.; Hicklin, D. J.; Platts-Mills, T. A. E. N. Engl. J. Med. 2008, 358, 1109.
11. Reubi, J. C.; Maecke, H. R. J. Nucl. Med. 2008, 49, 1735.
12. Chen, K.; Conti, P. S. Adv. Drug Deliv. Rev. 2010, 62, 1005.
13. Bray, B. L. Nat. Rev. Drug Discov. 2003, 2, 587.
28. Bates, R. C.; Bellovin, D. I.; Brown, C.; Maynard, E.; Wu, B.; Kawakatsu, H.;
Sheppard, D.; Oettgen, P.; Mercurio, A. M. J. Clin. Invest. 2005, 115, 339.
29. Breuss, J. M.; Gallo, J.; DeLisser, H. M.; Klimanskaya, I. V.; Folkesson, H. G.;
Pittet, J. F.; Nishimura, S. L.; Aldape, K.; Landers, D. V.; Carpenter, W.; Gillette,
M.; Sheppard, D.; Matthay, M. A.; Albelda, S. M.; Kramer, R. H.; Pytela, R. J. Cell
Sci. 1995, 108, 2241.
14. Dubikovskaya, E.; Thorne, S.; Pillow, T.; Contag, C.; Wender, P. Proc. Natl. Acad.
Sci. U.S.A. 2008, 105, 12128.
30. Kawashima, A.; Tsugawa, S.; Boku, A.; Kobayashi, M.; Minamoto, T.; Nakanishi,
I.; Oda, Y. Pathol. Res. Pract. 2003, 199, 57.
15. Yoneda, Y.; Steiniger, S. C. J.; Capková, K.; Mee, J. M.; Liu, Y.; Kaufmann, G. F.;
Janda, K. D. Bioorg. Med. Chem. Lett. 2008, 18, 1632.
31. Hamidi, S.; Salo, T.; Kainulainen, T.; Epstein, J.; Lerner, K.; Larjava, H. Br. J.
Cancer 2000, 82, 1433.
16. Ryppa, C.; Mann-Steinberg, H.; Biniossek, M. L.; Satchi-Fainaro, R.; Kratz, F. Int.
J. Pharm. 2009, 368, 89.
32. Hazelbag, S.; Kenter, G.; Gorter, A.; Dreef, E.; Koopman, L.; Violette, S.; Weinreb,
P.; Fleuren, G. J. Pathol. 2007, 212, 316.
17. Papas, S.; Akoumianaki, T.; Kalogiros, C.; Hadjiarapoglou, L.; Theodoropoulos, P.
A.; Tsikaris, V. J. Pept. Sci. 2007, 13, 662.
33. Hecht, J.; Dolinski, B.; Gardner, H.; Violette, S.; Weinreb, P. Appl.
Immunohistochem. Mol. Morphol. 2008, 16, 543.
18. Huang, C.-M.; Wu, Y.-T.; Chen, S.-T. Chem. Biol. 2000, 7, 453.
19. Safavy, A.; Raisch, K. P.; Khazaeli, M. B.; Buchsbaum, D. J.; Bonner, J. A. J. Med.
Chem. 1999, 42, 4919.
20. Safavy, A.; Raisch, K.; Matusiak, D.; Bhatnagar, S.; Helson, L. Bioconjug. Chem.
2006, 17, 565.
34. Jones, J.; Watt, F. M.; Speight, P. M. J. Oral Pathol. Med. 1997, 26, 63.
35. Regezi, J.; Ramos, D.; Pytela, R.; Dekker, N.; Jordan, R. Oral Oncol. 2002, 38, 332.
36. Thomas, G.; Nystrom, M.; Marshall, J. J. Oral Pathol. Med. 2006, 35.
37. Li, S.; Lin, M.; Liu, Y.-H.; Sun, X.; McGuire, M. J.; Brown, K. C. Mol. Cancer Ther.
2009, 8, 1239.
21. Guillemard, V.; Nedev, H. N.; Berezov, A.; Murali, R.; Saragovi, H. U. DNA Cell
Biol. 2005, 24, 351.
38. Deutsch, H. M.; Glinski, J. A.; Hernandez, M.; Haugwitz, R. D.; Narayanan, V. L.;
Suffness, M.; Zalkow, L. H. J. Med. Chem. 1989, 32, 788.
22. Kim, Y.; Lillo, A. M.; Steiniger, S. C. J.; Liu, Y.; Ballatore, C.; Anichini, A.;
Mortarini, R.; Kaufmann, G. F.; Zhou, B.; Felding-Habermann, B.; Janda, K. D.
Biochemistry 2006, 45, 9434.
23. Oyama, T.; Sykes, K. F.; Samli, K. N.; Minna, J. D.; Johnston, S. A.; Brown, K. C.
Cancer Lett. 2003, 202, 219.
39. Mathew, A. E.; Mejillano, M. R.; Nath, J. P.; Himes, R. H.; Stella, V. J. J. Med. Chem.
1992, 35, 145.
40. Nicolaou, K.; Dai, W.-M.; Gut, R. Angew. Chem., Int. Ed. Engl. 1994, 33, 15.
41. Wang, S.; Zhelev, N. Z.; Duff, S.; Fischer, P. M. Bioorg. Med. Chem. Lett. 2006, 16,
2628.
24. Elayadi, A. N.; Samli, K. N.; Prudkin, L.; Liu, Y. H.; Bian, A.; Xie, X. J.; Roth, J. A.;
Wistuba, I. I.; McGuire, M. J.; Brown, K. C. Cancer Res. 2007, 67, 5889.
25. Prudkin, L.; Liu, D.; Ozburn, N.; Sun, M.; Behrens, C.; Tang, X.; Brown, K.; Bekele,
B.; Moran, C.; Wistuba, I. Mod. Pathol. 2009, 22, 668.
26. Ahmed, N.; Riley, C.; Rice, G. E.; Quinn, M. A.; Baker, M. S. J. Histochem.
Cytochem. 2002, 50, 1371.
42. Kumar, S. K.; Williams, S. A.; Isaacs, J. T.; Denmeade, S. R.; Khan, S. R. Bioorg.
Med. Chem. 2007, 15, 4973.
43. Ajaj, K. A.; Biniossek, M. L.; Kratz, F. Bioconjug. Chem. 2009, 20, 390.
44. Phelps, R. M.; Johnson, B. E.; Ihde, D. C.; Gazdar, A. F.; Carbone, D. P.; Linnoila,
P.; Matthews, M. J.; Bunn, P. A.; Carney, D.; Minna, J. D.; Mulshine, J. L. J. Cell.
Biochem. 1996, 32.
27. Arihiro, K.; Kaneko, M.; Fujii, S.; Inai, K.; Yokosaki, Y. Breast Cancer 2000, 7, 19.