Development of a peptide-drug conjugate for prostate cancer therapy

Article abstract of DOI:10.1021/mp200007b

TGX-221 is a highly potent phosphoinositide 3-kinase β (PI3Kβ) inhibitor that holds great promise as a novel chemotherapeutic agent to treat prostate cancer. However, poor solubility and lack of targetability limit its therapeutic applications. The objective of this present study is to develop a peptide-drug conjugate to specifically deliver TGX-221 to HER2 overexpressing prostate cancer cells. Four TGX-221 derivatives with added hydroxyl groups were synthesized for peptide conjugation. Among them, TGX-D1 exhibited a similar bioactivity to TGX-221, and it was selected for conjugation with a peptide promoiety containing a HER2-targeting ligand and a prostate specific antigen (PSA) substrate linkage. From this selection, the peptide-drug conjugate was proven to be gradually cleaved by PSA to release TGX-D1. Cellular uptake of the peptide-drug conjugate was significantly higher in prostate cancer cells compared to the parent drug. Moreover, both the peptide-drug conjugate and its cleaved products demonstrated comparable activities as the parent drug TGX-D1. Our results suggest that this peptide-drug conjugate may provide a promising chemotherapy for prostate cancer patients.

Full text of DOI:10.1021/mp200007b

ARTICLE
pubs.acs.org/molecularpharmaceutics
Development of a PeptideꢀDrug Conjugate for
Prostate Cancer Therapy
Wanyi Tai,† Ravi S. Shukla,† Bin Qin,† Benyi Li,‡ and Kun Cheng*,†
†Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri—Kansas City, 2464 Charlotte Street, Kansas City,
Missouri 64108, United States
‡Department of Urology, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, United States
ABSTRACT: TGX-221 is a highly potent phosphoinositide
3-kinase β (PI3Kβ) inhibitor that holds great promise as a novel
chemotherapeutic agent to treat prostate cancer. However, poor
solubility and lack of targetability limit its therapeutic applica-
tions. The objective of this present study is to develop a
peptideꢀdrug conjugate to specifically deliver TGX-221 to
HER2 overexpressing prostate cancer cells. Four TGX-221
derivatives with added hydroxyl groups were synthesized for
peptide conjugation. Among them, TGX-D1 exhibited a similar bioactivity to TGX-221, and it was selected for conjugation with a
peptide promoiety containing a HER2-targeting ligand and a prostate specific antigen (PSA) substrate linkage. From this selection,
the peptideꢀdrug conjugate was proven to be gradually cleaved by PSA to release TGX-D1. Cellular uptake of the peptideꢀdrug
conjugate was significantly higher in prostate cancer cells compared to the parent drug. Moreover, both the peptideꢀdrug conjugate
and its cleaved products demonstrated comparable activities as the parent drug TGX-D1. Our results suggest that this peptideꢀdrug
conjugate may provide a promising chemotherapy for prostate cancer patients.
KEYWORDS: TGX-221, prostate cancer, prodrug, peptide ligand, HER2, PSA
’ INTRODUCTION
prostate cancer progression.16 The chemical inhibition or genetic
knockout of PI3Kβ is believed to prevent AR-dependent gene
expression and tumor growth. Given all of the evidence described
above, PI3Kβ is considered an attractive target for the develop-
ment of novel anticancer agents for prostate cancer, which is the
most common malignance in American men.
TGX-221 is a novel, potent, isoform-specific, and cell-perme-
able small molecule inhibitor of PI3Kβ. The inhibition effect of
TGX-221 on PI3Kβ is about 1000-fold over PI3KR and PI3Kγ,
and 20-fold over PI3Kδ.17 TGX-221 has been successfully used
in animal models to suppress the PI3Kβ activity in vivo.18 However,
the therapeutic potential of TGX-221 in cancer therapy is halted
due to its poor solubility and the wide distribution of PI3Kβ in vivo.
Organic solvent is generally required for intravenous administration
of TGX-221, which may cause significant toxicities.18 Addition-
ally, widely distributed PI3Kβ plays important roles in cell metabo-
lism, growth and development.19 For example, knockdown of
PI3Kβ leads to early embryonic lethality,20 glucose intolerance15
and platelet disaggregation.21 As a result, an improved solubility
and the targeted delivery of TGX-221 to tumor cells are two
essential prerequisites for its successful application in prostate
cancer therapy.
Phosphoinositide 3-kinases (PI3Ks) are lipid enzymes that phos-
phorylate the 30-OH of the inositol ring of phosphoinositides. It
has been established that PI3Ks play a pivotal role in cell growth,
proliferation, and survival,1 and PI3Ks have been divided into
three classes (I, II, and III) according to their different structural
features and in vitro lipid substrate specificities.2,3 Interestingly,
class I PI3Ks can be further divided into two subclasses (class IA
p110R/β/δ and class IB p110γ).1 Recent emerging evidence
indicates that PI3K isoforms (p110R/β/δ) possess an oncogenic
potential through gain-of-function mutations or gene amplifi-
cations.4 Their major PI3K downstream signaling effector, pro-
tein kinase B (PKB, also known as Akt), has been found to be
overactivated in human cancers, such as prostate cancer.5ꢀ7 How-
ever, a negative regulator of the PI3K signaling pathway, phos-
phatase and tensin homologue (PTEN), has long been reported
to be inactive in most human cancers, including prostate cancer.8ꢀ10
Conditional knockout of PTEN in mouse prostate epithelium
resulted in intraepithelial neoplasia.11,12 PTEN-null cancers repre-
senta significant portion (30ꢀ50%) of all prostate cancer cases,13
and they are always associated with an unfavorable prognosis.14
In PTEN-null prostate cancers, PI3Kβ (PI3K-p110β), rather
than PI3KR, has been proven to contribute to tumorigenesis and
androgen-independent progression.15 Recently, for the first time,
we have demonstrated that PI3Kβ is essential for the androgen-
stimulated AR (androgen receptor) transactivation and cell pro-
liferation.16 We also observed high expression of this isoform in
malignant prostate tissues compared to nonmalignant compart-
ments, and the expression level was significantly correlated with
Peptideꢀdrug conjugation is a promising strategy for the
targeted delivery of chemotherapy drugs. Unlike an antibodyꢀdrug
Received: January 5, 2011
Accepted: April 21, 2011
Revised:
April 13, 2011
Published: April 21, 2011
r
2011 American Chemical Society
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ARTICLE
conjugate, a peptideꢀdrug conjugate is small in size, which results
in excellent cell permeability and a high drug-loading capability.
In our approach, a HER2 (human epidermal growth factor recep-
tor 2)-specific peptide was conjugated to TGX-D1, a TGX-221
derivative, and the drug was specifically delivered to the prostate
cancer cells. HER2, a well-known membrane receptor in breast
cancer, is also overexpressed in many prostate cancers.22 It has
been revealed that 25% of untreated primary prostate tumors,
59% of localized tumors after hormone treatment, and 78% of
castrate metastatic tumors overexpress the HER2 protein.23ꢀ25
Although the monoclonal anti-HER2 antibody has not shown a
significant therapeutic effect in prostate cancer,26 the overexpressed
HER2 on prostate cancers does provide a promising molecular
target for targeted drug delivery systems. To further increase the
tumor specificity, a PSA (prostate specific antigen) cleavable peptide
(SSKYQ) was incorporated as a linker between the anti-HER2
peptide and TGX-D1. PSA is a prostate tissue-specific serine pro-
tease that is often overexpressed in prostate cancer cells, but not
in other normal cells.27 In this study, the peptide conjugated TGX-
D1 was specifically delivered to prostate cancer cells, and then
cleaved by PSA to release the parent drug, which led to high
cellular uptake of TGX-D1 by tumor cells.
TGX-221: 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.61 (s,
1H), 7.13 (m, 2H), 6.68 (t, 1H), 6.45 (d, 2H), 5.63 (s, 1H), 5.15
(q, 1H), 3.80 (m, 4H), 3.65 (m, 5H), 2.25 (s, 3H), 1.54 (d, 3H);
ESI-MS calcd for C21H24N4O2 364.4, found 365.4.
TGX-D2. 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 7.57 (s,
1H), 7.08 (d, 1H), 7.00 (t, 1H), 6.68 (t, 1H), 6.25 (d, 1H), 5.65
(s, 1H), 5.18 (m, 1H), 3.80 (m, 6H), 3.65 (m, 6H), 2.63 (s, 1H),
1.6 (d, 3H); ESI-MS calcd for C23H28N4O3 408.5, found 409.3.
TGX-D3. 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), δ7.57
(s, 1H), δ7.09 (t, 1H), δ6.68 (d, 1H), δ6.55 (s, 1H), δ6.34 (d,
1H), δ5.66 (s, 1H), δ5.12 (m, 1H), δ3.93 (m, 1H), δ3.79 (m,
4H), δ6.67 (m, 4H), δ3.60 (m, 2H), δ3.27 (s, 3H), δ1.50 (d,
3H); ESI-MS calcd for C22H26N4O3 394.5, found 395.3.
1
TGX-D4. H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.60
(s,1H), 6.94 (d, 2H), 6.41 (d, 2H), 5.63 (s, 1H), 5.09 (q, 1H),
3.80 (m, 6H), 3.65 (m, 4H), 2.71 (t, 2H), 2.30 (s, 3H), 1.58 (d,
3H); ESI-MS calcd for C23H28N4O3 408.5, found 409.4.
Synthesis of Compound 5. Methyl bromoacetate (5 mmol)
and N,N-diisopropylethylamine (5 mmol) were added into a
solution of TGX-221 (364 mg, 1 mmol) in CH3CN (50 mL), and
the reaction mixture was stirred at 85 °C for 24 h. After cooling to
room temperature, the reaction mixture was concentrated under
vacuum. The remaining brown residue was dissolved with CHCl3,
then washed with water and brine, and dried over Na2SO4. After
concentration, the residue was purified by silica gel chromatog-
’ EXPERIMENTAL SECTION
1
raphy (chloroform/methanol, 100/1, yield ≈60%): H NMR
Materials. All starting reagents listed below were obtained
from commercial sources and used without further purification.
2-Amino-3-bromo-5-methylpyridine was purchased from Oak-
wood Products, Inc. (West Columbia, SC). Piperidine, malonyl
dichloride, methanesulfonyl chloride, aniline, 4-aminophenethyl
alcohol, 2-aminophenethyl alcohol, 3-aminobenzyl alcohol and
butyl vinyl ether were obtained from Sigma-Aldrich (St Louis,
MO). Morpholine was purchased from Ricca chemical company
(Arlington, Texas). Triisopropylsilane (TIPS), trifluoroacetic
acid (TFA), triethylamine, N,N-diisopropylethylamine (DIPEA),
4-dimethyl-aminopyridine (DMAP), and dichloro 1,10-bis-
(diphenylphosphino)ferrocene palladium (PdCl2(dppf)) were
purchased from Acros Organics (Morris Plains, NJ). 2-(7-Aza-1
H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU) was ordered from Genscript Inc.
(Piscataway, NJ). Thiazolyl Blue was obtained from RPI Corp.
(Prospect, IL). All the amino acids used in solid phase peptide
synthesis were obtained from Anaspec Inc. (Fremont, CA). All the
organic solvents for synthesis and HPLC were ordered from
Fisher Scientific Inc. and used as received.
(400 MHz, CDCl3) δ 8.65 (s, 1H), 7.58 (s, 1H), 7.10 (t, 2H),
6.70 (t, 1H), 6.46 (d, 2H), 5.64 (s, 1H), 5.13 (q, 1H), 4.20 (s, 3H),
3.80 (m, 4H), 3.65 (m, 6H), 2.25 (s, 3H), 1.57 (d, 3H); ESI-MS
calcd for C24H28N4O4 436.5, found 437.3.
Synthesis of TGX-D1. LiCl (5 mmol) and NaBH4 (5 mmol)
were added to a solution of compound 5 (1 mmol) in THF
(10 mL). After stirring at room temperature for 10 min, 20 mL of
anhydrous ethanol was added to the reaction mixture. After
twelve hours, an additional 5 mmol of LiCl and NaBH4 were added
to the reaction mixture and stirred for another 12 h. The reaction
was quenched with saturated NH4Cl aqueous solution and then
extracted with CHCl3. The organic layer was washed with water
and brine and dried over Na2SO4. After concentration, the residue
was purified by silica gel chromatography (chloroform/methanol,
50/1, yield ≈50%): 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H),
δ 7.43 (s, 1H), δ 7.20 (t, 2H), δ 6.86 (d, 2H), δ 6.76 (t, 1H), δ 5.60
(s, 1H), δ 3.81 (t, 1H), δ 3.60 (m, 5H), δ 3.51 (m, 2H), δ 3.42 (t,
2H), δ 3.37 (m, 2H), δ 2.33 (s, 3H), δ 1.63 (d, 2H); ESI-MS
calcd for C23H28N4O3 408.5, found 409.5.
Synthesis of TGX-221, TGX-D2, TGX-D3 and TGX-D4.
Compounds 2, 3, and 4 were synthesized from commercially avail-
able 2-amino-3-bromo-5-methylpyridine (1) following Jackson’s
report.28 For the synthesis of TGX-221 and its derivatives, com-
pound 4 (1 mmol) was suspended in 20 mL of dry toluene. After
addition of 3 g of molecular sieves (4 Å) and 3 mmol of aniline or
its derivatives, the reaction was refluxed for 4 h under nitrogen.
The molecular sieves were removed, and the filtrate was concen-
trated under vacuum. The residue was redissolved in methanol,
followed by addition of 20 mg of NaBH4 at 0 °C. The reaction
was continuously stirred for another hour at room temperature.
After the reaction was quenched with 10 mL of saturated NH4Cl
aqueous solution, the product was extracted with CHCl3. The
organic phase was then washed with brine, dried and concen-
trated under vacuum. The final product was purified by silica gel
chromatography (CHCl3/methanol, 50/1) to give a yield of
50ꢀ80%.
Synthesis of NH2-L-TGX. Fmoc-Leu-OH (100 mg) and EDCI
(50 mg) were added to TGX-D1 (1 mmol) in dichloromethane
(DCM), and the reaction was initiated by adding DMAP and
stirring at room temperature overnight. After that time, water was
added to the reaction, and the organic phase was separated, washed,
and dried with Na2SO4. The intermediate was purified by silica
gel chromatography (CHCl3/methanol, 100/1). To remove the
Fmoc protecting groups, the intermediate was dissolved in
1.8 mL of DCM, followed by adding 200 μL of piperidine. The
reaction mixture was stirred at room temperature for 30 min. Finally,
the solvent was evaporated under vacuum, and the residue was
purified with silica gel chromatography (CHCl3/ethanol, 50/2.5):
ESI-MS calcd for C29H39N5O4 521.6, found 522.4.
Synthesis of NH2-SL-TGX. HATU (10 mg) and Boc-Ser-
(OtBu)-OH (20 mg) were dissolved in 5 mL of dried DMF under
nitrogen, followed by adding 10 μL of DIPEA. After 10 min, NH2-
L-TGX was added to initiate the coupling reaction. After 12 h,
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ARTICLE
the organic solvent was evaporated and the residue was purified
by silica gel chromatography. The purified intermediate was then
dissolved in the mixture of TFA/DCM/TIPS (v/v/v, 50/45/5).
The mixture was stirred for 3 h, followed by concentration and puri-
fication with HPLC (abs 268 nm). ESI-MS: calcd for C32H44N6O6
608.7, found 609.6.
R1881 (1.0 nM). TGX-221 and TGX-D1 were added to the cells
and incubated for 24 h. After washing with PBS, the cells were
lysed, and the total protein concentration of the cell lysate was
measured by a BCA protein assay (Pierce Biotech., IL). Lucifer-
ase activity of each sample was determined and normalized to the
protein concentration.
Synthesis of the PeptideꢀDrug Conjugate. Undeprotected
Peptide Synthesis. All undeprotected peptide intermediates were
synthesized by an Fmoc solid-phase peptide synthesis method.
The resin used for peptide synthesis was 2-chlorotrityl resin. HATU
was used as the coupling reagent, and DIPEA served as the base.
The undeprotected peptides were cleaved from the resin by acetic
acid/trifluoroethanol/DCM (v/v/v, 10/20/70) at room tem-
perature after 2 h. After removal of the resin by filtration, the reaction
was concentrated under vacuum at room temperature. The crude
products were purified by small silica gel chromatography (CHCl3/
methanol, 50/2.5ꢀ50/10).
In Vitro Release of TGX-D1 from the PeptideꢀDrug Con-
jugate. Drug Release from NH2-SL-TGX. Stock solution of NH2-
SL-TGX (10 mM in DMSO, 0.5% formic acid) was diluted to
a final concentration of 100 μM with buffer (50 mM Tri-Cl,
100 mM NaCl, pH 7.4) and then incubated at 37 °C. At selected
time intervals, a 50 μL aliquot was removed and mixed with 50 μLof
0.1% TFA to quench the reaction. The samples were stored at
ꢀ30 °C for HPLC analysis.
Drug Release from Ac-SSKYQSL-TGX. Ac-SSKYQSL-TGX was
mixed with freshly prepared PSA solution (10 μg/mL) in buffer
(50 mM Tri-Cl, 100 mM NaCl, pH 7.4) to give a final concentration
of 100 μM. The mixture was incubated at 37 °C, and 50 μL aliquots
were quenched with an equal volume of 0.1% TFA in methanol at
selected time intervals. After incubation at room temperature for
10 min, the samples were centrifuged at 12000g for 10 min, and
the supernatant was collected for HPLC analysis.
Coupling and Deprotection. The undeprotected peptides
(0.02 mmol) and purified NH2-L-TGX-D1 (0.01 mmol) were
dissolved in 3 mL of dried DMF. Two equivalents of HATU
(0.02 mmol) and four equivalents of DIPEA (0.04 mmol) were
added to the solution. After stirring at room temperature overnight,
the reaction was quenched by a saturated NH4Cl aqueous sol-
ution. The solvent was then removed, and the brown residue was
reconstituted in 20 mL of CHCl3, followed by washing with water.
After concentration under vacuum, the crude product was purified
by silica gel chromatography (CHCl3/methanol, 50/2.5ꢀ50/10).
The residual protecting groups in the peptideꢀdrug conjugate
were removed by acidic cleavage. Briefly, the purified compound
was dissolved in a mixture of DCM and TFA (1 mL/1 mL) and
stirred for 2 h at room temperature. To increase the yield, 50 μL
of scavenging agent such as triisopropylsilane (TIPS) was added
into the reaction. The reaction mixture was then concentrated,
and the final product was purified by reverse phase HPLC. The
molecular weight was confirmed by LC/MS.
The samples were assayed by a Waters HPLC system (717
plus autosampler, 600 controller pump, 486 tunable absorbance
detector) on a C-18 reverse phase column (4.6 ꢁ 250 mm, 5 μm).
The mobile phase was methanol/water (65/35, v/v) in 0.1% formic
acid, and the flow rate was set at 1.0 mL/min. Samples were tested as
30 μL injections, and the drug concentration was monitored by
UV detector at 268 nm.
Immunostaining. LNCaP cells cultured on 96-well plates
were fixed in ice-cold acetone/methanol (1/1, v/v) for 10 min,
followed by blocking with 1% BSA for 1 h. The cells were then
incubated with the biotinylated peptides for 1 h in TBST buffer
(0.1% Tween-20 in 0.1 M Tri-Cl, pH 7.4). After washing to
remove the unbound peptides, streptavidinꢀFITC (Pierce,
Rockford, IL) was added, and the cells were examined with a
Leica DMI3000 fluorescence microscope (Leica Microsystems
GmbH, Wetzlar, Germany).
Aqueous Solubility Assay. The aqueous solubilities of TGX-
221, TGX-D1 and KCC-TGX were assayed by the thermody-
namic method. Briefly, 1 mg of TGX-221, TGX-D1, or KCC-TGX
was suspended in 100 μL of water, and the mixture was shaken at
room temperature for 3 days. The undissolved drug crystals were
removed by centrifugation at 12000g for 10 min. The supernatant
was diluted, and the drug concentration was quantified by HPLC.
Stability Assay of KCC-TGX. The stability of KCC-TGX was
analyzed in PBS, cell culture medium and mouse serum. KCC-
TGX was diluted to 5 μM in PBS (pH 7.5), cell culture medium
(10% FBS in RMPI 1640) or 50% mouse serum. Next, the
solutions were incubated at 37 °C for their indicated time interval. A
20 μL aliquot was mixed with 60 μL of acetonitrile containing
0.1% formic acid to precipitate the proteins. The samples were
centrifuged at 12000g for 10 min, and 50 μL of the supernatant
was collected for HPLC quantification.
Cellular Uptake Study. LNCaP cells were plated in 24-well
plates at a density of 2 ꢁ 105 cells/well and incubated for 12 h
before the study. The cells were washed once with PBS and in-
cubated with freshly prepared drug solutions in RPMI-1640
medium at 37 °C. One hour after the incubation, the drug was re-
moved and the cells were washed three times with cold PBS. The
cells were then harvested and kept at ꢀ80 °C for the LCꢀMS/
MS assay. The total protein concentration of the cell lysate was
Ac-SSKYQSL-TGX: ESI-MS calcd for C60H85N13O16 1244.4,
found 1245.2.
Ac-KCCYSLGGGSSKYQSL-TGX-D1 (KCC-TGX): ESI-
MS calcd for C96H141N23O27S2 2113.4, found 705.5 (M þ 3)3þ
,
1056.4 (M þ 2)2þ
.
Cytotoxicity Assay. LNCaP cells were seeded in 96-well plates at
a density of 1 ꢁ 104 cells/well (DU145 cells were seeded at a
density of 3 ꢁ 103 cells/well due to its fast growth rate). After
24 h, TGX-221 and its derivatives (TGX-D1, TGX-D2, TGX-D3
and TGX-D4) were added, and the cells were incubated at 37 °C
for 72 h. The relative viable cell numbers were determined
using a MTT assay. IC50 was calculated by fitting a concentra-
tionꢀresponse curve using Graphpad Prism 5 (GraphPad Soft-
ware, Inc.).
Androgen Induced Proliferation Assay. LNCaP cells were
seeded in 96-well plates 12 h before the assay. The medium was
changed to serum-free medium containing 1 nM R1881 (a synthetic
androgen), followed by adding TGX-221 and its derivatives.
After incubation for 72 h, the proliferation rate was examined
by the Alamar Blue-based proliferation assay (Promega Corp.,
Madison, WI).
Androgen Induced Gene Expression Assay. The luciferase
reporter plasmid controlled by a synthetic androgen response
element (ARE-LUC) has been described previously.16 LNCaP
cells were seeded in 6-well plates and transfected with 1.0 μg of
ARE-LUC using Lipofectamine on the following day. After 24 h,
the medium was changed to RPMI-1640 containing 2% FBS and
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Figure 1. (A) Structure of the peptideꢀdrug conjugate KCC-TGX. KCC-TGX contains a HER2-targeting peptide, a GGG spacer, a PSA cleavable
substrate, a self-cyclizing linker and the parent drug TGX-D1. (B) The activation mechanism of KCC-TGX. KCC-TGX is expected to be cleaved at the
Gln-Ser site by PSA to release NH2-SL-TGX. The free amino group of Ser (S) in NH2-SL-TGX attacks the ester carbonyl group and releases the parent
drug TGX-D1. The dipeptide Ser-Leu self-cyclizes to form diketopiperazines (DKP).
quantified by a BCA protein assay kit (Pierce, Rockford, IL), and
the cellular uptake was normalized to the total amount of protein
for each sample.
ions were 409.1/272.1 for TGX-D1, and 365.1/272.1 for TGX-221.
The turbo ion spray setting and collision gas pressure were optimized
(IS voltage of 5500 V; temperature of 300 °C; nebulizer gas of
40 psi; curtain gas of 40 psi). Ion source parameters employed a
declustering potential (DP) of 31 V; collision energy (CE) of 16 V;
entrance potential (EP) of 5 V; and collision cell exit potential
(CXP) of 4 V. The detection limit of TGX-D1was 10 nM.
Statistical Analysis. Data were expressed as the mean ( stan-
dard deviation (SD). The difference between any two groups was
determined by ANOVA. P < 0.05 was considered statistically sig-
nificant.
In Vitro Competitive Receptor Binding Assay. To inhibit
the uptake of KCC-TGX by the anti-HER2 ligand Ac-KCCYSL,
LNCaP cells were preincubated with the indicated concentra-
tions of Ac-KCCYSL at 37 °C for 1 h. KCC-TGX was then added
and incubated with the cells for another 30 min. The cells were
finally washed and lysed as described above. Cellular uptake of
KCC-TGX was analyzed by LCꢀMS/MS.
LCꢀMS/MS Analysis of TGX-221 Derivatives and the Peptideꢀ
Drug Conjugate. Drug molecules were extracted from the
cells using a liquidꢀliquid extraction technique as reported.29
TGX-221 was used as the internal standard during the extraction
and analysis. Ethyl acetate (AcOEt) was utilized to extract the
drug from the aqueous layer. All samples were hydrolyzed to release
the parent drug TGX-D1 for LCꢀMS/MS analysis. Briefly, the
cells were lysed by three cycles of a freezeꢀthaw procedure, and
100 μL of the clear cell suspension was incubated with 34 μL of
4 N NaOH for 1 h. Phosphate buffer (68 μL, 2N) was then added
to neutralize the pH. After addition of the internal standard TGX-
221, each sample was vortexed with 200 μL of AcOEt for 5 min.
The organic layer was separated from the aqueous layer by cen-
trifugation, and 150 μL of the organic layer were transferred to a
tube and dried under vacuum. The residue was reconstituted with
50 μL of acetonitrile for LCꢀMS/MS analysis.
’ RESULTS
Structure of the PeptideꢀDrug Conjugate. The chemical
structure of the peptideꢀdrug conjugate (KCC-TGX) is de-
picted in Figure 1A. This peptideꢀdrug conjugate consists of five
parts: (i) the parent drug TGX-D1; (ii) a self-cyclizing linker (SL);
(iii) the PSA cleavable peptide (SSKYQ); (iv) a peptide spacer
(GGG); and (v) a HER2-targeting peptide (KCCYSL). The
peptideꢀdrug conjugate could be activated by PSA cleavage to
release the parent drug TGX-D1.
Figure 1B illustrates the release of TGX-D1 from KCC-TGX
upon binding to HER2 on prostate cancer cells. Briefly, KCC-TGX
was specifically delivered to the prostate cancer cells, and cleaved
by PSA to form NH2-SL-TGX, which was further cleaved by self-
cyclization to release the active parent drug TGX-D1. PSA’s en-
zymatic activity only exists in the microenvironment of the prostate
cancer cells, but not in systemic circulation due to the presence of
PSA inhibitors (R1-antichymotrypsin and R2-macroglobulin) in
LCꢀMS/MS analysis was performed on a QTrap 3200 LC/
MS/MS mass spectrometer equipped with a Shimadzu Promi-
nence HPLC system. Detection was operated in the multiple-
reaction monitoring (MRM) mode. The precursor and product
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Figure 2. Synthesis of TGX-221 and its derivatives. Reagents and conditions: (a) malonyl dichloride, DCM, rt; (b) CH3SO2Cl, Et3N, morpholine;
(c) (I) PdCl2(dppf), DIPEA, butyl vinyl ether; (II) 1 N HCl, rt; (d) (I) 4 Å MS, toluene, reflux; (II) NaBH4, methanol; (e) methyl bromoacetate,
DIPEA, CH3CN; (f) LiCl, NaBH4, THF/EtOH.
the blood.30 Therefore, the PSA-cleavable linker was expected to
remain stable in systemic circulation.
(IC50 = 2.67 μM) in LNCaP cells. Both TGX-221 and TGX-D1
showed a negligible activity in DU145 cells (IC50 > 100 μM).
TGX-D2, TGX-D3 and TGX-D4 also demonstrated activities in
the LNCaP cells with IC50 of 6.9 μM, 8.8 μM, and 17.5 μM, re-
spectively. DU145 cells did not respond to these derivatives with
the exception of TGX-D3, which had an IC50 of 52.9 μM.
PI3Kβ has been reported to be essential for androgen-stimulated
AR transactivation and cell proliferation in prostate cancer cells.16
Androgen-stimulated cell proliferation and gene expression are
dramatically reduced by silencing the PI3Kβ gene with siRNA or
inhibiting its activity with the PI3Kβ-selective inhibitor TGX-
221.16 TGX-D1 demonstrated comparable effects as TGX-221 on
the inhibition of androgen-induced cell proliferation (Figure 3C)
and gene expression (Figure 3D), suggesting that TGX-D1 has a
similar potency to TGX-221 to specifically inhibit PI3Kβ activity.
As a result, TGX-D1 was selected for conjugation with the pep-
tide promoiety.
Synthesis of the PeptideꢀDrug Conjugate. Synthesis of
NH2-SL-TGX is depicted in Figure 4A. Fmoc-Leu-OH was con-
jugated to the hydroxyl group of TGX-D1 via an ester bond. After
deprotection, Boc-Ser(tBu)-OH was coupled to the amino group
of the NH2-L-TGX. The dipeptideꢀdrug conjugate (NH2-SL-
TGX) was obtained by deprotecting the peptide and purifying it
by HPLC using acidic conditions. Neutral and basic environ-
ments were avoided for NH2-SL-TGX because of its instability.
For the synthesis of KCC-TGX, undeprotected peptide KCC-
YSLGGGSSKYQS including the PSA substrate, a GGG spacer,
and the HER2-specific peptide was synthesized independently, and
then coupled to NH2-L-TGX (Figure 4B). The undeprotected
Synthesis of the TGX-221 Derivatives. Because TGX-221
does not contain any functional groups for conjugation with pep-
tides, we synthesized four TGX-221 derivatives (TGX-D1, TGX-
D2, TGX-D3, and TGX-D4) with additional hydroxyl groups, as
illustrated in Figure 2. Compounds 2, 3, and 4 were synthesized
from compound 1, as reported.28 TGX-221 was synthesized ac-
cording to the same report with a modification. In line with the
reported procedure,18 the Schiff base was generated successfully
after refluxing in toluene. However, the reported conditions (NaBH4
in toluene) could not reduce the Schiff base to the amine, even
after increasing the temperature to 100 °C. This result is pro-
bably due to the poor solubility of NaBH4 in toluene. After changing
the solvent from toluene to methanol, a complete conversion was
achieved in 30 min at room temperature. TGX-D2, TGX-D3 and
TGX-D4 were also prepared by the same route. Compound 5
was synthesized from TGX-221 by substituting the secondary
aniline with a methyl acetate. To carry out this conversion, the
ester group of compound 5 was converted to a primary alcohol
(TGX-D1) by LiCl/NaBH4 with a yield of 40ꢀ60%.
Bioactivity Screening of the TGX-221 Derivatives. We next
examined the bioactivities of these TGX-221 derivatives in two
prostate cancer cell lines, LNCaP and DU145. LNCaP represents
a PTEN-null prostate cancer cell line that is sensitive to TGX-221
treatment, while DU145 is PTEN-wild and unresponsive to TGX-
221. As shown in Figures 3A and 3B, TGX-221 exhibits the
highest activity in LNCaP cells with an IC50 of 1.07 μM. Among
the four TGX-221 derivatives, TGX-D1 showed the best activity
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Figure 3. Bioactivity screening of TGX-221 and its derivatives. TGX-D1 showed comparable effects as TGX-221 in cytotoxicity,the inhibition of
androgen-induced cell proliferation and gene expression. (A) Structures of the TGX-221 derivatives and their IC50 values in LNCaP (PTEN-null) and
DU145 (PTEN-wild) cells. (B) Cytotoxicities of TGX-221 and TGX-D1 are examined on LNCaP cells by MTT assay. (C) Both TGX-221 and TGX-D1
inhibit androgen-induced cell proliferation. LNCaP cells were treated with TGX-221 and TGX-D1 in R1881-containing serum-free mediumfor 3 days
before the Alarm Blue assay. (D) Both TGX-221 and TGX-D1 inhibit androgen-induced gene expression. The ARE-LUC reporter was transfected into
LNCaP cells and the cells were stimulated with R1881 (1.0 nM) in the presence or absence of TGX-221 or TGX-D1 (5μM and 10μM) for 24h before
being subjected to the luciferase assay. The results are represented as the mean ( SD (n = 3).
peptide KCCYSLGGGSSKYQS was constructed on 2-Cl-Trt-
resin using solid-phase synthesis. The N-terminal of the peptide
was protected with an acetyl group to enhance the stability of the
peptideꢀdrug conjugate. Coupling reagent HATU was used to
increase the yield and suppress the racemization. The undepro-
tected peptideꢀdrug conjugate (KCC-TGX) was isolated by silica
gel column, followed by deprotection and purification by HPLC.
The peptideꢀdrug conjugate exhibited good solubility, and no
organic solvent was required in its drug release and bioactivity
studies.
Drug Release from the PeptideꢀDrug Conjugate. Release
of the parent drug from the peptideꢀdrug conjugate is essential
to elicit the therapeutic effect in prostate cancer cells. According
to our hypothesis, KCC-TGX (Ac-KCCYSLGGGSSKYQSL-TGX)
would be delivered to prostate cancer cells and cleaved on the cell
surface by PSA. Additionally, the released NH2-SL-TGX would
undergo a self-cyclization reaction to release the parent drug TGX-
D1 in physiological pH (Figure 5A). To test this hypothesis, we
monitored the release of TGX-D1 from NH2-SL-TGX in vitro by
HPLC. The data presented in Figures 5B and 5C illustrated that
TGX-D1 dissociated readily from the dipeptideꢀdrug conjugate
NH2-SL-TGX in PBS buffer (pH 7.4) at 37 °C. The release
profile followed zero-order kinetics with a half-life of 8 h.
We further examined the hypothesis using Ac-SSKYQSL-TGX
that only contained the PSA cleavable peptide sequence SSKYQ.31
PSA enzymes expressed in prostate cancers recognize SSKYQ
and cleave it between the residues Gln (Q) and Ser (S) to form
the dipeptideꢀdrug conjugate (NH2-SL-TGX) which undergoes
a self-cyclization reaction to release the parent drug TGX-D1 in a
physiological pH (Figure 6A). Our selection of the dipeptide SL
as the linker between the PSA substrate and TGX-D1 was not
only for the self-cyclizaiton reaction, it was also because the most
efficient PSA cleavage occurs between Gln (Q) and Ser (S) that
are followed by Leu (L).32 To monitor the PSA cleavage and drug
release, the peptideꢀdrug conjugate Ac-SSKYQSL-TGX was
incubated with PSA at 37 °C and then assayed by HPLC. As
Figures 6B and 6C depict, Ac-SSKYQSL-TGX was gradually cleaved
by PSA to release the intermediate NH2-SL-TGX. The genera-
tion of NH2-SL-TGX by PSA cleavage was faster than the self-
cyclization rate at the beginning, leading to an increase of NH2-
SL-TGX. However, a gradual and steady release of the parent
drug TGX-D1 was also observed, suggesting that the peptideꢀ
drug conjugate could readily release the active TGX-D1 in pro-
state cancer cells.
Cytotoxicity of the PeptideꢀDrug Conjugates. We next
examined whether the released parent drug TGX-D1 was still active
to induce cytotoxicity in the prostate cancer cells. As demonstrated
in Figure 7, NH2-SL-TGX, Ac-SSKYQSL-TGX and KCC-TGX
induced similar cytotoxicities in the LNCaP cells. The IC50 values
for NH2-SL-TGX, Ac-SSKYQSL-TGX and KCC-TGX obtained
were 5.54 μM, 4.20 μM and 5.27 μM, respectively, which are close
to the IC50 (2.67 μM) of the parent drug TGX-D1. These results
clearly indicated the efficient release of the parent drug TGX-D1
from the peptideꢀdrug conjugate.
High Affinity of the HER2-Targeting Peptide with LNCaP
Cells. It has been demonstrated that HER2 is highly expressed in
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Figure 4. Synthesis of the peptideꢀdrug conjugate. (A) Synthesis of NH2-SL-TGX. Reaction conditions: (a) Fmoc-Leu-OH, EDCI, DMAP (cat),
DCM; (b) 20% piperidine in DCM; (c) Boc-Ser(OtBu)-OH, EDCI, DMAP (cat), DCM; (d) 50% TFA in DCM. (B) Synthesis of KCC-TGX. Reaction
conditions: (a) AcOH/trifluoroethanol/DCM (10/20/70), rt; (b) HATU, DIPEA, NMP; (c) TFA/DCM/TIPS (47.5/47.5/5), rt.
Figure 5. In vitro drug release from the intermediate NH2-SL-TGX. NH2-SL-TGX underwent self-cyclization to release the parent drug TGX-D1. The
dipeptide Ser-Leu self-cyclizes to form DKP in a neutral pH. (A) Illustration of the self-cyclization reaction. (B) HPLC chromatogram monitored at
268 nm. (C) Release profile of TGX-D1 from NH2-SL-TGX. The results are represented as the mean ( SD (n = 3).
a significant proportion of prostate cancer cells including LNCaP
cells.33,34 KCCYSL is a HER2-targeting peptide that was gener-
ated against HER2-positive breast cancer cells using phage display
technology.35 KCCYSL is relatively small, and its affinity to the
HER2 receptor is well-defined.35,36 To verify its affinity to LNCaP
cells, C-terminus biotinylated KCCYSL was incubated with LNCaP
cells and then stained with streptavidinꢀFITC. KCCYSL exhibited
high-affinity binding on the LNCaP cells at a low concentration
(10 μM) in comparison to the control peptide DPRATPGSK
(Figure 8). No significant fluorescence was observed for this control
peptide even when the concentration was increased to 100 μM
(data not shown). These results further support the rationale of
using the HER2-targeting peptide for targeted drug delivery to
prostate cancer cells.
Cellular Uptake of KCC-TGX. We finally investigated wheth-
er the HER2-targeting peptide enhanced the delivery of the
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Figure 6. PSA-mediated drug release from the peptideꢀdrug conjugate (Ac-SSKYQSL-TGX). Ac-SSKYQSL-TGX was cleaved by PSA to release the
intermediate NH2-SL-TGX, which underwent self-cyclization to release the parent drug TGX-D1. (A) Illustration of PSA activation of the
peptideꢀdrug conjugate. (B) HPLC chromatogram monitored at 268 nm. (C) Release profile of TGX-D1 from Ac-SSKYQSL-TGX. The results
are represented as the mean ( SD (n = 3).
Figure 7. Cytotoxicity of NH2-SL-TGX (A), Ac-SSKYQSL-TGX (B) and KCC-TGX (C) in LNCaP cells. LNCaP cells were incubated with the
peptideꢀdrug conjugates for 72 h, and cytotoxicity was measured by a MTT assay. The results are represented as the mean ( SD (n = 3).
a high uptake. By contrast, KCC-TGX, which contains the HER2
targeting peptide, exhibited the highest uptake, indicating that the
HER2-specific peptide (KCCYSL) dramatically increased the cel-
lular uptake of KCC-TGX. The time course of drug uptake in
LNCaP cells was also studied (Figure 9B). While KCC-TGX
showed the highest uptake extent, the uptake rate was comparable
to that of TGX-D1 and NH2-SL-TGX, suggesting that coupling
Figure 8. Immunostaining of LNCaP cells with biotinylated anti-HER2
peptide. After blocking with 1% BSA in PBS, the fixed LNCaP cells were
of the anti-HER2 peptide increases the drug affinity to prostate
cancer cells without compromising its uptake rate.
incubated with the biotinylated peptide for 1 h in TBST buffer. After
It is interesting to note that NH2-SL-TGX demonstrated a higher
washing with TBS, the cells were incubated with streptavidinꢀFITC for
cellular uptake in comparison to Ac-SSKYQSL-TGX and TGX-D1.
20 min and the nucleus was stained with DIPA for cell tracking. The cells
The possible explanation might be that NH2-SL-TGX can be taken
were visualized using a Leica DMI3000 microscope at a 200ꢁ magni-
up through peptide transporters that are expressed on many cancer
cells.37 In line with this hypothesis, Ac-SSKYQSL-TGX did not
show increased uptake either because only di- and tripeptide can
be actively transported by peptide transporters.38 However, to
test this hypothesis, LNCaP cells were incubated with 0.5 μM of
NH2-SL-TGX in the presence of dipeptide Ser-Leu (0, 3.16, 10,
and 100 μM) at 37 °C for 1 h. As Figure 9C revealed, the uptake
fication.
peptideꢀdrug conjugate, KCC-TGX, to prostate cancer cells.
Cellular uptake of TGX-D1 and its peptide conjugates were first
evaluated on LNCaP cells at a concentration of 0.5 μM after 1 h
of incubation (Figure 9A). Both TGX-D1 and Ac-SSKYQSL-TGX
showed low uptake in LNCaP cells, while NH2-SL-TGX showed
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Figure 9. Cellular uptake of the peptideꢀdrug conjugates in LNCaP cells. (A) Cellular uptake in LNCaP cells at a concentration of 0.5 μM. (B) The
time-course of cellular uptake in LNCaP cells. (C) Dipeptide Ser-Leu concentration-dependent inhibition of the uptake of NH2-SL-TGX. (D). The
HER2- specific ligand AcKCCYSL inhibits the uptake of KCC-TGX in LNCaP cells. The results are represented as the mean ( SD (n = 3). (* P < 0.05; **
P < 0.01).
Table 1. Solubilities of TGX-221, TGX-D1 and KCC-TGX
aqueous solubility
compd
μg/mL
μM
TGX-221
TGX-D1
KCC-TGX
147.5 ( 17.6
33.5 ( 6.6
405.2 ( 48.2
82.1 ( 16.2
g9899.1 ( 465.4
g4684.9 ( 465.4
of NH2-SL-TGX showed a concentration-dependent decrease
after coincubation with the dipeptide Ser-Leu, which competed
for the same transporters on the prostate cancer cells. The peptide
transporters were almost completely saturated by Ser-Leu at 10 μM,
and the uptake of NH2-SL-TGX was accordingly inhibited by 75%.
To demonstrate whether the uptake of KCC-TGX by LNCaP
cells is mediated by the anti-HER2 peptide ligand, we preincubated
the cells with the anti-HER2 peptide and then applied KCC-TGX
to the cells. As Figure 9D revealed, the uptake of Ac-KCCYSL was
significantly inhibited by the preincubation with the HER2-specific
ligand, which confirmed the specific binding of KCC-TGX to
LNCaP cells via the recognition of the anti-HER2 peptide.
Aqueous Solubility and Stability of KCC-TGX. The aqueous
solubility of KCC-TGX was assayed by the thermodynamic
method at room temperature. The aqueous solubility of TGX-221
is approximately 147.5 μg/mL, which is considered to be water-
insoluble according to the United States Pharmacopeia (USP)
(Table 1). Surprisingly, the solubility of TGX-D1 is only 33.5 μg/mL
which is much lower than that of TGX-221. However, after con-
jugating to the peptide promoiety, its solubility increased to more
than 9.895 mg/mL, which is 60-fold higher than that of TGX-D1
in terms of molarity. The substantial increase in aqueous solubility is
probably due to the hydrophilic peptide promoiety Ac-KCCYS-
LGGGSSKYQSL-OH in KCC-TGX.
Figure 10. The stability of KCC-TGX in PBS, cell culture medium and
mouse serum. Results were represented as mean ( SD (n = 3).
The stability of KCC-TGX was measured in three different
solutions. KCC-TGX was found to be relatively stable in PBS and
cell culture medium at 37 °C. After 4 h of incubation, 7% and 12%
of KCC-TGX were found to be degraded in PBS and cell culture
medium, respectively (Figure 10). However, KCC-TGX was quickly
degraded in mouse serum with a half-life of 15 min, and nearly
83% of the KCC-TGX molecules were degraded in mouse serum
after 60 min. The quick degradation of KCC-TGX is probably due to
the use of the labile ester linker between the peptide and TGX-D1.
However, it should be noted that the hydrolysis rate of the ester
linker is species dependent, and the stability is much higher in human
plasma than that in mouse serum.39 Therefore, the degradation
of KCC-TGX in human plasma is expected to be much slower.
’ DISCUSSION
In this study, we conjugated a peptide promoiety that con-
tains a HER2-specific peptide and a PSA-cleavable substrate to
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TGX-221, a highly potent PI3Kβ inhibitor that shows great pro-
mise for prostate cancer therapy. Despite the essential role of PI3Kβ
in prostate cancer development, the therapeutic application of
TGX-221 is limited due to its poor solubility and lack of specificity
to prostate cancer cells. Therefore, the aim of this study is to over-
come these two problems by attaching a peptide promoiety to
TGX-221. Targeted delivery of this peptide-conjugated drug was
achieved by considering two mechanisms. First, the HER2-specific
peptide binds to HER2 on the surface of prostate cancer cells.
Second, PSA, which exists in the microenvironment of prostate
cancer cells, cleaves the peptide and releases the active parent drug.
Because TGX-221 does not contain a functional group for pep-
tide conjugation, we first synthesized four TGX-221 derivatives
(Figure 2) by attaching a ꢀOH group at different sites. Among
them, TGX-D1 was selected for peptide conjugation due to its
similar activity compared to TGX-221 (Figure 3).
to 10ꢀ30 min when 340 μg/mL of PSA was used.32 In
comparison, the self-cyclization of NH2-SL-TGX might be the
rate-limiting step in the release of the parent drug in vivo.
One interesting observation was that the dipeptideꢀdrug con-
jugate NH2-SL-TGX exhibited a higher cellular uptake than TGX-
D1 and Ac-SSKYQSL-TGX (Figure 9A). In other words, the
peptide transporters that are believed to be widely expressed in
various cancers could take up NH2-SL-TGX readily.37 In line with
this hypothesis, we have found that coincubation with the dipeptide
Ser-Leu suppressed the cellular uptake of NH2-SL-TGX (Figure 9C).
Therefore, we reasonably believe that PSA enzymes cleave the
KCC-TGX bound on the cell surface to release NH2-SL-TGX,
which is then transported into the cells through the peptide
transporters. However, KCC-TGX can also be directly taken up
by HER2 receptor-mediated endocytosis.
In summary, a multicomponent peptideꢀdrug conjugate was
synthesized for TGX-221. This peptideꢀdrug exhibits high cellular
uptake in LNCaP cells. PSA enzymes activate the peptideꢀdrug
conjugate by cleaving the PSA substrate to release NH2-SL-TGX,
which undergoes a self-cyclization to release the parent drug
TGX-D1. This peptideꢀdrug conjugate exhibits a much higher
cellular uptake in prostate cancer cells in comparison to the parent
drug, indicating its tremendous potential as a targeted therapy for
prostate cancer patients.
HER2 has been reported to be overexpressed in many cancers
such as breast cancer and prostate cancer. Although the role of its
overexpression in prostate cancer is not fully understood, the HER2
receptor has been successfully exploited as a molecular target for
targeted drug delivery to prostate cancer.33,40 Anti-HER2 anti-
body is the first type of targeting moiety used for an antibodyꢀdrug
conjugate. However, its large size compromises its cellular uptake,
and the antibodyꢀdrug conjugate is unable to reach the tumor
interior readily. Furthermore, one antibody molecule can only be
conjugated with 1ꢀ3 drug molecules, which leads to a low drug
’ AUTHOR INFORMATION
loading (<1%). As a result, only highly potent drugs (IC50
=
10ꢀ9ꢀ10ꢀ11 M) can be used for this antibodyꢀdrug approach.41
By contrast, a peptide ligand is a more appropriate targeting moiety
due to its excellent cell permeability, small molecular weight, ease
of production, and flexibility in chemical conjugation.42 The size
of the peptideꢀdrug conjugate is small enough to efficiently pen-
etrate tissues, and drug loading can be as high as 20ꢀ30%. In this
study, a 16-mer peptide (KCCYSLGGGSSKYQSL) was conjugated
to TGX-D1 to form the peptideꢀdrug conjugate (MW 2113.4),
in which a drug loading of 19.4% was achieved. This 16-mer
peptide promoiety is composed of a HER2-targeting peptide, a
GGG spacer, a PSA substrate peptide and a self-cycling dipeptide
(Figure 1A).
Corresponding Author
*Division of Pharmaceutical Sciences, School of Pharmacy,
University of Missouri—Kansas City, 2464 Charlotte Street,
Kansas City, MO 64108. Phone: (816) 235-2425. Fax: (816)
235-5779. E-mail: chengkun@umkc.edu.
’ ACKNOWLEDGMENT
This project has been supported by awards from the National
Cancer Institute (NCI), NIH (1R21CA143683-01) and Na-
tional Institute of Alcohol Abuse and Alcoholism (NIAAA), NIH
(1R21AA017960-01A1) to K.C., and also partially by a Depart-
ment of Defense grant (W81XWH-09-1-0455) and KU Masonic
Foundation to B.L. The authors also would like to express thanks
for the financial support from a start-up package at the University
of Missouri—Kansas City.
PSA-activated peptideꢀdrug conjugates have been well stu-
died in recent years.32,43 For example, L-377202, a peptideꢀ
doxorubicin prodrug hydrolyzed by PSA, has already entered clinical
trials.44 However, none of these peptideꢀdrug conjugates in-
clude tumor-specific ligands, which causes accumulation in the
cancer cells. Here, we reported the first application of a HER2-
specific ligand to deliver the PSA-activated TGX-221 prodrug to
prostate cancer cells. Uptake of the peptideꢀdrug conjugate
KCC-TGX in prostate cancer cells is approximately 10-fold that of
the peptideꢀdrug conjugate (Ac-SSKYQSL-TGX) that only
contains the PSA cleavage linker (Figure 9A). In addition, the
peptide-mediated cellular uptake is fast, and equilibrium is
quickly reached in less than 10 min, demonstrating a rapid
uptake of the prodrug in prostate cancer cells (Figure 9B). This
rapid uptake is critical to minimize the loss of the peptideꢀdrug
conjugate caused by kidney excretion after systemic administra-
tion. Compared to the quick uptake, PSA cleavage of the peptideꢀ
drug conjugate is slow in in vitro studies (Figure 6). However, a much
more efficient cleavage in vivo is expected because the prostate cancer
interstitial fluid contains a substantial level of enzymatically active
PSA (50ꢀ500 μg/mL), which is 5ꢀ50 times higher than we used
in the in vitro cleavage study.45 As demonstrated in Garsky’s
study, the half-life of a similar PSA substrate peptide was reduced
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