Dendritic hexadecapeptide as a cathepsin B degradable carrier for delivery of HSP90 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2015.06.012

Biodegradable vehicles that degrade specifically at tumor sites are highly desirable since they can cause selective exposure of highly toxic drugs at tumor sites whereas keep the conjugates stable during blood circulation. Here, we evaluate the utility of

Full text of DOI:10.1016/j.bmcl.2015.06.012

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3744–3747
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dendritic hexadecapeptide as a cathepsin B degradable carrier
for delivery of HSP90 inhibitor
⇑
Vidula Kolhatkar, Jose Suárez, Rohit Kolhatkar
Department of Biopharmaceutical Sciences, University of Illinois, Chicago, 1601 Parkview Ave, Rockford, IL 61107, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Biodegradable vehicles that degrade specifically at tumor sites are highly desirable since they can cause
selective exposure of highly toxic drugs at tumor sites whereas keep the conjugates stable during blood
circulation. Here, we evaluate the utility of a dendritic hexadecapeptide comprised of four arms, each
having a tetrapeptide sequence recognized by an enzyme cathepsin B as a carrier system for heat shock
protein 90 (HSP90) inhibitor geldanamycin (GDM). We report the synthesis of a carrier having GDM con-
jugated to the terminal end of each arm (>55% wt/wt drug). We further report the stability of the GDM
containing peptidic dendrimer in various buffers and in the presence of serum along with its ability to
release free drug in the presence of cathepsin B, the enzyme overexpressed in a variety of tumors.
Using androgen-independent prostate cancer cell line (DU-145) we further demonstrate that the gel-
danamycin containing peptidic dendrimer has antiproliferative property similar to the free drug
derivative.
Received 5 May 2015
Revised 28 May 2015
Accepted 2 June 2015
Available online 11 June 2015
Keywords:
Geldanamycin
Degradable polymer
HSP90 inhibitor
Cathepsin B
Peptidic dendrimer
Ó 2015 Elsevier Ltd. All rights reserved.
Synthetic polymers offer several advantages for delivery of
small molecular weight anticancer therapeutics.1,2 Dendrimers,
recognized as new class of synthetic polymers have been widely
accepted as drug carriers because of their unique architecture
and low polydispersity.3 However, one of the limitations of cur-
rently used dendrimers is that they are nondegradable.4 Because
degradable systems are more biocompatible than their nondegrad-
able counterparts5 there is a great interest in developing physio-
logically degradable dendrimers. A few degradable dendrimers
reported so far have relied on the chemical hydrolysis in a
biological environment;6 majority of them having ester bonds that
can degrade nonspecifically.4 In contrast, using dendrimers that
can be degraded by enzymes is an attractive choice because of
degradation specificity associated with the selected enzyme.
Enzymatic trigger has been used successfully for releasing the drug
from a polymeric backbone.7 The use of enzymatically degradable
polymeric backbone has also been proven useful compared to
traditional N-(2-hydroxypropyl methacrylamide) (HPMA) copoly-
meric conjugates.8,9 However, the use of enzymes to make
biodegradable polymers is relatively unexplored.10
previously demonstrated that a dendritic hexadecapeptide (pep-
tidic dendrimer) synthesized using the tetrapeptide GFLG, which
is a substrate for cathepsin B, is stable in serum but degrades
rapidly and completely in the presence of cathepsin B.12 In this let-
ter we demonstrate the utility of the peptidic dendrimer for the
delivery of the heat shock protein 90 (HSP90) inhibitor gel-
danamycin (GDM).
HSP90 inhibitors are highly toxic; thus, the use of a delivery sys-
tem to facilitate their tumor specific exposure will be highly benefi-
cial.13–16 Several preclinical and clinical studies have demonstrated
the utility of polymeric carriers in reducing toxicity associated with
small molecular weight anticancer drugs such as topoisomerase I
and II inhibitors (doxorubicin, camptothecin), microtubule inhibi-
tors (taxanes) and platinates.17–19 Comparatively very few studies
have been reported to evaluate the use of polymeric conjugates
for HSP90 inhibitors, despite HSP90 being regarded as a validated
target for anticancer therapeutics.20,21 Thus, in this letter we evalu-
ate the utility of a completely degradable peptidic dendrimer for the
delivery of geldanamycin, a potent inhibitor of HSP90.
Figure 1 shows the structure of GDM and aminohexyl gel-
danamycin (AHGDM) that was used to conjugate to the tetrapep-
tide GFLG. 1,6 Diaminohexane was chosen as a linker based on
the optimization studies reported previously using HPMA copoly-
mers.22 We synthesized aminohexylgeldanamycin (AHGDM) and
p-nitrophenyl ester of BocGFLG (BocGFLGONp) as described
previously.12,22 AHGDM was conjugated to BocGFLGONp to obtain
BocGFLGAHGDM that was deprotected under acidic conditions to
We intend to use the enzyme cathepsin B as a trigger for com-
plete degradation of a dendritic carrier. Cathepsin B is a cysteine
protease overexpressed in several tumor tissues.11 We have
⇑
Corresponding author. Tel.: +1 815 395 5922; fax: +1 815 395 5936.
E-mail address: rohitk@uic.edu (R. Kolhatkar).
http://dx.doi.org/10.1016/j.bmcl.2015.06.012
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

V. Kolhatkar et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3744–3747
3745
Figure 1. Structure of geldanamycin (1) (GDM), aminohexane geldanamycin (2) (AHGDM) and monomeric peptides GFLGAHGDM (3) and DGFLGAHGDM (4).
Table 1
yield GFLGAHGDM having free terminal amine group. To obtain
Characteristics of monomers and dendrimers
drug containing peptidic dendrimers we reacted GFLGAHGDM
with tetrakis-(p-nitrophenyl ester) of EDTA (5) (Scheme 1).
Briefly, GFLGAHGDM (85 mg, 0.083 mmol) was dissolved in anhy-
drous DMSO. EDTA(ONp)4 (10.7 mg, 0.014 mmol) and DIPEA
(0.23 mL, 1.2 mmol) were added to it. The reaction mixture was
stirred for 72 h at room temperature. DMSO was then evaporated
under vacuum and the reaction mixture was precipitated in ether.
LCMS analysis of the precipitate revealed that it was a mixture of
three products having two, three, or four arms of GFLGAHGDM
attached to the central core (EDTA). The mixture was separated
by preparative HPLC to isolate compound 6 (three peptidic arms
conjugated to the core; yield 7%) and 7 (four peptidic arms conju-
gated to the core; yield 25%). Formation of heterogeneous mixture
(two, three, and fours arms conjugated to EDTA core) might be
responsible for the lower yields that can be improved in future
by using higher equivalents of GFLGAHGDM. The molecular weight
of HPLC purified peptidic dendrimers was confirmed using MS and
MALDI and the purity of the compounds was determined using
HPLC. Table 1 describes characteristics of the AHGDM containing
peptides. Both the compounds (6 and 7) had very high drug con-
tent (>55% wt/wt).
Previously we have reported the stability of a peptidic den-
drimer EDTA(GFLGOH)4 lacking drug.12 Here, we report the stabil-
ity of drug containing polymer (Fig. 2) as well as drug release
profile (Fig. 2B). Thus, compound 7 was incubated in PBS (pH
7.4), and PBS with serum (10%) for 24 and 48 h and intact polymer
was quantified by HPLC. The stability of 7 was also checked in acet-
ate buffer to mimic acidic conditions used during studies per-
formed to determine drug release in the presence of cathepsin B.
More than 97% of the intact polymer was detected after 24 h incu-
bation with PBS and serum (10% serum in PBS). Further increase in
incubation time from 24 to 48 h did not change the percentage of
intact polymer detected suggesting the stability of the polymer.
Intact peptidic dendrimer (7) detected in acetate buffer was
slightly lower (94% and 93% at 24 and 48 h, respectively). Overall,
Sr.
no.
Compound
Mw
calcd
Mw
MALDI
HPLC ret.
time (min) content%
wt/wt
Drug
1
3
4
5
6
EDTA(GFLGOH)4
EDTA(GFLGAHGDM)3 3293.78 3321.28 18.66
EDTA(GFLGAHGDM)4 4294.34 4320
AHGDM
1788.87 1791.87 27.08
—
58.74
60.08
—
17.83
3.31
4.09
644.38 645.38
1018.57 1019.57
GFLGAHGDM
56.92
the stability of the drug containing peptidic dendrimer (7) was
similar to the one without drug as reported previously. When 7
was incubated with cathepsin
intact polymer was detected after 24 h and 48 h, respectively.
Previously we have reported complete degradation of the peptidic
dendrimer (without the drug) within 4 h incubation with cathep-
sin B.12 These findings suggest that conjugation of drug hinders
accessibility of cathepsin B.
The release of AHGDM was quantified by LCMS after incubation
of the compound 7 with cathepsin B (Fig. 2B) following the
procedure as described before. 14% and 17% of AHGDM release
was determined after 24 h and 48 h incubation, respectively.
GDM containing HPMA copolymers have been reported to release
20–25% of AHGDM.23,24 Comparatively the peptidic dendrimers
in this study showed lower drug release. LCMS profile also indi-
cated the presence of a peak corresponding to Gly-AHGDM.
Borgman et al. have reported the release of 27–36% Gly-AHGDM
from HPMA copolymers depending on the presence or absence of
targeting moiety.23 In this study we found only 8% and 10% release
of Gly-AHGDM after 24 and 48 h, respectively. Total release of
AHGDM and GlyAHGDM was 22% and 27% after 24 and 48 h,
respectively, which corresponds well with 73% and 66% of the
intact polymer observed from HPLC analysis.
B only 73% and 66% of the
Next, we evaluated the growth inhibition properties of GDM
(Fig. 3, Table 2), AHGDM, GFLGAHGDM, and GDM containing
Scheme 1. Schematic representation of the synthesis of drug containing peptidic dendrimers using EDTA as a core.

3746
V. Kolhatkar et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3744–3747
peptidic dendrimers. HSP90 is actively pursued as a therapeutic tar-
get for prostate cancer treatment and HSP90 inhibitors have been
demonstrated to inhibit the growth of androgen independent
prostate cancer cell line DU-145. Thus, we chose DU-145 cell line
for our studies to determine their growth inhibition after 48 h incu-
bation in the presence of test compounds as described previously.25
IC50 values (concentrations to inhibit growth by 50%) were calcu-
lated from the graphs obtained by cell viabilities at different free
drug equivalent doses of test compounds (Fig. 3, Table 2). GDM
was highly toxic with an IC50 value of 30 nM. AHGDM and
GFLGAHGDM were less active than GDM exhibiting IC50 values in
micromolar range (2.5 lM and 1.2 lM). GFLGAHGDM was about
2-folds more active than AHGDM. Conjugation of GFLGAHGDM to
the core yielded compounds (6 and 7) with significantly lower
activity than GFLGAHGDM (p<0.005 for IC50 values). Compounds
6 (three arms) and 7 (four arms) had similar IC50 values (13.1 vs
13.6 lM) and both the compounds were 5-folds less active than
AHGDM. This could be because of lower drug release from the
peptidic dendrimers. We have previously demonstrated that a con-
trol peptidic dendrimer having no drug (synthesized from core and
GFLGOH) did not exhibit any cytotoxicity up to 66
centration tested).
lM (highest con-
Because AHGDM release was low at earlier time points (up to
48 h) we evaluated the antiproliferative properties of compound
7 and small molecules after 72 h and 96 h treatment (Table 2).
No change in IC50 was observed for AHGDM and GFLGAHGDM.
However, IC50 value for 7 decreased significantly (p<0.02) to
Figure 2. (A) Stability of peptidic dendrimers in PBS, FBS, and acetate buffers and
(B) degradation and drug release profile catalyzed by cathepsin (B).
125
100
75
GDM
AHGDM
GFLGAHGDM
EDTA(GFLGAHGDM)4
3.5 lM after 72 h incubation (4-folds decrease compared to 48 h
period) but did not decrease any further after 96 h treatment. For
all the compounds IC50 values were similar after 72 and 96 h treat-
ment period.
50
Overall, 7 exhibited good antiproliferative activity after longer
incubation time. One of the limitations noticed during growth inhi-
bitions studies was the limited solubility of 7, probably because of
its higher drug content (>50% wt/wt). Although final solutions con-
tained less than 1% DMSO while testing, stock solutions of drug
containing peptidic dendrimers were required to be made in
DMSO. To increase the solubility we decided to add a polar amino
acid (aspartic acid) at the amine terminus of the tetrapeptide GFLG.
Thus, p-nitrophenyl ester of aspartic acid that has alpha-acidic
group protected as tert-butyl ester was reacted with
GFLGAHGDM to form a pentapeptide that was deprotected under
basic conditions to yield the expected product DGFLGAHGDM (8)
as shown in Scheme 2. It should be noted that the use of methyl
ester instead of tert-butyl ester as an alpha-acid protecting group
led to the formation of a pentapeptide but with an unwanted imide
bond between the two acidic groups of aspartic acid and an amine
from GFLGAHGDM.
25
0
-25
-50
-75
-8
-7
-6
-5
-4
conc (log M)
Figure 3. Growth inhibition effect on DU-145 cells after treatment at various
concentrations with free drugs (GDM, AHGDM) monomeric peptide (GFLGAHGDM)
and peptidic dendrimer for 96 h.
Table 2
IC50 values of compounds evaluated using DU-145 cell line
Compound
IC50 (lM)
48 h
72 h
96 h
Geldanamycin (GDM)
AHGDM
GFLGAHGDM
DGFLGAHGDM
EDTA(GFLGAHGDM)3
EDTA(GFLGAHGDM)4
0.03
0.01
0.01
2.5 0.2
1.2 0.1
8.2 0.3*
13.1 0.5*
13.8 3.9*
2.6 0.1
1.2 0.1
2.2 0.2
—
2.4 0.0
1.5 0.1
1.9 0.1
—
The solubility of GFLGAHGDM and DGFLGAHGDM in PBS was
determined from the absorbance of saturated solutions and
relating them to the calibration curve obtained from known con-
centrations of the compounds in DMSO (Fig. 4A). DGFLGAHGDM
3.4 0.5#
3.5 0.0
#
Significant difference (p <0.02) compared to 48 h value for the same compound.
Significant difference (p <0.005) compared to GFLGAHGDM.
*
Scheme 2. Synthesis of AHGDM conjugated pentapeptide DGFLG.

V. Kolhatkar et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3744–3747
3747
containing peptide (compound 6) further suggests that the remain-
ing free arm can be used for conjugation of hydrophilic polymer
such as polyethylene glycol to increase water solubility. Further
studies are currently under progress to target these constructs to
tumor sites to avoid non-specific toxicity associated with HSP90
inhibitors.
Acknowledgments
Authors would like to thank Dr. Lee, Director of Protein
Research lab, Research Resource Center at UIC for assistance with
MALDI analysis. Part of this work was supported by the
Department of Defense Breast Cancer Research Program
Concept Award (W81XWH-09-1-0687) to R.K. The work was also
supported by start-up funds to R.K. from the Department of
Biopharmaceutical Sciences, University of Illinois Chicago.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
012.
References and notes
Figure 4. (A) Solubility (mg/mL) of GDM, AHGDM and peptide conjugated AHGDM
in PBS. # The solubility of GDM was below detection limit. (B) Growth inhibition
effect of tetrapeptide and pentapeptide conjugated AHGDM on DU-145 cells after
48 h treatment.
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