Switch off/switch on regulation of drug cytotoxicity by conjugation to a cell targeting peptide

Article abstract of DOI:10.1016/j.ejmech.2014.07.073

Bi-nuclear amino acid platforms loaded with various drugs for conjugation to a peptide carrier were synthesized using simple and convenient orthogonally protective solid-phase organic synthesis (SPOS). Each arm of the platform carries a different anticancer agent linked through the same or different functional group, providing discrete chemo-and bio-release profiles for each drug, and also enabling switch off/switch regulation of drug cytotoxicity by conjugation to the platform and to a cell targeting peptide. The versatility of this approach enables efficient production of drug-loaded platforms and determination of favorable drug combinations/modes of linkage for subsequent conjugation to a carrier moiety for targeted cancer cell therapy. The results presented here potentiate the application of amino acid platforms for targeted drug delivery (TDD).

Full text of DOI:10.1016/j.ejmech.2014.07.073

European Journal of Medicinal Chemistry 85 (2014) 139e146
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
“Switch off/switch on” regulation of drug cytotoxicity by conjugation
to a cell targeting peptide
,
,
, c
Yossi Gilad a c, Michael A. Firer b, Alex Rozovsky a c, Elena Ragozin a, Boris Redko a
,
Amnon Albeck c, Gary Gellerman a
,
*
a Department of Biological Chemistry, Ariel University, Ariel, 40700, Israel
b Department of Chemical Engineering, Ariel University, Ariel, 40700, Israel
c The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar Ilan University, Ramat Gan, 52900, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Bi-nuclear amino acid platforms loaded with various drugs for conjugation to a peptide carrier were
synthesized using simple and convenient orthogonally protective solid-phase organic synthesis (SPOS).
Each arm of the platform carries a different anticancer agent linked through the same or different
functional group, providing discrete chemo- and bio-release profiles for each drug, and also enabling
“switch off/switch on” regulation of drug cytotoxicity by conjugation to the platform and to a cell tar-
geting peptide. The versatility of this approach enables efficient production of drug-loaded platforms and
determination of favorable drug combinations/modes of linkage for subsequent conjugation to a carrier
moiety for targeted cancer cell therapy. The results presented here potentiate the application of amino
acid platforms for targeted drug delivery (TDD).
Received 26 May 2014
Received in revised form
19 July 2014
Accepted 21 July 2014
Available online 22 July 2014
Keywords:
Amino acid platform
Controlled drug release
Peptideedrug conjugates
Prodrug
© 2014 Elsevier Masson SAS. All rights reserved.
Solid phase organic synthesis
1. Introduction
exemplified by antibodyedrug conjugates (ADC): (a) Gemtuzumab
ozogamicin (Mylotag), contains Gemtuzumab, a recombinant hu-
Targeting drugs through biomolecular carriers with high affinity
to receptors on cancer cells can overcome two major problems in
anticancer therapy: the lack of target cell specificity of most anti-
cancer drugs and improvement of their toxicology [1]. Although
many targeted drug delivery approaches are being tested, the
linkage of several and different drugs to a single carrier molecule
might further enhance their therapeutic efficacy, particularly if the
drugs are engineered for variable time release.
Over the past two decades carrieredrug conjugates have been
developed for target cell delivery of potent anticancer drugs with
the aim of eliminating the morbidity-causing non-specific side ef-
fects common to conventional chemotherapy. Usually the carriers
are macromolecules such as monoclonal antibodies and other
proteins, or smaller molecular carriers like polynucleotide seg-
ments and peptides [2].
manized IgG4 monoclonal antibody (mAb) targeting the cell
membrane protein CD33, which is linked to several copies of cal-
icheamycin; (b) Vedotin (Adcetris), a chimeric antibody directed to
CD30, is conjugated to the antimitotic compound monomethyl
auristatin E; (c) Trastuzumab emtansine (T-DM1) is a conjugate of
the well-studied antibody Trastuzumab (Herceptin) that targets the
HER-2 cell surface protein, and maytansine derivative DM1; (d)
Inotuzumab ozogamicin, a conjugate of an IgG4 monoclonal anti-
body targeting the CD22 antigen linked to calicheamicin. Despite
these advances, ADCs still have several important limitations
including target cell specificity, conjugation chemistry, tumor
penetration and product heterogeneity [4]. Peptideedrug conju-
gates (PDCs) could overcome many, if not all, of these shortcomings
as we have described elsewhere [5], although they are yet to reach
the clinic. Several recent publications presented biologically active
peptideedrug conjugates, manifesting improvement of drug-like
features of the linked drugs. Sherz and coworkers reported on se-
lective accumulation and prolonged retention of the RGD analog
c(RGDfK) linked to fluorescent bacteriochlorophyll derivative in the
tumor necrotic domain in MDA-MB-231-RFP bearing mice. This
construct enables early detection of tumor growth and foster
prognosis and the development of novel modes of treatment [6].
Despite advances in these areas, the biomolecularedrug con-
jugates reported so far are limited to carry one drug type, although
drug conjugation chemistry is well elaborated [3]. This point is
* Corresponding author.
E-mail address: garyg@ariel.ac.il (G. Gellerman).
http://dx.doi.org/10.1016/j.ejmech.2014.07.073
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
Kostenich et al. developed accurate detection of small-cell lung
cancer (SCLC) with fluorescence-based imaging by preparing novel
backbone cyclic somatostatin analogs conjugated with fluorescein
and rhodamine [7]. We recently reported an increased efficacy of
multidose chlorambucilepeptide conjugates on murine leukemic B
cells through receptor-mediated endocytosis [8]. The most clini-
cally advanced peptideedrug-conjugate is GRN1005, an angio-
peptin-2-paclitaxol conjugate that targets lipoprotein receptor
protein-1, a cell surface molecule overexpressed on solid tumor
cells. The conjugate is under clinical assessment for treatment of
advanced solid tumors, in particular in patients with brain metas-
tases [9]. Although diverse, the above examples share one common
feature: they all describe conjugation of just one drug molecule (or
a few copies of the same drug) to the carrier. Nanoparticle systems
represent an approach that could be utilized for multiple drug
delivery [10]. Liposomes were demonstrated to be efficient vehicles
for the delivery of two drugs with very different solubility prop-
erties [11]. However, they suffer various limitations such as stability
and loading efficiency [12]. Cross-linked multilamellar liposomal
vesicles were studied in order to address those limitations [13].
Thus, there are no examples yet of well-defined unimolecular
platforms containing a few different drugs e a cocktail.
The chemistry of carrier-drug attachment has received much
attention. Important parameters include selection of a linker
attachment site that retains carrier activity, linker length and
composition, and the design of drug analogs for attachment to the
linker [14]. For ADCs, two methods are now commonly used for
conjugating drugs to antibodies: alkylation of reduced inter chain
cysteine disulfides through a non-cleavable maleimido linker and
acylation of lysine residues by cleavable linear amino acids [15].
Cathepsin-cleavable linkers are also utilized (for example Val-Cit,
or Phe-Lys) bound to self-emulative moiety PABA (p-aminobenzyl
alcohol), enabling selective drug release in cancer cells [14c].
Spacers are usually essential extensions of the drug linkage and are
responsible for avoiding the shielding of the active site of the
antibody as well as improving solubility properties of ADCs (for
example by the use of polyethylene glycol [16]).
carbonates; (3) Drugs can be linked to MAAPs through these same
types of moieties; (4) Linker-tagged drugs will be enzymatically
cleaved in vivo in the same manner as prodrugs; (5) The release
profile of the drugs will depend on the nature of the linker, linking
moiety and drug itself. By optimizing these parameters in the
design of loaded MAAPs that are subsequently conjugated to a
target specific carrier, we hope to release the payload specifically in
the target cancer cells and thereby avoid exposure of benign tissues
to the cytotoxic treatment [18]. We anticipate that MAAP technol-
ogy can be integrated into the rational design and application of
targeted drug delivery strategies and ultimately to a broader basket
of more effective therapies for cancer patients.
In this communication we describe the synthesis and initial
evaluation of first generation (G1) MAAPs (Fig. 1), linked to known
chemotherapy drugs, as well as the controllable release of the
payload. Several fundamental tasks were achieved during this
research: (1) Development of a novel synthetic protocol of orthog-
onally protective SPOS for the facile synthesis of heterogeneous
L-
Lys, -Ser and -Tyr based G1 MAAPs with selective drug loading
L
L
capabilities; (2) Measurements of chemostability at various pHs and
bio-stability in mouse liver homogenate for linker-containing drugs
and loaded MAAPs; (3) Screening the activity of free drugs vs pep-
tide carrier-conjugated drugs on cancer cells in vitro.
2. Results and discussion
2.1. Synthesis
Initially, we loaded MAAPs with four known anticancer drugs
and drug candidates and one previously reported 9-aminoacridine
anticancer compound YG-42 [19] (Fig. 2). These agents act through
different cellular pathways, therefore their combination on MAAPs
present a model for delivery of chemotherapeutic “cocktails”.
First, the DNA-Topo II intercalating inhibitor Azatoxin (AZA) [20],
the DNA-Topo I intercalating inhibitor Camptothecin (CAMP) and the
synthetic corticosteroid drug Prednisone (PRED) were functional-
ized for linkage to MAAP according to Scheme 1, forming activated
carbonate functionalities through the free phenolic or aliphatic OH.
Chlorambucil (CLB), a well known DNA mustard alkylator, and YG-42
were used without modification due to the presence of an existing
carboxylic group suitable forlinkage. Thereactions of CAMP, AZA and
PRED with pNO2PhOCOCl [21], when carried out under standard
conditions in heated pyridine or in DCM at room temperature in the
presence of excess of DMAP, afforded the preactivated drugs 1e3
respectively. These building blocks were used after standard workup
procedure without any further purification.
We have previously demonstrated that Ligand Drug Conjugates
(LDCs) can be successfully employed for the targeted delivery of
drugs and toxins to receptor-positive murine leukemic cells [8,17].
In particular, the use of multifunctional dendrone linkers that bear
several covalently bound DNA alkylating Chlorambucil molecules
to one peptide carrier have enhanced efficacy of inhibition of target
cancer cell growth [8]. Based on these results, we envision sub-
stantial therapeutic potential for drugecarrier conjugates that
consist of several different cytotoxic compounds linked via biode-
gradable linkages to Multifunctional Amino Acid Platforms (MAAP,
Fig. 1). The rationale behind this concept is based on the following:
(1) Amino acids are not toxic in vivo, (2) Prodrugs can be prepared
with enzymatically cleavable moieties such as esters, carbamates,
Acid-sensitive Cl-Trt polystyrene resin was chosen as solid
support for the synthesis of loaded G1 MAAPs for two reasons: (1)
The resin allows utilization of mild acidic cleavage conditions for
avoiding decomposition of the linked drugs; (2) The system yields
cleavage products of loaded MAAPs with a free carboxylic group
that can act as an anchor point for conjugation chemistry to a
carrier.
Y1
Drug1
Next, orthogonally protected Fmoc-(L)-Lys(Alloc)-OH was
loaded on to the resin using standard methods (DIEA, DMF then
MeOH for capping) [22]. After Fmoc release (20% piperidine/DMF,
2 ꢀ 20 min), the pre-made active ester of CLB (PyBoP, DMF) was
X
reacted with the deprotected L-Lys(Alloc) peptidyl residue at rt for
90 min, yielding the adduct 6. This compound was further depro-
tected to 5 by homogenous catalysis using Pd(PPh3)4 in the pres-
ence of barbituric acid ligand. At this point, adduct 5 was divided
into three parts, and each one was reacted with preactivated YG-42,
AZA or CAMP (see Scheme 2), yielding, after mild acidic cleavage
(3%TFA in DCM), the corresponding 4aec compounds in good yields
and purity (see Supplementary Information). In particular,
Y2 Drug2
X = CO2H, (CH2)nNH2, (CH2)SH; Y1,2= amide, ester, carbamate,
carbonate, sulfonamide, ureido, disulfide, etc
Fig. 1. General structure of multifunctional amino acid platform (MAAP) for loading of
two drugs.

Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
141
Cl
N
HN
N
O
O
MeO
HO
N
N
Cl
O
O
HO2C
O
HO
MeO
Chlorambucil (CBL)
DNA alkylator
Azatoxin (AZA)
Topo II inhibitor
Camptothecin (CAMP)
Topo I inhibitor
OH
O2N
HN
CO2H
O
OH
O
H
H
H
O
N
Prednisone (PRED)
Corticosteroid
YG-42
Fig. 2. Anticancer substances acting through different oncogenic mechanisms.
pharmaceutically active components YG-42 and CLB in 4a are
linked to the -Lys platform through amide bonds, while AZA and
(5 eq) was successfully coupled in two steps to the deprotected
Ser peptidyl 11a (EDC/DMAP), affording after cleavage the desired
L-
L
CAMP in 4b,c are linked through phenolic and aliphatic carbamates,
G1 MAAP 8, that bears two molecules of CLB linked to
L-Ser by two
respectively.
different biodegradable moieties, namely amide at the a-amine and
In the following step,
the synthesis of G1 MAAPs. These amino acids have two different
functionalities for drug linkage: -amine and a primary or a
phenolic OH respectively on the side chain. The purpose of this
stage was to extend the linkage repertoire for coupling of two
bioagents, and to measure the drug release rate vs linkage mode.
L
-serine and
L
-tyrosine were employed in
ester on the side chain hydroxyl. Being a hetero-tethering amino
acid, -Ser was found useful for loading of another combination of
L
a
drugs through a different set of biodegradable moieties. For
demonstrating this, 12a first underwent esterification with active
ester of CLB (EDC, DMAP) yielding 13, and then Fmoc removal
afforded deprotected Ser(CLB) residue on the solid support.
Importantly, no intramolecular migration of CLB from the side
Initially, Fmoc-(L)-Ser-OH was loaded onto Cl-Trt resin (Scheme 3)
using the same procedure as for 7. Notably, reacting the
a
-amino
chain to the a-amine was observed by HPLC and NMR analysis (data
protected Ser without side chain protection did not disrupt the
regioselective loading toward 12a, most probably due to the
reduced nucleophilicity of OH under the coupling conditions. This
significantly simplified the synthesis by avoiding the use of a sec-
ond orthogonally protected group. Subsequently, after removal of
Fmoc under standard conditions, the premade active ester of CLB
not shown). The resulting peptidyl was coupled with the activated
carbonate derivative of PRED 3 (DIEA, DMF, rt, 90 min; twice) to
give, after subsequent cleavage, the desired MAAP 10 in good yield
and purity (see Supplementary Information). In the resulting MAAP
10, CLB is linked through a biodegradable ester while PRED is linked
through a biodegradable aliphatic carbamate.
O2N
O
O
O
O2N
O
O
O
O
O2N
OMe
MeO
HN
O
AZA
a
CAMP
O
b
O
Cl
N
O
N
N
O
O
b
PRED
1 (94%)
O
2 (92%)
O
NO2
O
O
OH
O
H
H
H
3 (95%)
O
(a) Pyridine, 90oC, 1h; (b) DMAP (6eq.) DCM, 00C.
Scheme 1. Chemical activation of CAMP, AZA and PRED by p-nitrophenyl chloroformate.

142
Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
Scheme 2. Synthesis of Lys-diheteronuclear platforms 4aec.
2.2. Chemo-stability of drug-linkers
The results of these experiments indicate that carbamate and
ester chemodegradable linkages are superior to amides. This served
For screening of degradable linker structures appropriate for
construction of drug-loaded MAAPs, several drug-linker derivatives
were initially synthesized (Fig. 3).
as a basis for preparing peptideemultidrug conjugates.
2.3. Bio-stability of bis-CLB MAAPs
According to a previous report, modification of the 20-hydroxy
group in the lactone ring moiety of CAMP contributes to
enhancing its stability [23]. Therefore, CAMP derivative 15, with a
carbamate functionality substituting the aliphatic OH was prepared
[24]. Additionally, AZA derivative 16 [25], which includes activated
phenolic carbamate, was obtained, and the amidated DNA inter-
calator AHMA derivative 17 was obtained from AHMA [26] pre-
cursor and glutaric anhydride [27]. The chemo-stability studies of
15e 17 were performed at three pH values (pH 2.0, 5.0 and 7.4).
Aliquots taken at selected time intervals were analyzed by analyt-
ical HPLC and ES-MS. The results are shown in Fig. 4.
All three tested compounds showed high stability at pH 2 and pH
5 (>6 days). However, in tris buffer (pH 7.4) at 37 ꢁC, compounds 15
and 16 exhibited pseudo first- order kinetics drug release profile of
CAMP (t1/2 ¼ 76.3 h) and AZA (t1/2 ¼ 9.4 h) respectively. These results
are consistent with the expected stability of the phenolic carbamate
in AZA, which is known to degrade more rapidly than the aliphatic
carbamate in CAMP. AHMA derivative 17 remained stable (>6 days).
The chemo-stability of 8 and 9 was then also tested (Fig. 4),
showing the same trend observed for 15 and 16. Thus, the aliphatic
ester of 8 was more stable than the phenolic ester of 9 (t1/2 ¼ 92.4
and 6.9 h, respectively). As expected, the amide bond with the
second CLB compound in 8 was much more stable than the ester
bond with the first CLB compound, as demonstrated by the iden-
tical kinetics of the formation of CLB (the product of the ester bond
hydrolysis) and of Ser-CLB (containing the amide bond). The sta-
bility of the phenolic ester bond of 9 was pH dependent, being
much more labile at pH 2 (t1/2 ¼ 2.8 h). All experiments were
monitored by HPLC against premade standards.
We further characterized the bio-stability of simple L-Ser and L-
Tyr G1 MAAP 8 and 9 respectively in liver homogenates (LH) (see
Supplementary Information), loaded with two equivalents of CLB,
for measurement of CLB release rate vs linkage moiety. The results
revealed an unstable phenolic ester linkage on
L-Tyr platform 9, (t1/
¼ 5.8 min, (LH)), whereas the same drug linked to
L
-Ser platform 8
2
through an alkyl ester moiety exhibited prolonged stability (t1/
¼ 22.3 min (LH), Fig. 5). In both experiments the mono-adduct,
2
namely a-amidated AA-CLB, was observed and subsequently un-
derwent decomposition. Interestingly, the half-life of the mono-
adduct of -Ser-CLB was longer than that exhibited by -Tyr-CLB,
most probably because of the higher stability of G1 -Ser MAAP.
Notably, released CLB exhibited a significant accumulation profile,
especially in -Tyr G1 MAAP, with slow CLB degradation rate.
L
L
L
L
These results indicate that drug e MAAP linkages can be based
on ester, carbamate and amide functionalities, with esters being the
most bio-cleavable moieties. This information allowed us to pro-
ceed to the biological tests of the compounds.
2.4. “Switch off/switch on” of drug cytotoxicity against cancer cells
CAMP is a potent inhibitor of DNA topoisomerase I (Topo I). By
stabilizing the covalent binding of Topo I to DNA, it induces irre-
versible and lethal strand breaks of DNA during its replication [28].
Unfortunately, its unfavorable properties such as non-specific
toxicity and negligible water solubility [29] have resulted in sus-
pension of its clinical use in cancer therapy. To overcome these
drawbacks, significant efforts have been made to develop various

Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
143
Scheme 3. Synthesis of Ser and Tyr-dinuclear platforms 8e10.
modifications such as CAMP prodrugs [30] and its combination
with drug delivery systems [31], some of which have been assessed
in clinical trials [32].
To test the “switch off” hypothesis, the growth of MBP and BCL1
hybridoma cancer cell lines was assessed following their culture for
48 h with free- and pro-drugs over a concentration range of
In light of these results, we asked whether the cytotoxic activity
of CAMP could be controlled by chemical modification and appro-
priate delivery. Could this activity be switched off by converting the
compound to a prodrug and then switched on again by conjugating
the prodrug to a carrier, which would deliver it to the target cancer
cell where it would undergo activation?
5e50 mM. MBP is a cell line expressing a surface antibody specific to
a 13 amino acid fragment of the Myelin Basic Protein (MBP). BCL-1
is a cell line of similar lineage expressing a surface antibody to a
different antigen. We previously used these cell lines to demon-
strate the specificity of other peptideedrug conjugates for anti-
cancer therapy [17]. More recently, we demonstrated that
O
O
O
N
O
HN
OH
N
N
H
N
O
O
O
OH
HN
N
O
O
O
O
O
NH
O
NH
17
16
15
CO2H
HO2C
Fig. 3. CAMP, AZA and AHMA derivatives 15e17, respectively, with various carboxylated linkers.

144
Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
compound 8
compound 9
100
80
60
40
20
0
80
70
60
50
40
30
20
10
0
8
pH 7.4
pH 5.0
pH 2.0
CLB
Ser-CLB
0
5
10
15
20
25
30
0
50
100
time (h)
150
200
time (h)
compound 15
compound 16
90
80
70
60
50
40
30
20
10
100
80
60
40
20
0
15
16
CPT
AZA
0
5
10
15
20
25
30
35
40
0
50
100
150
200
time (h)
time (h)
Fig. 4. Chemo-stability of drug-linker of compounds 8, 9, 15 and 16. Stock solutions were made of 2e5 mg of the measured compound dissolved in 500 ml DMSO. 100 ml of stock
solution were diluted to 2.5 ml with PBS buffer pH 7.4. The samples were incubated at 37 ꢁC. Aliquots were taken at various time points along the incubation, filtered and analyzed
by HPLC. Chemo-stability of 9 is also shown at pH 5.0 and 2.0. The points correspond to the experimental data, and the lines represent fitting to first order kinetics.
multifunctional Lys platforms bearing several CLB molecules
covalently bound to a single copy of the MBP peptide fragment
induced enhanced growth inhibition in target cells compared to
PDCs carrying a single CLB molecule [8].
Fig. 6A shows that the only cytotoxic compound was free CAMP.
As free CLB alone was not effective, the results for the mixture of
free CAMP and free CLB represent the effect of CAMP. The effect of
the drug was non-specific as it equally inhibited the growth of both
MBP and BCL-1 cells. Significantly, the CAMP þ CLB MAAP prodrug
4c was not active against either cell line. During the 48 h of
incubation, there was probably some degradation of the prodrug
and release of free drug, but this seems to be of consequence to the
cells only at higher concentrations (50 mM). These results support
the first hypothesis, namely that CAMP activity can be “switched
off” by transforming it into a prodrug as a MAAP conjugate.
In contrast, Fig. 6B demonstrates how CAMP activity can be
“switched on” again, following specific delivery to target cells. The
linkage of CLB and CAMP to the Lys MAAP (4c) and subsequent
conjugation to the MBP carrier to produce MBP-Lys(CLB)CAMP 14
(Scheme 4) is described in the Supplementary Information.
Fig. 5. Bio-stability of G1 MAAPs L-Ser 8 (left) and L-Tyr 9 (right). General procedure: to prepare the stock solution, 5 mg of 8 or 9 were dissolved in 500 ml DMSO. 50 ml of stock
solution were diluted with 2 ml of fresh mouse liver homogenate. The mixture was incubated at 37 ꢁC. Aliquots of the mixture were collected at 10, 30, 60, 90 and 120 min and
immediately mixed with 2.5 volumes of ethanol. To remove the precipitate the samples were centrifuged at 14,000 for 15 min. Supernatants were collected carefully, filtered and
analyzed by HPLC. Each graph point represents the mean and SD of at least three measurements.

Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
145
Fig. 6. “Switch off/switch on” regulation of CLB þ CAMP “cocktail” cytotoxicity by conjugation to a cell targeting MBP peptide. A: Free or platform-linked drugs. B: MBP-conjugated
drugs. The effect of free-, platform-linked and peptide-conjugated drugs on cancer cells growth was studied. MBP or BCL-1 hybridoma cells were cultured for 48 h either alone or
together with different compounds in increasing concentrations (5e50 mM). At the end of the culture period, cell growth was estimated by XTT assay. After 4 h incubation of the
cells with the XTT reagent, optical density (OD) of the reduced XTT product was measured at 480 and 680 nm. Percentage of growth inhibition by a test compound was calculated by
comparison of the treated culture versus a control culture (free of any compound). The result shown for each concentration point represents the mean standard error calculated
from 3 different experiments. In each experiment the compounds were tested in triplicates.
The results clearly support the second hypothesis, that CAMP
activity can be “switched on” again by conjugating the prodrug to a
carrier peptide that delivers the peptideedrug conjugate (PDC) to
the target cells. The MBP-CAMP/CLB PDC was extremely specific,
suitable for combinatorial synthesis of loaded MAAPs, signifi-
cantly accelerating the discovery of favorable parameters
matching essential for defining desired release profiles of the
drugs from the platforms.
being non-toxic to BCL-1 at 5 and 10
m
M. The results shown for the
Our results showed high variability in the bio-stability of the
drugs linked through various moieties and therefore strongly
support our hypothesis that exposure to cleavage enzymes will lead
to differing kinetics of drug release from the loaded MAAP.
We have demonstrated that linkage of a cytotoxic drug cocktail
consisting of DNA alkylator CLB and Topo I inhibitor CAMP to the
Lys platform and subsequent conjugation to the MBP carrier gains
“switch off/switch on” capabilities, specifically activating the
cocktail in the target MBP hybridoma cells. In this study, a cocktail
of free CLB and CAMP was non-specifically cytotoxic to hybridoma
cell lines. Platform Lys(CLB)CAMP was not cytotoxic to any cell line
(switch off), while MBP peptide e Lys(CBL)CAMP conjugate showed
MBP-CLB PDC are consistent with our previous report in which we
compared the efficacy of Lys platforms bearing 1, 2 or 4 CLB mol-
ecules covalently bound to the MBP peptide [8].
3. Conclusions
The versatility of the synthetic approach presented here
suggest that utilization of other L and D amino acids like Thr, Asp,
Glu and Cys may also be successful for construction of MAAPs.
Bearing functional side chains suitable for drug linkage, these
amino acids can form remarkable architectures with highly ver-
satile and tunable drug linkage/release capabilities. Moreover,
other semi-orthogonal and orthogonal combinations of protect-
ing groups are applicable in such methodology, for instance: Dde/
Fmoc, o-Nosyl/Fmoc, Teoc/Fmoc, Acm/Fmoc. More acid sensitive
resins can also be employed leading, after cleavage, to various
non-carboxylic anchor points such as amines or thiols for
conjugation properties. Importantly, the developed facile SPOS is
remarkable specific cytotoxicity (90% growth inhibition/1 mM) on
MBP hybridoma (switch on).
Based on these results we are now constructing and assessing
more sophisticated MAAPs bearing diverse chemotherapeutic
“cocktails” in different linkage variations. These are being conju-
gated to peptide carriers of clinical significance for preclinical
cancer therapy assessment.
O
N
O
N
O
O
HN
O
O
a
4c
NH2
-HPTRPTVINKFFHV-GABA-NH2
H2N-HPTRPTVINKFFHV-GABA
O
b,c
HN
Rink
Amide
protected MBP Peptide
MBP Peptide
Cl
N
14 MBP-(CLB+CAMP) (47%)
Cl
(a) Fmoc Chemistry SPPS; (b) 4c, PyBOP, DIPEA/DMF, rt, 90 min; (c) TFA/H2O/TIPS/DCM (3:2:2:93), 0oC-rt, 90 min.
Scheme 4. Solid phase synthesis of MBP-(CLB þ CAMP) 14 on Rink Amide resin.

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Y. Gilad et al. / European Journal of Medicinal Chemistry 85 (2014) 139e146
[15] (a) M.M.C. Sun, K.S. Beam, C.G. Cerveny, K.J. Hamblett, R.S. Blackmore,
Acknowledgment
M.Y. Torgov, F.G.M. Handley, Reduction-alkylation strategies for the modifi-
cation of specific monoclonal antibody disulfides, Bioconjugate Chem. 16
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The authors thank Mrs. Cherna Moskowitz for generous sti-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.073.
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