Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting

Article abstract of DOI:10.3762/bjoc.14.29

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified α_Vβ₃ receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of α_Vβ₃ integrin expression: human glioblastoma U87 (α_Vβ₃+), human lung carcinoma A549 (α_Vβ₃?) and breast adenocarcinoma MDA-MB-468 (α_Vβ₃?). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of α_Vβ₃ integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (α_Vβ₃+, α_Vβ₅+, α_Vβ₆?, α₅β₁+) and MDA-MB-468 (α_Vβ₃?, α_Vβ₅+, α_Vβ₆+, α₅β₁?) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC₅₀ was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only α_Vβ₃, but also α_Vβ₅, α_Vβ₆, and α₅β₁. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from α_Vβ₃ (e.g., α_Vβ₅).

Full text of DOI:10.3762/bjoc.14.29

Synthesis and biological evaluation of RGD and isoDGR
peptidomimetic-α-amanitin conjugates for tumor-targeting
Lizeth Bodero‡1, Paula López Rivas‡2, Barbara Korsak3, Torsten Hechler3,
Andreas Pahl3, Christoph Müller3, Daniela Arosio4, Luca Pignataro2, Cesare Gennari*2
and Umberto Piarulli*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 407–415.
1Dipartimento di Scienza e Alta Tecnologia, Via Valleggio, 11, 22100,
doi:10.3762/bjoc.14.29
Como, Italy, 2Dipartimento di Chimica, Università degli Studi di
Milano, Via C. Golgi, 19, I-20133, Milan, Italy, 3Heidelberg Pharma
Research GmbH, Schriesheimer Strasse 101, 68526, Ladenburg,
Germany and 4CNR, Istituto di Scienze e Tecnologie Molecolare
Received: 30 November 2017
Accepted: 31 January 2018
Published: 14 February 2018
(ITSM), Via C. Golgi, 19, 20133, Milan, Italy
This article is part of the Thematic Series "Peptide–drug conjugates".
Guest Editor: N. Sewald
Email:
Cesare Gennari* - cesare.gennari@unimi.it; Umberto Piarulli* -
umberto.piarulli@uninsubria.it
©
2018 Bodero et al.; licensee Beilstein-Institut.
*
Corresponding author ‡ Equal contributors
License and terms: see end of document.
Keywords:
antitumor agents; cancer; drug delivery; integrins; peptidomimetics
Abstract
RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers
and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αVβ3 receptor,
retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was
evaluated in three cell lines possessing different levels of αVβ3 integrin expression: human glioblastoma U87 (αVβ3+), human lung
carcinoma A549 (αVβ3−) and breast adenocarcinoma MDA-MB-468 (αVβ3−). In the U87, in the MDA-MB-468, and partly in the
A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were
found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the
cytotoxicity and the expression of αVβ3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-
amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried
out in which the conjugates were tested with U87 (αVβ3+, αVβ5+, αVβ6−, α5β1+) and MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+, α5β1−)
cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line,
a fivefold increase of the IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly
bind not only αVβ3, but also αVβ5, αVβ6, and α5β1. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conju-
gates are possibly internalized by a process mediated by integrins different from αVβ3 (e.g., αVβ5).
407

Beilstein J. Org. Chem. 2018, 14, 407–415.
Introduction
α-Amanitin is a bicyclic octapeptide toxin belonging to the receptor ligand – avoids the drawbacks of ADCs such as high
amatoxin family, found in Amanita Phalloides (death cap mush- manufacturing costs, unfavorable pharmacokinetics (low tissue
room), see Figure 1 [1]. Its mechanism of action consists in the diffusion and low accumulation rate) and possible elicitation of
inhibition of cellular transcription by an effective blocking of immune response [6]. By conjugation to a specific cell-mem-
RNA polymerase II, which is present in the nuclei of eukary- brane-receptor ligand, the toxin can be delivered at the tumor
otic cells and is responsible for the transcription of DNA to site and internalized through receptor-mediated endocytosis. In
mRNA [1,2]. Despite this strong inhibitory activity, α-amanitin 2013, Reshetnyak and co-workers conjugated α-amanitin to
exhibits only a micromolar cytotoxicity and low cellular uptake pHLIP (pH low insertion peptide) via linkers of different
in most mammalian cells, due to its strong polarity and poor hydrophobicities [7]. The results indicated that pHLIP could
membrane permeability [2]. One notable exception are human deliver α-amanitin into cells and induce cell death in 48 h by a
hepatocytes, where the transporting protein OATP1B3 internal- pH-mediated direct translocation across the membrane and
izes amatoxins resulting in high liver toxicity [2,3].
cleavage of the disulfide linker in the cytoplasm. In another ex-
ample, Perrin and co-workers conjugated the N-propargylas-
paragine of an amanitin analog to a cycloRGD integrin ligand
(
cyclo[RGDfK]) using a copper-catalyzed azide–alkyne cyclo-
addition [8]. The conjugates were tested in the U87 glioblas-
toma cell line, but only a slight enhancement in toxicity over
α-amanitin was observed.
The transmembrane receptor αVβ3 integrin is widely expressed
on the blood vessels of several human cancers (for example,
breast cancer, glioblastoma, pancreatic tumor, prostate carci-
noma) but not on the vasculature of healthy tissues [9-11], and
therefore constitutes a suitable therapeutic target in the field of
SMDCs. Integrin αVβ3 recognizes endogenous ligands by the
tripeptide arginine-glycine-aspartate [12] (RGD) and also by the
related sequence isoaspartate-glycine-arginine (isoDGR) [13-
Figure 1: α-Amanitin.
This strong toxicity in the presence of endocytosis mediators 20]. Many synthetic peptides or peptidomimetics containing
allowing cell permeation, aroused interest towards the use of these sequences have been prepared and show low nanomolar
α-amanitin as a payload for targeted cancer therapy. In 1981, IC50 values for integrin αVβ3 binding [21-27]. A number of
Davis and Preston reported the synthesis of the antibody–drug cyclic RGD and isoDGR ligands containing a bifunctional dike-
conjugate (ADC) α-amanitin-anti-Thy 1.2 IgG, which was topiperazine (DKP) scaffold have been developed by the
4
7-fold more toxic than the unconjugated α-amanitin in the Gennari and Piarulli groups in the last decade [24-27]. Among
murine T lymphoma S49.1 cell line [4]. In 2012, a new ADC them, the cyclo[DKP-RGD] 1 [25] and cyclo[DKP-isoDGR] 3
containing α-amanitin and a chimerized anti-EpCAM (epithe- [26] (Figure 2) showed a binding affinity for the purified recep-
lial cell-adhesion molecule) monoclonal antibody was prepared tor αVβ3 in the low nanomolar range and a good selectivity for
by Moldenhauer and co-workers [5]. The cytotoxicity of this this integrin in comparison with integrin αVβ5 (33–34 times, see
conjugate was tested in EpCAM-overexpressing cancer cell Table 1).
lines obtaining IC50 values from 2.5 × 10−10 to 2.0 × 10−12 M.
Promising results were also observed in mice bearing BxPc-3 Furthermore, these ligands were shown to inhibit the FAK
pancreatic xenograft tumors, with complete tumor regression in (focal adhesion kinase) and Akt (protein kinase B) signaling
9
0% of the cases after two injections of the α-amanitin-anti- cascade and the tumor cell infiltration process, performing as
EpCAM ADC at a dose of 100 μg/kg with respect to α-amani- true integrin antagonists [27].
tin. In these two examples, the internalization of the mono-
clonal antibody and subsequent release of the toxin leads to the Ligands 1 and 3 were also functionalized with an aminomethyl
enhancement of α-amanitin activity on the targeted cells.
group (-CH2NH2) as a handle for conjugation to cytotoxic drugs
Figure 2, ligands 2 and 4) [28-30]. Conjugates of the function-
(
An alternative approach to the antibody targeted therapy is alized ligands 2 and 4 with paclitaxel (PTX) via a 2’-carbamate
represented by small molecule–drug conjugates (SMDCs), with a self-immolative spacer and the lysosomally cleavable
where the small molecule – usually a peptide or peptidomimetic Val-Ala linker [31] were synthesized (Figure 2, cyclo[DKP-
408

Beilstein J. Org. Chem. 2018, 14, 407–415.
Figure 2: Structure of the ligands cyclo[DKP-RGD] (1), NH2CH2-cyclo[DKP-RGD] (2), cyclo[DKP-isoDGR] (3), NH2CH2-cyclo[DKP-isoDGR] (4) and
the related SMDCs cyclo[DKP-RGD]-Val-Ala-PTX (5) and cyclo[DKP-isoDGR]-Val-Ala-PTX (6).
Table 1: Inhibition of biotinylated vitronectin binding to αvβ3 and αvβ5 receptors.
ligand
structure (name)
IC50 (nM)a αVβ3
IC50 (nM)a αVβ5
1
3
cyclo[DKP-RGD]
4.5 ± 1.1
9.2 ± 1.1
149 ± 25
312 ± 21
cyclo[DKP-isoDGR]
aIC50 values were calculated as the concentration of compound required for 50% inhibition of biotinylated vitronectin binding as estimated by
GraphPad Prism software. All values are the arithmetic mean ± the standard deviation (SD) of triplicate determinations.
RGD]-Val-Ala-PTX 5 and cyclo[DKP-isoDGR]-Val-Ala-PTX Results and Discussion
6
). Their tumor targeting ability was assessed in vitro in anti- In the present paper, we report the synthesis and biological
proliferative assays comparing an αVβ3 positive with an αVβ3 evaluation of two cyclo[DKP-RGD]-α-amanitin and three
negative cell line [29,30]. The cyclo[DKP-isoDGR]-Val-Ala- cyclo[DKP-isoDGR]-α-amanitin conjugates. In these conju-
PTX conjugate 6 displayed a remarkable targeting index gates, the integrin ligands are bound to α-amanitin via a 6’-ether
(
TI = 9.9), especially when compared to the strictly related with two different linkers: an “uncleavable” six carbon aliphat-
cyclo[DKP-RGD]-Val-Ala-PTX conjugate 5 (TI = 2.4) [30]. ic chain (Figure 3, compounds 7 and 8) and a lysosomally
409

Beilstein J. Org. Chem. 2018, 14, 407–415.
Figure 3: Structure of the α-amanitin conjugates: cyclo[DKP-RGD]-uncleavable-α-amanitin (7), cyclo[DKP-isoDGR]-uncleavable-α-amanitin (8),
cyclo[DKP-RGD]-Val-Ala-α-amanitin (9), cyclo[DKP-isoDGR]-Val-Ala-α-amanitin (10) and cyclo[DKP-isoDGR]-PEG-4-Val-Ala-α-amanitin (11).
cleavable Val-Ala linker bound to a self-immolative spacer Integrin receptor competitive binding assays
(
Figure 3, compounds 9, 10 and 11). Integrin receptor competi- Conjugates 7–11 were evaluated for their ability to inhibit
tive binding assays and cell proliferation assays with an αVβ3 biotinylated vitronectin binding to the purified αVβ3 receptor.
positive (U87) and two αVβ3 negative cell lines (A549 and The calculated half-maximal inhibitory concentrations (IC50)
MDA-MB-468) were performed for all the conjugates.
are listed in Table 2. Screening assays were performed by incu-
bating the immobilized integrin receptor with solutions of the
cyclo[DKP-RGD]-α-amanitin and cyclo[DKP-isoDGR]-α-ama-
Synthesis
Cyclo[DKP-RGD]-α-amanitin and cyclo[DKP-isoDGR]-α-ama- nitin conjugates 7–11 at different concentrations (10−12 to
nitin conjugates 7–11 were synthesized as described in 10−5 M) in the presence of biotinylated vitronectin (1 µg mL−1)
Scheme 1 and Scheme 2, by joining the functionalized ligands and measuring bound vitronectin.
H2NCH2-cyclo[DKP-RGD] (2) [18,29] and H2NCH2-
cyclo[DKP-isoDGR] (4) [30] to α-amanitin via a 6’-ether with It was found that the cyclo[DKP-RGD]-α-amanitin conjugates 7
various linkers and spacers. Details are reported in Supporting and 9 and cyclo[DKP-isoDGR]-α-amanitin conjugates 8, 10 and
Information File 1.
11 retain good binding affinity for αVβ3 integrin, in the same
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Beilstein J. Org. Chem. 2018, 14, 407–415.
Scheme 1: Synthesis of α-amanitin-cyclo[DKP-RGD] and α-amanitin-cyclo[DKP-isoDGR] conjugates 7–10. Reagents and conditions: a) 1. glutaric
anhydride, DMAP, iPr2NEt, DMF, overnight, 2. DIC, N-hydroxysuccinimide, DMF, overnight; b) di-N-succinimidyl glutarate, iPr2NEt, rt, DMF, 6 h;
c) H2NCH2-cyclo[DKP-RGD] (2), PBS/MeCN or PBS/DMF (pH 7.5), overnight; d) H2NCH2-cyclo[DKP-isoDGR] (4), PBS/DMF (pH 7.5), overnight.
DMAP: 4-dimethylaminopyridine; DIC: diisopropylcarbodiimide; PBS: phosphate-buffered saline; PAB: p-aminobenzyl.
range as the free ligands (cf. Table 2 with Table 1). These while A549 cells (human lung carcinoma) and MDA-MB-468
results encouraged us to proceed with cell viability assays in (breast adenocarcinoma) [32] were used as αVβ3 negative.
αVβ3 positive and αVβ3 negative cell lines, to study the ability The expression of αVβ3 integrin on the cell membrane was
of the conjugates to selectively target αVβ3 expressing tumor assessed by flow cytometry (see Supporting Information File 1,
cells.
Figure S1), and the results were in good agreement with the lit-
erature for U87 and A549 [33-35]. In the case of MDA-MB-
468, while the presence of the β3 integrin subunit is still quite
Cell viability assays
The antiproliferative activity of the conjugates was evaluated in controversial in the literature [36-38], our FACS analysis could
three cell lines expressing different levels of αVβ3 integrin. not detect any αVβ3 expression (see Supporting Information
U87 cells (human glioblastoma) were selected as αVβ3 positive, File 1).
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Beilstein J. Org. Chem. 2018, 14, 407–415.
Scheme 2: Synthesis of cyclo[DKP-isoDGR]-PEG-4-Val-Ala-α-amanitin conjugate 11. Reagents and conditions: a) 4-pentynoic acid N-hydroxysuccin-
imidyl ester, iPr2NEt, DMF, overnight; b) N3-PEG-4-cyclo[DKP-isoDGR], sodium ascorbate, CuSO4·5H2O, DMF/water, rt, overnight.
Table 2: Inhibition of biotinylated vitronectin binding to αvβ3 receptor.
compound
structure (name)
IC50 (nM)a αVβ3
7
8
9
cyclo[DKP-RGD]-uncleavable-α-amanitin
cyclo[DKP-isoDGR]-uncleavable-α-amanitin
cyclo[DKP-RGD]-Val-Ala-α-amanitin
11.6 ± 2.4
6.8 ± 4.3
14.7 ± 6.6
6.4 ± 1.9
3.8 ± 0.3
10
1
cyclo[DKP-isoDGR]-Val-Ala-α-amanitin
cyclo[DKP-isoDGR]-PEG-4-Val-Ala-α-amanitin
1
aIC50 values were calculated as the concentration of compound required for 50% inhibition of biotinylated vitronectin binding as estimated by
GraphPad Prism software. All values are the arithmetic mean ± the standard deviation (SD) of triplicate determinations.
The cell lines were treated with different concentrations of the and MDA-MB-468 (αVβ3−) cell lines (2.1–2.8 times, cf. entry 1
free drug α-amanitin and conjugates 7–11 for 96 hours. The cell with entry 6 in Table 3), while in A549 cell line (αVβ3−) it
viability was evaluated with the CellTiterGlo 2.0 assay and the turned out to be less potent (1.4 times) than the free drug.
calculated IC50 are shown in Table 3.
Cyclo[DKP-RGD]-uncleavable-α-amanitin (7), cyclo[DKP-
isoDGR]-uncleavable-α-amanitin (8) and cyclo[DKP-RGD]-
The cyclo[DKP-isoDGR]-α-amanitin conjugate bearing the Val-Ala-α-amanitin (9) proved less potent than α-amanitin in all
lysosomally cleavable Val-Ala linker 10 proved slightly cell lines (see Table 3, cf. entry 1 with entries 2–4). In general,
more potent than α-amanitin in the U87 (αVβ3+) cell line, as one can conclude that the “uncleavable” compounds 7 and 8 are
well as in the A549 and MDA-MB-468 (αVβ3−) cell lines much less cytotoxic than free α-amanitin, while the lysoso-
(
2.4–3.1 times, cf. entry 1 with entry 5 in Table 3). The mally cleavable compounds 9–11 show variable results, with
cyclo[DKP-isoDGR]-α-amanitin conjugate bearing the lysoso- RGD compound 9 behaving worse than the free drug but better
mally cleavable Val-Ala linker and a PEG-4 spacer 11 proved than the corresponding uncleavable conjugate 7. The isoDGR
slightly more potent than α-amanitin in both the U87 (αVβ3+) motif gives generally better results, with 10 and 11 behaving
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Beilstein J. Org. Chem. 2018, 14, 407–415.
Table 3: Evaluation of anti-proliferative activity of α-amanitin and α-amanitin conjugates 7–11 in U-87, MDA-MB-468 and A549.
entry
structure (name)
IC50 (nM)a
U87 (αVβ3+)
MDA-MB-468 (αVβ3−)
A549 (αVβ3−)
1
2
3
4
5
6
α-amanitin
347 ± 132.5b
2552 ± 37.6
3355 ± 19.1
1446 ± 83.9
143 ± 33.8
165 ± 4.0
185 ± 49.6b
1111 ± 228.4
2200 ± 96.2
202 ± 10.3
59 ± 23.4
518 ± 305b
n.d.c
cyclo[DKP-RGD]-uncleavable-α-amanitin (7)
cyclo[DKP-isoDGR]-uncleavable-α-amanitin (8)
cyclo[DKP-RGD]-Val-Ala-α-amanitin (9)
cyclo[DKP-isoDGR]-Val-Ala-α-amanitin (10)
cyclo[DKP-isoDGR]-PEG-4-Val-Ala-α-amanitin (11)
n.d.c
2160 ± 23.3
217 ± 98.3
720 ± 98.1
66 ± 24.1
aIC50 values were calculated as the concentration of compound required for 50% inhibition of cell viability. All cell lines were treated with different
concentrations of α-amanitin and compounds 7–11 for 96 hours. The samples were measured in triplicate. bAverage values from three independent
experiments. cn.d.: these data could not be determined.
much better than the corresponding uncleavable conjugate 8 and emerges between the expression of integrin αVβ3 and cytotoxic-
slightly better than the free drug. Apparently, for all the α-ama- ity. However, it should be noted that these cell lines overex-
nitin conjugates there is no direct correlation between the cyto- press other integrins (α5β1 in U87, αVβ6 in MDA-MB-468, and
toxicity and the expression of αVβ3 integrin [39].
αVβ5 in both) [34-36]. Thus, the increase of the IC50 of conju-
gates 10 and 11 (up to 5.5 times) may be possibly due to the
To determine whether the increased cytotoxicity of cyclo[DKP- block of other integrins with excess cilengitide, which is known
isoDGR]-α-amanitin conjugates 10 and 11 is governed by an to efficiently bind not only αVβ3 (IC50 = 0.6 nM) [23], but also
integrin-mediated binding and internalization process, competi- αVβ5 (IC50 = 8.4 nM) [23], αVβ6 (IC50 = 82.8 nM) [41], and
tion experiments [40] were carried out in which conjugates 10 α5β1 (IC50 = 14.9 nM) [23].
and 11 were tested on U87 (αVβ3+, αVβ5+, αVβ6−, α5β1+)
[
[
[
(
34-36] and on MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+, α5β1−) Conclusion
34-36] cells in the presence of 50-fold excess of cilengitide In this paper, two cyclo[DKP-RGD]-α-amanitin and three
23], with the aim of blocking integrins on the cell surface cyclo[DKP-isoDGR]-α-amanitin conjugates were prepared in
Table 4, see also Supporting Information File 1, Biological good yields following a straightforward synthetic route. Conju-
assays). Using the U87 cell line, a modest IC50 increase of gates 7–11 retain good binding affinities for the purified αVβ3
conjugate 11 from 91 nM (without cilengitide) to 143 nM (with receptor, in the same range as the respective free ligands. Cell
excess cilengitide) was observed (Table 4, entry 2). Using the proliferation assays were performed with three cell lines
MDA-MB-468 cell line, a more pronounced IC50 increase was possessing different levels of integrin expression: human
observed for both conjugates 10 (from 47 nM without cilengi- glioblastoma U87 (αVβ3+), human lung carcinoma
tide to 259 nM with excess cilengitide; Table 4, entry 1) and 11 A549 (αVβ3−) and breast adenocarcinoma MDA-MB-468
(
from 65 nM without cilengitide to 340 nM with excess cilengi- (αVβ3−). With all these cell lines the cyclo[DKP-isoDGR]-α-
tide; Table 4, entry 2). From these results, no correlation amanitin conjugate 10 proved slightly more potent than
Table 4: Competition experiments of conjugates 10 and 11 in the presence of a 50-fold excess of cilengitide in U-87 and MDA-MB-468.
entry
compound
IC50 (nM)a
MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+, α5β1−)
U87 (αVβ3+, αVβ5+, αVβ6−, α5β1+)
1
0
107 ± 26.8
106 ± 11.6
47 ± 21.1
1
2
1
0 + 50-fold excess of cilengitide
259 ± 55.2
11
91 ± 30.6
65 ± 17.6
1
1 + 50-fold excess of cilengitide
143 ± 59.3
340 ± 210.3
aIC50 values were calculated as the concentration of compound required for 50% inhibition of cell viability. Both cell lines were treated with different
concentrations of compounds 10 and 11 in the presence of a 50-fold excess of cilengitide during 96 hours. The samples were measured in triplicate.
413

Beilstein J. Org. Chem. 2018, 14, 407–415.
α-amanitin, whereas conjugate 11 showed enhanced potency Acknowledgements
compared to the free drug only on U87 and MDA-MB-468 We thank the European Commission (Marie Skłodowska-Curie
cells.
ITN MAGICBULLET 642004) for PhD fellowships (to L.B.,
P.L.R., B.K.) and financial support.
Apparently, for all these α-amanitin conjugates there is no
correlation between the cytotoxicity and the expression of αVβ3 ORCID® iDs
integrin. To determine whether the slightly increased cytotoxici-
ty of cyclo[DKP-isoDGR]-α-amanitin conjugates 10 and 11 is
governed by an integrin-mediated binding and internalization
process, competition experiments were carried out in which
conjugates 10 and 11 were tested with U87 (αVβ3+, αVβ5+,
αVβ6−, α5β1+) and MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+,
α5β1−) cells in the presence of 50-fold excess of cilengitide,
Lizeth Bodero - https://orcid.org/0000-0003-2552-0064
Paula López Rivas - https://orcid.org/0000-0002-0539-8682
Daniela Arosio - https://orcid.org/0000-0001-5486-3504
Luca Pignataro - https://orcid.org/0000-0002-7200-9720
Cesare Gennari - https://orcid.org/0000-0002-7635-4900
Umberto Piarulli - https://orcid.org/0000-0002-6952-1811
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tide. Therefore, it appears that blocking integrins with excess
cilengitide, which is known to strongly bind not only αVβ3, but
also αVβ5, αVβ6, and α5β1, results in an increase (up to
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