β-Glucuronidase triggers extracellular MMAE release from an integrin-targeted conjugate

Article abstract of DOI:10.1039/c9ob00617f

A non-internalizing α_vβ₃ integrin ligand was conjugated to the anticancer drug MMAE through a β-glucuronidase-responsive linker. In the presence of β-glucuronidase, only the conjugate bearing a PEG4 spacer inhibited the proliferation

Full text of DOI:10.1039/c9ob00617f

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β-Glucuronidase Triggers Extracellular MMAE Release from an
Integrin-Targeted Conjugate
Received 00th January 20xx,
Accepted 00th January 20xx
Paula López Rivas,a Christoph Müller,b Christian Breunig,b Torsten Hechler,b Andreas Pahl,b Daniela
Arosio,c Laura Belvisi,a,c Luca Pignataro,a Alberto Dal Corso a and Cesare Gennari*a,c
DOI: 10.1039/x0xx00000x
observations, new classes of antigens (collagen IV,10 fibrin,11
A non-internalizing αvβ3 integrin ligand was conjugated to the
anticancer drug MMAE through a β-glucuronidase-responsive
linker. In the presence of β-glucuronidase, only the conjugate
bearing a PEG4 spacer inhibited the proliferation of integrin-
expressing cancer cells at low nanomolar concentrations,
indicating important structural requirements for the efficacy of
these therapeutics.
carbonic anhydrase IX,12 oncofetal fibronectin13 and tenascin
C14) have been explored as targets for non-internalizing
conjugates and new linkers for the selective extracellular
release of the payload have been proposed.
Since 2012, our group has explored the conjugation of
different anticancer drugs to the peptidomimetic compound
cyclo(DKP-RGD)15 (compound
1 in Figure 1), a low-nanomolar
ligand of integrin αvβ3.16 The latter is a heterodimeric
transmembrane glycoprotein overexpressed in a variety of
cancer types (e.g. melanoma, glioblastoma, renal cell
carcinoma, and tumours of lung, ovary, breast, prostate, and
colon), where it is involved in disease progression.17
Among the current pharmacological approaches for cancer
treatment, cytotoxic agents are still frequently administered to
patients with early-stage or advanced disease, as single agents
or in combination with radiation, before or after surgery.1 The
covalent conjugation of these drugs to specific ligands
(antibodies, peptides or small molecules) capable of selective
binding to tumour-overexpressed receptors, has been
proposed as a suitable strategy to increase the tumour
accumulation of these therapeutics, sparing healthy tissues
and reducing dose-limiting toxicities.2 In particular, the so-
called Antibody-Drug Conjugates (ADCs)3 and Small Molecule-
Drug Conjugates (SMDCs)4 consist of a targeting unit and a
cytotoxic payload covalently assembled through a linker, which
should be perfectly stable in plasma and be cleaved at the
tumour site. It has been originally postulated that, in order to
minimize side-toxicity in healthy cells, the payload should be
preferentially released within the intracellular compartments
(e.g. lysosomes) of the targeted cancer cell, upon receptor-
mediated endocytosis of the conjugate. More recently, the
observation of the high mutation rates of cancer cells (which
may generate different cell populations, showing different
levels of antigen expression and drug resistance)5 and of the
role on disease progression played by cancer-associated host
cells (e.g. tumour endothelial cells,6 fibroblasts,7 infiltrating
immune cells8) stimulated the hypothesis that the extracellular
release of the drug and its diffusion within the tumour mass
may increase the treatment efficacy.9 In line with these
Figure 1. Molecular structure of the αvβ3 integrin ligand cyclo(DKP-RGD) (1) and the
conjugate cyclo(DKP-RGD)-NPV-PTX (2).
In contrast with literature reports on similar ligands,18 confocal
microscopy studies recently showed that ligand
on the surface of αvβ3 integrin-expressing cancer cells, without
promoting
significant receptor-mediated endocytosis.19
1 accumulates
a
a.Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19 I-20133,
Milan (Italy). E-mail: cesare.gennari@unimi.it
While this observation may further stimulate the existing
debate about the link between the agonist/antagonist
behaviour and the internalization of integrin ligands,20 we
focused on the development of non-internalizing conjugates
b.Heidelberg Pharma Research GmbH, Schriesheimer Strasse 101, 68526,
Ladenburg, Germany.
c. CNR, Istituto di Scienze e Tecnologie Molecolari (ISTM), Via C. Golgi, 19 I-20133,
Milan (Italy).
based on ligand 1. In particular, we recently developed a new
Electronic Supplementary Information (ESI) available: synthetic procedures, 1H and
13C NMR spectra, HPLC and MS data, procedures for biological and biochemical
assays. See DOI: 10.1039/x0xx00000x
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SMDC (compound
2 in Figure 1), in which the linker moiety exhibited a higher antiproliferative activity against renal cell
consisted of a substrate of elastase (the Asn-Pro-Val peptide carcinoma cells 786-O upon addition oDfOeI:la10st.1a0s3Ve9ie/wCin9AOrtticBhl0ee0O6nc1lei7nlFel
sequence), a serine protease released in the extracellular medium, indicating that
tumour environment by tumour-infiltrating neutrophils in against inflamed tumours.
response to inflammation stimuli.19 The resulting conjugate
2 may be therapeutically efficient
Figure 2. Molecular structures of the αvβ3 integrin-targeted conjugates 3a and 3b, featuring the cyclo(DKP-RGD) peptidomimetic as targeting unit, a glucuronide linker, the
anticancer drug monomethyl auristatin E (MMAE) and different spacers (glutaric acid in 3a, a triazole and PEG4 spacer in 3b) between the linker and the targeting unit. The
mechanism of MMAE release upon linker cleavage mediated by β-glucuronidase is depicted.
Other enzymes may be suitable effectors of non-internalizing affinity and linker cleavage),25 conjugates 3a and 3b featured
SMDCs. For instance, β-glucuronidase is a well-known tumour- two different spacer units (a glutaric acid derivative in
associated enzyme, responsible for the hydrolysis of β-
D-
conjugate 3a and a triazole + PEG4 spacer in 3b). The non-
glucuronic acid residues in glycosaminoglycans. Despite its optimized synthetic procedures for the preparation of
primary expression in intracellular compartments (lysosomes), conjugates 3a,b are described in Scheme 1. Firstly, a glycosidic
β-glucuronidase is often abundant in tumour extracellular bond between bromide
areas, shed by apoptotic or necrotic cancer cells as well as by intermediate . Upon aldehyde reduction, the nitro group of
tumour-infiltrating monocytes and neutrophils.21 For these was reduced via catalytic hydrogenation. Aniline
was used as
reasons, β-glucuronidase-responsive linkers have been a common intermediate for the preparation of both final
installed in a large variety of anticancer biotherapeutics, conjugates. For the synthesis of 3a was Fmoc-protected and
4 and phenol 5 was formed, affording
6
7
8
,
8
designed to release anticancer drugs both inside22 and reacted with 4-nitrophenylchloroformate, affording carbonate
outside23,24 cancer cells.
9. The latter was treated with MMAE and HOBt, enabling
carbamate bond formation with the secondary amine of the
free drug. Basic aqueous conditions allowed protecting group
removal, and the resulting free aniline 10 was reacted with
di(succinimidyl) glutarate. Following a well-known procedure,
the N-hydroxysuccinimidyl ester 11 was reacted with the
Inspired by these studies, we designed two new integrin-
targeted conjugates (3a and 3b in Figure 2) equipped with
ligand 1, a glucoronide linker, a self-immolative spacer and the
potent microtubule-disruptor monometyl auristatin E (MMAE),
the well-known payload of the marketed ADC Adcetris®. Since
the spacer between the ligand and the linker moieties may be
crucial for different aspects of the final SMDC assembly (e.g. to
modulate the conjugate flexibility, solubility, ligand binding
benzylic amine of the cyclo(DKP-RGD) ligand 1516a to afford 3a
.
Conjugate 3b was prepared following a similar synthetic plan.
However,
a
significant improvement consisted in the
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derivatization of with 4-pentynoic acid, which avoided the cycloaddition. The conjugates were purifiedViewbAyrticlesOenmlinie-
8
installation/removal of protecting groups at the low-reactive preparative HPLC, characterized by DhOigIh: 1-0re.1s0o3l9u/Cti9oOnB0m06a17sFs
aniline moiety and the conjugation of the azide-bearing ligand spectrometry and lyophilized before biological analysis.
16 16c,f
via a chemoselective copper-catalyzed Huisgen
,
Scheme 1. Synthesis of conjugates 3a and 3b. Reagents an conditions: a) Ag2O, molecular sieves, MeCN, overnight; b) Silica gel, NaBH4, iPrOH/CHCl3, 2.5 h; c) H2, Pd/C, EtOAc,
EtOH, MeOH, overnight; d) Fmoc-OSu, iPr2NEt, DMAP, CH2Cl2, 3 h; e) 4-nitrophenylchloroformate, pyridine, THF, 3 h; f) MMAE, HOBt, iPr2NEt, pyridine, DMF, 2 h; g) LiOH, 1:1
MeOH/H2O, 2 h; h) Di(succinimidyl)glutarate, iPr2NEt, DMAP, DMF, 3 h; i) 4-pentynoic acid, HATU, HOBt, iPr2NEt, DMF, overnight; j) PBS pH 7.5/DMF, 3 h; k) CuSO4·5H2O, sodium
ascorbate, DMF/H2O.
Table 1. Inhibition of biotinylated vitronectin binding to the αvβ3 receptor.
The affinity of conjugates 3a,b for the purified αvβ3 integrin
receptor was estimated. The conjugates were found to inhibit
[a]
Compound
αvβ3 IC50 [n
22.0 ± 9.6
76.7 ± 5.8
4.5 ± 1.1
M]
the binding of the biotinylated vitronectin at nanomolar
concentrations, as shown in Table 1. The IC50 values of both
the MMAE−RGD conjugates 3a and 3b were comparable, albeit
cyclo(DKP-RGD)-A-Gluc-MMAE (3a
)
)
cyclo(DKP-RGD)-B-Gluc-MMAE (3b
a bit higher, to that of the free cyclo(DKP-RGD) ligand
1. These
15
cyclo(DKP-RGD) (1)
data reassured us that the enormous increase of steric
hindrance in the MMAE−RGD conjugates did not significantly [a]: IC50 values were determined as the concentration of compound required for 50%
inhibition of biotinylated vitronectin binding as estimated by GraphPad Prism software.
influence the high affinity for the αvβ3 integrin receptor.
All values are the arithmetic mean
determinations.
± the standard deviation (SD) of duplicate
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Following the protocol reported by Papot and coworkers,23 the upon enzyme addition, with IC50 values in the same low-
cytotoxic activity of 3a,b was examined by incubating the RGD- nanomolar range of free MMAE (FDigOuI:r1e0.1033VB9ie/wCa9AnOrtidcBl0e0O36nD1li7n)Fe.
MMAE conjugates with αvβ3-expressing cancer cells, in the Noticeably, this enhanced cytotoxicity was not observed for
presence or absence of β-glucuronidase. This experiment aims conjugate 3a, which showed a micromolar activity regardless
at simulating the tumour microenvironment in vivo, where a of the β-glucuronidase addition. Conceivably, the lack of
high extracellular expression of β-glucuronidase is expected to anticancer activity of 3a can be ascribed to its structural
promote MMAE release from the non-internalizing conjugates, features. In particular, the glutarate spacer (Spacer A in Figure
and the free drug can then enter cancer cells by passive 1) establishes a suboptimal distance between the glucuronide
diffusion. Human glioblastoma U87MG26 and renal cell linker and the bulky integrin ligand, affecting the efficiency of
carcinoma 786-O19 cells were chosen, and their high αvβ3 the β-glucuronidase-mediated cleavage. On the contrary, the
surface expression was confirmed by flow cytometry analysis triazole + PEG4 spacer (Spacer B) in conjugate 3b allows a
(Figure S1 in the Supporting Information).
perfect drug release process. Finally, in the absence of the
extracellular enzyme, conjugate 3b inhibits the proliferation of
αvβ3-positive cells at submicromolar/low micromolar
concentrations (Figure 3A and 3C). This observation confirms
the non-internalizing behaviour of the cyclo(DKP-RGD) ligand.
The modest cytotoxic activity of conjugate 3b under these
conditions may be due to a poor internalization leading to drug
release mediated by lysosomal β-glucuronidase.
Conclusions
The design of new conjugates for the selective release of
therapeutics at the diseased site represents a highly attractive
pharmacological approach for the future treatment of cancer
and other indications. However, the modular nature of these
products requires a fine tuning of their individual components,
from the ligand to the linker, from the drug to the spacers. In
this work, we developed a non-internalizing, αvβ3-targeted
conjugate (compound 3b), capable of releasing the highly
potent MMAE payload upon extracellular cleavage of a
glucoronide linker. Conjugate 3b inhibited the proliferation of
integrin-expressing U87MG and 786-O cancer cells at low
nanomolar concentrations. These data suggest that 3b may be
therapeutically active in vivo against solid tumours expressing
both integrin αvβ3 (required for the conjugate accumulation at
the tumour site) and extracellular β-glucuronidase (responsible
for the drug release). From the structural point of view, the
use of a long and hydrophilic PEG4 spacer, connecting the
integrin ligand to the linker moiety, was found to be
fundamental for the overall conjugate efficiency, conceivably
due to a diminished steric hindrance around the cleavable
glycosidic bond. Once released from 3b in its active form,
MMAE can penetrate the tumour cell membrane by passive
diffusion. According to this mechanism, MMAE and other
lipophilic drugs (e.g. duocarmycins, maytansinoids) should be
considered as ideal payloads, rather than more hydrophilic
agents (MMAF, α-amanitin), as it was recently demonstrated
for other non-internalizing conjugates.27 As future perspective,
the quantitative biodistribution analysis of the cyclo(DKP-RGD)
ligand in tumour-bearing mice would unveil the therapeutic
Figure 3. Antiproliferative activity of conjugates 3a, 3b and free MMAE in αvβ3-
expressing human glioblastoma (U87MG) and renal cell carcinoma (786-O) cells, after
incubation for 96 hours in the absence (experiments A and C) or presence (experiments
B and D) of β-glucuronidase. Data points are averages of 3 experiments. Error bars potential of this class of conjugates.
indicate SDs. Calculated IC50 values (M) are listed below each experiment.
As shown in Figure 3A and 3C, in the absence of β-
glucuronidase, conjugates 3a,b proved poorly cytotoxic,
Conflicts of interest
whereas free MMAE inhibited cell proliferation at very low
The authors declare no conflict of interest.
concentration (IC50 = ca. 86 p
M against U87MG and ca. 2 nM
against 786-O cells). Interestingly, in both cell lines the activity
of conjugate 3b was found to be a bit higher (IC50 4.1-7.8 times
lower) than that of analogue 3a. On the other hand, the
anticancer activity of conjugate 3b increased dramatically
Acknowledgements
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We thank the European Commission (Marie Skłodowska-Curie
ITN MAGICBULLET 642004) for a PhD fellowship (to P.L.R.) and
financial support. We also gratefully acknowledge Ministero
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DOI: 10.1039/C9OB00617F
dell’Università
e
della Ricerca (PRIN 2015 project
20157WW5EH) for financial support.
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Organic & Biomolecular Chemistry
Communication
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DOI: 10.1039/C9OB00617F
β-Glucuronidase Triggers Extracellular MMAE Release from an Integrin-Targeted
Conjugate
P. López Rivas, C. Müller, C. Breunig, T. Hechler, A. Pahl, D. Arosio, L. Belvisi, L. Pignataro, A. Dal Corso and C. Gennari
A non-internalizing conjugate targeting αvβ3 integrin inhibits the proliferation of integrin-expressing cancer cells in the
presence of β-glucuronidase.