Design, Synthesis, and Use of MMP-2 Inhibitor-Conjugated Quantum Dots in Functional Biochemical Assays

Article abstract of DOI:10.1021/acs.bioconjchem.6b00065

The development of chemically designed matrix metalloprotease (MMP) inhibitors has advanced the understanding of the roles of MMPs in different diseases. Most MMP probes designed are fluorogenic substrates, often suffering from photo- and chemical instabi

Full text of DOI:10.1021/acs.bioconjchem.6b00065

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Article
Design, synthesis, and use of MMP-2 inhibitor-conjugated
quantum dots in functional biochemical assays.
Erika Bourguet, Kristina Brazhnik, Alyona Sukhanova, Gautier Moroy, Sylvie Brassart-
Pasco, Anne-Pascaline Martin, Isabelle Villena, Georges Bellon, Janos Sapi, and Igor Nabiev
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00065 • Publication Date (Web): 01 Mar 2016
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Design, synthesis, and use of MMP-2 inhibitor-conjugated quantum dots in
functional biochemical assays.
*
,†
‡,§
‡,§
ơ
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Erika Bourguet , Kristina Brazhnik , Alyona Sukhanova , Gautier Moroy , Sylvie Brassartꢀ
┴
┴,#
#
┴
†
‡,§
Pasco , AnneꢀPascaline Martin , Isabelle Villena , Georges Bellon , Janos Sapi , Igor Nabiev
†
Institut de Chimie Moléculaire de Reims, UMR 7312ꢀCNRS, SFR CapꢀSanté, UFR de
Pharmacie, Université de Reims ChampagneꢀArdenne, 51 rue Cognacq Jay, 51100 Reims,
France
‡
Laboratoire de Recherche en Nanosciences, LRN ꢀ EA4682, UFR de Pharmacie, Université de
Reims ChampagneꢀArdenne, 51 rue Cognacq Jay, 51100 Reims, France
§
Laboratory of NanoꢀBioengineering, National Research Nuclear University MEPhI (Moscow
Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russian Federation
ơ
Molécules Thérapeutiques In Silico, INSERM UMRꢀS 973, Université Paris Diderot, Sorbonne
Paris Cité, 35 rue Hélène Brion, 75013 Paris, France
┴
Laboratoire de Biochimie et de Biologie moléculaire, MEDyC, UMR CNRS/URCA 7369, SFR
CapꢀSanté, UFR de Médecine, Université de Reims ChampagneꢀArdenne, 51 rue Cognacq Jay,
5
1100 Reims, France.
#
Laboratoire de ParasitologieꢀMycologie, EA3800, SFR CapꢀSanté, UFR de Médecine,
Université de Reims ChampagneꢀArdenne, 51 rue CognacqꢀJay, 51100 Reims, France.
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ABSTRACT: The development of chemically designed matrix metalloprotease (MMP) inhibitors
has advanced the understanding of the roles of MMPs in different diseases. Most MMP probes
designed are fluorogenic substrates, often suffering from photoꢀ and chemical instability and
providing fluorescence signal of moderate intensity, which is difficult to detect and analyze when
dealing with crude biological samples.
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Here, an MMP inhibitor that selectively inhibits MMPꢀ2 more strongly than Galardin has been
synthesized and used for enzyme labelling and detection of the MMPꢀ2 activity. A complete MMPꢀ
2
recognition complex consisting of a biotinylated MMP inhibitor tagged with the streptavidinꢀ
quantum dot (QD) conjugate have been prepared. This recognition complex, which is characterized
by a narrow fluorescence emission spectrum, long fluorescence lifetime, and negligible
photobleaching, has been demonstrated to specifically detect MMPꢀ2 in in vitro sandwichꢀtype
biochemical assays with sensitivities orders of magnitude higher than those of the existing “gold
standards” employing organic dyes.
The approach developed can be used for specific in vitro visualization and testing of MMPꢀ2 in
cells and tissues with sensitivities significantly exceeding those of the best existing fluorogenic
techniques.
KEYWORDS: matrix metalloproteinases, matrix metalloproteinase inhibitors, selective MMPꢀ
2 inhibition, molecular modelling, quantum dots.
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INTRODUCTION
Studying biological processes at the molecular level within a living cell is a major challenge
1
,2
for cell biology investigation. Similarly, spatiotemporal tracking of small molecules in ex vivo
or in vivo environment gives a better insight into their interactions with proteins, rendering target
identification more efficacious and, in fine, accelerating drug development. The existing
techniques based on spectroscopic and biooptical methods using organic dyes are mostly limited
by the difficulties in imaging individual molecules in the optically noisy cellular environment
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and accessing directly the interior of living cells. As promising alternative tools, semiꢀconductor
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nanocrystals quantum dots (QDs) have emerged for numerous biomedical applications, such as
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cellular labeling, biochemical sensing, probing biocatalyzed reactions, and drug delivery.
QDs are characterized by their narrow, composition and size dependent, emission
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wavelength , extreme brightness, rockꢀsolid photostability and chemical robustness. QD
fluorescence covers the optical spectrum from the nearꢀUV to the IR, which provides a unique
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possibility for multiplexing, unlike with the use of organic dyes. Even if QDs possess optical
properties for biological applications, nanoparticles need to be functionalized to achieve a
convenient solubility in aqueous media and in fine hydrophilic ligands may be used as anchor
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points for biomolecules such as peptides, DNA or drugs. The hydrophobic outer shell of QDs
may be exchanged or chemically reacted with biꢀ or multifunctional molecules containing
1
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capping ligands, cationic polymers or liposomes bearing cell penetrating peptide (CPP)
, or
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with amphiphilic polymers.
Biomolecules may be attached to their surface by covalent
binding via different surface functional groups (amino, thiol, carboxyl…), or through nonꢀ
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covalent streptavidin/biotin, electrostatic or ionic interactions.
Matrix metalloproteinases (MMPs) belong to the family of zincꢀdependent proteases involved
in the degradation and remodelling of extracellular matrix proteins. The activity of MMPs is
controlled by endogenous tissue inhibitors of metalloproteinases (TIMPs). The finely tuned
MMPs/TIMPs balance found in normal physiological processes (tissue modeling, angiogenesis,
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wound healing, embryonic growth…) is disrupted in several pathologies such as atherosclerosis,
aneurysm, pulmonary emphysema and cancer... Among more than 25 MMPs, MMPꢀ2
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(
gelatinase A) plays an important role in tumorigenesis, particularly in cancer progression
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rendering this enzyme a therapeutic and diagnostic target for drug development and in vivo
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detection of tumour growth or invasion. Thus, the conception of potent and MMPꢀ2 selective
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inhibitors may constitute a valuable therapeutic approach for melanoma.
In the past two decades, several pseudoꢀdipeptideꢀtype inhibitors, such as batimastat,
®
marimastat, and ilomastat (Galardin ) were synthesized with the aim to regulate imbalanced
proteolytic activity (Figure 1). Although, first generation of MMP inhibitors, in spite of their
high potency and preliminary clinical trials, have been withdrawn before advanced clinical
experiments due to severe musculoskeletal side effects. They may be considered as starting
points for the design and development of selective MMP inhibitors with better physicochemical
and pharmacological profiles. These efforts took two directions: (1) exploration of structural
differences in the substrateꢀbinding regions around the zinc cation and (2) design of new zincꢀ
binding groups (ZBG).
O
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Batimastat
Marimastat
Ilomastat (Galardin®)
Figure 1. The structure of highly efficient inhibitors: batimastat, marimastat, and ilomastat.
Although matrix metalloproteinases have a great homology of sequence, the size, shape,
depth, and conformational mobility of side pockets (denoted S1, S2, S3 for the nonꢀprimed side
and S’1, S’2, S’3 for the primed side around the zincꢀcontaining catalytic site), are determinant
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for inhibitory potency and selectivity.
Studies on structureꢀactivity relationship have shown
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that the subsite selectivity decreases in the following order: S’1 > S2, S’3, S3 > S1 > S’2.
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Thus, attempts at obtaining selectivity among MMP enzymes have concentrated on the S’1
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subsite, called the specificity pocket.
The S’1 pocket of MMPꢀ2 is hydrophobic, forms a
large, nearly bottomless channel, and the S’1 loop is flexible to accommodate more bulky
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ligands. Thus, modifications of P’1 group in MMP inhibitors (MMPIs) ensure selectivity
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among MMPꢀfamily members. Since the succinyl hydroxamate backbone is a common feature in
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the most efficient firstꢀgeneration MMP binders , pharmacomodulation was carried out at this
structural unit.
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For this purpose, Miller et al. observed the maximum specificity of the inhibitor for MMPꢀ2
in comparison to MMPꢀ1 with addition of a C12 alkyl chain as the P’1 group into the structure of
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batimastat (IC50 = 1 and 50000 nM, respectively). Broadhurst et al. have described a
marimastat analog containing a C9 linear chain displaying a good MMPꢀ2 inhibitory activity
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(
IC50 < 0.15 nM). However, Miller et al. described the best selectivity for MMPꢀ2 versus
MMPꢀ1 with a compound containing a C16 alkyl chain (IC50 = 0.6 and 5000 nM, respectively).
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Levy et al. modified ilomastat by elongating its alkyl chain to eight carbon atoms. Neither
inhibitory activity nor selectivity were ameliorated.
MMP inhibitors contain hydroxamic acid as a zincꢀbinding group (ZBG). Although this
function has proved to be a very strong ZBG, toxicity, low bioavailability, and in vivo unstability
of this chemical entity associated with severe side effects have resulted in the development of
alternative ZBG such as carboxylic acid, a precursor of the hydroxamate group.
Batimastat and marimastat analogs bearing a C16 alkyl chain in P’1 position with carboxylic
acid type ZBG showed a lowest MMPꢀ2 inhibitory activity (IC = 50 and 30 nM, respectively).
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Reliable techniques are crucial for hit detection, biological evaluation of compound
collections, or monitoring enzyme inhibitory activities. By the past, conventional techniques
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included gel electrophoresis , fluorescence (nearꢀinfrared)
, or magneticꢀresonanceꢀbased
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approaches
were used to detect MMPs activity, but recently much attention has been paid to
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Förster resonance energy transfert (FRET) for assay of proteases.
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QDꢀFRETꢀbased protease sensors exploiting QDs as donor and rhodamine or gold
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nanoparticle as acceptor have also been developed to survey collagenase activity
in normal
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and cancer cells. QDs can also act as energy acceptors in bioluminescence resonance energy
transfer (BRET) from a protein energy donor, such as a mutant form of Renilla luciferase, to
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detect protease activity.
QDꢀbased methods have also been used for multiplexed detection
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and imaging of several MMPs (MMPꢀ2 and MMPꢀ7)
and multiplexed protease inhibition and
In these methods, the MMP activity is identified
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competition (folic acid and MMPꢀ7) assays.
by changes in QD fluorescence resulting from the cleavage of a specific enzymeꢀrecognizing
peptide attached to the QDs. Other approaches employ QDs coupled to antibodies or MMPꢀ9ꢀ
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siRNA to study the enzymatic activity of MMPꢀ9 or the MMPꢀ9 gene expression in the brain.
Herein, we describe the synthesis and preliminary biological evaluation of a range of
ilomastat derivatives bearing carboxylic acid as a ZBG and alkylidene chains of different lengths
at the P’1 position. Additional pharmacomodulations were made at the Cꢀ2 position of the indole
ring to evaluate the impact of a bulky phenyl substitution, P’2 group, on MMP inhibition
activity. Among these derivatives, the most selective MMPꢀ2 inhibitor (4k) was conjugated to
QDs to use it as probe to identify MMPꢀ2 enzyme activity. The advanced optical properties of
streptavidinꢀQD conjugates, the high binding constant for streptavidinꢀbiotin interaction and the
technical simplicity of mixing streptavidinꢀQD nanoparticles with the biotinylated ligand (4k)
make this approach an attractive tool to generate imaging probes with potential in vitro
applications in advanced biochemical MMPꢀ2 assays (Scheme 1).
QDots
Streptavidin
+
Biotin
Ligand
QDots
Streptavidin
Ligand
Scheme 1. Nonꢀcovalent coupling of a biotinylated compound and a fluorescent QDꢀstreptavidin
conjugate.
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RESULTS AND DISCUSSION
Influence of the P’1 part of ilomastat on gelatinase inhibition
For a decade, we have been involved in the pharmacomodulation of ilomastat, focusing
recently our efforts on designing analog derivatives with a modified P’1 succinic component.
First, incorporation of one unsaturation at the P’1 position aiming at increasing the
hydrophobicity and conformational rigidity was considered. Replacement of the isobutyl group
of ilomastat with an isobutylidene function 1 with the E geometry improved the selectivity for
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MMPꢀ2 versus MMPꢀ3 (IC50 = 1.3 and 179 nM, respectively) (Figure 2). Pursuing these
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pharmacomodulations, we synthesized other dehydro and didehydro analogs (2a-d).
P'1
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P'1 = -(CH ) -CH -(CH ) -Ph, -CH=CH-Me, -CH=CH-Ph
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Figure 2. Pharmacomodulation of the P’1 group.
The inhibitory activities of these analogs for all MMPs were decreased as compared to
®
ilomastat (Galardin ). However, analog 2a with a C7 alkyl chain displayed an interesting
4
selectivity for MMPꢀ2 compared to MMPꢀ9 (IC = 123 and > 10 nM, respectively) indicating
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that the S’1 pocket of MMPꢀ2 is sufficiently deep to accommodate long alkyl chains.
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Influence of the P’2 part of ilomastat on gelatinase inhibition
Introduction of an aminoꢀalkyl chain at the C2 position of the indole ring leads to a decrease
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in the MMP inhibitory activity but greatly improves the selectivity for MMPꢀ2. In this line, a
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phenyl group was introduced at the C2 position through the Suzuki reaction
(Figure 3) and
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the obtained analog 3 was shown to have enhanced potency and selectivity for MMPꢀ2 versus
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MMPꢀ1 (IC = 0.092 and 0.244 nM, respectively).
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Figure 3. Pharmacomodulation of the P’2 group.
In order to clarify the mode of interaction between MMPꢀ2 and analog 3, molecular docking
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computations have been carried out using the AutoDock software.
As expected, the
hydroxamate group chelates the Zn atom in the lowestꢀenergy binding mode (Figure 4a).
Moreover, the carbonyl function of the hydroxamate group is involved in Hꢀbond interaction
with Ala192. The isobutyl group is located in the S’1 subsite forming van der Waals interactions
with Leu191, Ala192, Val400, His403, Pro423, and Tyr425. The modified indole ring is located in the
S’2 subsite and is oriented towards the solvent. In addition, four Hꢀbonds between compound 3
and MMPꢀ2 amino acid residues located in the S’3 subsite, namely, Gly , Leu , Pro , and
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423
Tyr425, (highlighted) stabilize this model (Figure 4b).
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Figure 4. Docking of analogue 3 in the structure of MMPꢀ2.
a) The binding mode of analogue 3 with MMPꢀ2;
b) A detailed view of the Hꢀbonds enabling the model stabilization.
It is wellꢀknown that the large and solventꢀexposed S’2 pocket is flexible to accommodate
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bulky and hydrophobic groups
and aromatic groups are in agreement with this model as
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supported by in vitro activity.
Influence of the P’1 and P’2 parts of ilomastat on gelatinase inhibition
In order to further improve the selectivity for MMPꢀ2, the alkyl chain was elongated by 8 to
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2
0 carbon atoms using a method described previously and a phenyl group was also introduced
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at the C2 position of the indole part through the Suzuki reaction (Figure 5). Replacement of the
very strong hydroxamic acid Znꢀchelating group by a less complexing carboxylate function may
also favour selectivity to the detriment of affinity. For comparison, ilomastat and compound 3b
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were also included in the series of biological evaluations (Table 1).
As shown in Table 1, the
carboxylate counterpart 3b is more selective for MMPꢀ1 (among other MMPs) than ilomastat,
although its inhibitory activity is decreased.
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n
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O
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N
H
N
H
Galardin®
3a R = Ph, X = NH-OH
b R = H, X = OH
4a-p
R = H, Ph
n = 6,7,8,9,10,11,14,18
3
Figure 5. Pharmacomodulation of the ZBG, the P’1 and P’2 groups.
In an agreement with the literature data, the inhibitory activity values for compounds 4a-p are
®
detrimental for affinity compared to ilomastat (Galardin ) but remain of the same order of
magnitude as that found for the carboxylicꢀacidꢀtype ilomastat analogue 3b.
Contrary to our expectations, elongation of the alkyl chain with an incorporated unsaturation
did not improve the inhibitory activity of the compounds towards MMPꢀ2. However, we found
an increased selectivity of the C10 analog 4c (n = 8, R = H) for MMPꢀ9 versus MMPꢀ1.
Furthermore, the ilomastat derivative 4k (n = 8, R = Ph) with a C10 alkyl chain and a phenyl
group at the P’2 position displayed the best selectivity for MMPꢀ2 inhibition (IC = 80 nM) in
5
0
comparison to other MMPs (Table 1), demonstrating that a carboxylate type ZBG may be an
alternative for a finely tuned balance between enzyme activity and selectivity. In addition, this
pharmacomodulation helps to avoid the toxicity and side effects known about the parent
ilomastatꢀtype molecules.
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2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. P’1ꢀ and P’2ꢀmodified ilomastat inhibitors. The IC values are expressed in nM.
50
Compounds
Ilomastat
n
R
H
H
H
H
H
H
H
H
H
H
MMPꢀ1 MMPꢀ2 MMPꢀ9 MMPꢀ13 MMPꢀ14
ꢀ
1.5
9.9
1.1
0.5
ꢀ
13.4
a
b
b
a
3
b
ꢀ
170
100
838
241
173
450
211
673
582
1260
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
5
5
5
5
5
5
5
5
5
4a
6
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
7570
458
247
249
351
655
762
992
1280
925
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
5
4
4
4
b
c
7
5
5
5
5
5
5
5
5
5
5
5
5
5
5
8
d
9
243
4e
10
11
14
18
6
244
4
4
4
f
515
g
h
155
5
5
> 10
670
976
80
> 10
297
339
320
554
581
768
982
1277
1573
1062
1068
459
4i
Ph > 10
4
4
j
7
Ph 5182
Ph 1190
Ph 3085
k
8
4l
9
501
459
568
1235
4
4
4
m
10 Ph 4151
555
5
n
o
11 Ph > 10
14 Ph > 10
18 Ph > 10
607
5
5
185
5
4p
> 10
1395
245
a
68
Unpublished results
b
Ki values are expressed in nM
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Bioconjugate Chemistry
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According to the docking computations, the lowestꢀenergy binding modes are characterized
by chelation of the Zn atom with the carboxylate group of 4k in the MMPꢀ2 active site. Four Hꢀ
bonds are formed between the pseudopeptide backbone of the 4k analog and MMPꢀ2 residues,
located at the upper (Gly189 and Leu191) and lower (Pro423 and Tyr425) rims of the S’3 subsite.
The modified P’2 group in the S’2 subsite is exposed to the solvent. The long alkyl chain is
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inserted into the S’1 pocket, inducing van der Waals interactions with Leu399, Val400, His403
,
Ala419, Leu420, Ala422, Pro423, Ile424, Tyr425, Thr426, Thr428, and Phe431 (Figure 6).
Figure 6. Docking of analogue 4k in the structure of MMPꢀ2.
(a) The binding mode of analogue 4k with MMPꢀ2;
(b) A detailed view of the Hꢀbonds enabling the model stabilization.
We have also investigated the binding mode of compound 4k with MMPꢀ9. In the lowestꢀ
energy docked conformation predicted by AutoDock, the Zn atom is chelated by the carboxyl
group and four Hꢀbonds are formed with Gly , Leu , Pro , and Tyr located in the S’3
186
188
421
423
subsite. The modified P’2 group in the S’2 subsite is exposed again to the solvent. The long alkyl
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2
2
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5
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5
5
5
6
group is stabilized in the S’1 subsite by several van der Waals interactions formed by the
following amino acids: Leu397, Val398, His401, Ala417, Leu418, Tyr420, Met422, Tyr423, Arg424, and
Thr426 (Figure 7).
0
1
2
3
4
5
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9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Docking of analogue 4k in the structure of MMPꢀ9.
(a) The binding mode of analogue 4k with MMPꢀ9;
(b) A detailed view of the Hꢀbonds enabling the model stabilization.
As a result of preliminary modelling and examination studies, the most appropriate
compound, 4k, was selected for further evaluation and estimation of its inhibitory activity and
effects on cell migration and invasion. MMPꢀ2 proteolytic activity was measured in the presence
or absence of the inhibitor using a fluorescent Dye Quenched substrate (DQ) gelatin.
Concanavalin A treatment of cells induces active MMPꢀ14 expression on the cell surface,
6
9
ensuring rapid MMPꢀ2 activation and release into the culture medium. In the presence of
®
compound 4k, the MMPꢀ2 activity was reduced by 20%, while ilomastat (Galardin ) decreased
the enzyme activity by 30% (Figure 8).
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Bioconjugate Chemistry
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*
*
60000
0000
40000
0000
20000
*
5
3
10
11
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13
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1
0000
0
-ConA +ConA +ConA +ConA
+4k +Galardin®
Figure 8. MMPꢀ2 proteolytic activity measurement.
HTꢀ1080 cells were treated overnight with concanavalin A. Cells were then preꢀincubated with
®
or without the effector (100 µM 4k or 10 µM Galardin ) for 1 h and then with Dye Quenched
substrate (DQ gelatin) (500 ng/300 µL per well). Fluorescence was measured at 525 nm. *:
p<0.05; **: p<0.01.
Figure 9. HTꢀ1080 cell invasion rate estimated by the ability of cells to migrate through
®
®
Matrigel ꢀcoated membranes upon treatment with compound 4k or ilomastat (Galardin ).
a) A representative photomicrograph showing cell invasion after 6 h of incubation.
b) A representative bar graph quantifying cell invasion. *: p<0.1. Scale bar, 50 µm.
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HTꢀ1080 cell invasion was also estimated in vitro in the presence of compound 4k or
®
ilomastat. HTꢀ1080 cells were tested for their ability to migrate through Matrigel ꢀcoated (20
µg/well) filters during 6 h. Compound 4k did not alter cell invasion (Figure 9), while ilomastat
inhibited cell migration by 25%.
0
1
2
3
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0
1
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0
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1
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0
During the invasion process, HTꢀ1080 cells produce different types of MMPs (MMPꢀ14,
MMPꢀ2, etc.) and serine proteinases (uꢀPA, tꢀPA, etc.) which are involved in cell migration.
Evidently, compound 4k mainly blocks the MMPꢀ2 activity and is a selective MMPꢀ2 inhibitor,
but its involvement in the migration process is probably insignificant, which explains the
®
absence of inhibitory effect on HTꢀ1080 cell invasion. Ilomastat (Galardin ) inhibits different
types of MMPs, including MMPꢀ14, which is known to degrade extracellular matrix, to activate
proMMPꢀ2 and to greatly favour tumor cell invasion. This could explain the observed inhibitory
effect on HTꢀ1080 cell invasion (25%).
Synthesis of a biotinylated inhibitor 4k
In line with our goal providing tools for imaging, we investigated the feasibility of MMP
inhibitorꢀQDs conjugates. The selected compound 4k was modified at the indole NH group in
order to insert the biotin compound into the inhibitor structure (Figure 10). Biotin interacts nonꢀ
1
5
ꢀ1
covalently with streptavidin with an extremely high affinity (K
D
~10 M ), resulting in the
formation of a highlyꢀstable specific biotinꢀstreptavidin complex, which is widely employed in
7
0,71
numerous biological approaches.
8
O
O
H
N
HO
NHMe
O
O
N
O
O
HN
NH
NH
N
H
S
5
spacer
biotin
Figure 10. Structure of the resultant biotinylated compound 4k.
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Bioconjugate Chemistry
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9
Modification of compound 4k with biotin included several steps. First, the tryptophan
72
derivative 8 obtained from derivative 7 using the Suzuki reaction was treated with tꢀ
butylacrylate (Michael reaction) to obtain compound 9 with a yield of 90%. The obtained
functionalized indole derivative 9 was subjected to transamidification and deprotected with a
yield of 80% and coupled to compound 11 to obtain compound 12 with a yield of 82% (Scheme
2).
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11
12
13
14
15
16
17
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1
) NBS, CCl4, 96%
O
O
O
O
H
H
1
) SOCl
2) (CF CO)
Et N
2
, EtOH
H
N
2) Pd(PPh 4, LiCl
PhB(OH)2, NaHCO3,
92%
3
)
N
F
3
C
N
F
3
C
H
2
N
3
2
O, F C
OEt
N
MeNH4 (40% aq.)
EtOH
3
OEt
OH
OEt
3
1) tBuOK, THF
O
O
O
72 %
2)
80%
COOtBu
90%
N
H
N
H
N
H
7
O
6
8
9
O
O
8
H
2
N
8
NH
O
O
8
O
O
H
HBTU,
CH Cl2
H
O
,
N
2
N
O
NHMe
2
,4,6-collidine
TFA, CH
Q
2
Cl
2
O
NHMe
OH
O
O
+
O
N
8
2%
O
O
N
O
N
O
O
10
11
12
13
O
OH
Scheme 2. Preparation of the substituted analogue 13.
The biotinylated diaminoalkyl spacer 16 was obtained by coupling monoꢀprotected
diaminopropan 14 and biotin 15 with a yield of 74% (Scheme 3).
O
HBTU,
,4,6-collidine,
DMF, CH Cl
O
O
O
HN
O
HN
O
HN
2
TFA, CH
2 2
Cl
NH
2
2
NH
NH
BocHN
NH
2
HO
BocHN
N
H
2
N
N
+
H
H
S
74 %
S
S
Q
1
4
15
16
17
Scheme 3. Preparation of the biotinylated linker 17.
In the next step of the synthesis, the deprotected pseudoꢀdipeptide 13 was reacted with the
deprotected biotinylated diaminoalkyl spacer 17 to obtain compound 18 with a yield of 38% in
two consecutive steps. From compound 18, the allylꢀprotecting group was removed upon
3 4
treatment with the catalyst Pd(Ph P) to obtain compound 5 with a yield of 72% (Scheme 4).
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5
5
5
5
5
6
HBTU,
2,4,6-collidine,
DMF, CH
17
Pd(PPh
morpholine
THF
3
)
4,
8
8
8
O
O
O
2
Cl2,
O
O
O
H
H
H
N
N
N
HO
O
NHMe
O
NHMe
NHMe
38 %
72 %
O
over two steps
O
O
biotin
spacer
NH
O
N
O
O
N
O
N
O
O
O
HN
HN
NH
N
NH
OH
NH
18
N
H
H
S
S
1
3
5
Scheme 4. Synthesis of the compound 5.
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Compound 5 was also submitted to MMPꢀ2 inhibition assay by using a classical gelatin
zymography made under the same conditions as described for compound 4k. Comparative study
showed that compound 5 ensured a 34% inhibition of the enzyme versus 45% inhibition with
compound 4k, indicating that biotinylation only slightly decreased the inhibitory activity towards
MMPꢀ2 (Figure 11). Subsequently, compound 5 served as a basis for our further experiments.
a)
Control
4K
5
b)
10000
9
8
7
000
000
000
6000
*
**
**
5
4
3
000
000
000
2000
000
1
0
4K
5
Control
Figure 11. Inhibition of MMPꢀ2 gelatinolytic activity.
a) Results of zymography analysis: 0.4 ng of active MMPꢀ2/well were resolved in gelatin
zymography. Each lane was incubated overnight in 50 mM Tris buffer, pH 7.5, with 200 mM
2
NaCl and 5 mM CaCl without (control) or with 10 µM inhibitor (compound 4k or compound 5);
b) Quantification of MMPꢀ2 gelatinolytic activity by densitometry. ***: p<0.001; **: p<0.005.
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MMP-2 inhibition with the designed inhibitor 5
The MMPꢀ2 activity was estimated in standard gelatinase/collagenase assay based on
digestion of an internally quenched fluorescent substrate with an activated enzyme. The
efficiency of substrate digestion resulting in an increase in dye fluorescence was measured
continuously at multiple time points. The fluorescence of the digested product increased
proportionally to the enzyme proteolytic activity and the amount of activated MMPꢀ2 (Figure
12a). In the presence of the designed inhibitor 5, the protease gelatinolytic activity decreased
proportionally to an increase in the concentration of inhibitor, resulting in quenching of the
fluorescence of the undigested substrate (Figure 12b). We have found that addition of 100 ng of
compound 5 to 50 ng of MMPꢀ2 reduces the protease activity by 50%. Increasing the amount of
the inhibitor leads to stronger enzyme inactivation.
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Figure 12. Influence of compound 5 on MMPꢀ2 activity as determined by Dye Quenched
substrate (DQ gelatin) digestion.
a) MMPꢀ2 activity at different concentrations of preꢀactivated enzyme resulting in an increase in
fluorescein fluorescence.
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b) Concentrationꢀdependent effect of compound 5 on MMPꢀ2 activity. Each analysis has been
performed in duplicate (p<0.001).
We have further coupled the MMPI biotinylated compound 5 to fluorescent QDꢀstreptavidin
conjugate in order to detect the MMPꢀ2 in different in vitro assays and to compare QDꢀbased
assays with those based on organic dyes.
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MMP-2 ultrasensitive detection with QD-tagged compound 5
According to the manufacturer’s (Invitrogen) protocol, the number of streptavidin molecules
per QD may be estimated as 5 to 10, depending on the QD batch, emission wavelength, and
steric effects of the bound streptavidin. Typically, a streptavidin molecule contains four biotin
7
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binding sites
enabling coupling from 20 to 40 biotinylated compounds per QDꢀstreptavidin
conjugate. However, it was shown that, due to the steric effects, only one or two binding sites are
7
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easily accessible for biotin on the surface of a QDꢀstreptavidin conjugate.
Dotꢀblotting of the biotinylated MMPꢀ2 inhibitor
Formation of an effective complex of compound 5 with streptavidinꢀQD565 conjugate was
visualized using the dotꢀblot technique. Biotinylated compound 5 diluted in methanol severely
damaged and dissolved nitrocellulose membrane and was only detectable in amounts above 2 ꢁg.
Depositing these samples on a polyvinylidene difluoride (PVDF) membrane allowed the
detection of 1 ꢁg of the inhibitor as the lowest detection limit (data not shown). When dissolved
in dimethyl sulfoxide (DMSO) and subsequently diluted in phosphate buffered saline (PBS), the
inhibitor can be loaded on a nitrocellulose membrane without any damaging and is detectable in
amounts of 0.2 to 1 ꢁg as the lowest detection limit (Figure 13).
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Figure 13. Detection of biotinylated compound 5 with the fluorescent streptavidinꢀQD 565 nm
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conjugate using the dotꢀblot technique.
In vitro sandwichꢀtype assay for MMPꢀ2 detection using the designed QDꢀcoupled inhibitor 5.
The formation of a complete complex consisting of MMPꢀ2 enzyme, biotinylated inhibitor 5,
and fluorescent conjugate of streptavidin with QD800 nm was performed by means of solidꢀstate
sandwichꢀtype analytic biochemical assay. Here, 50 ng of MMPꢀ2 was used in MMPꢀ2 activity
assays, since this amount of active enzyme was estimated to efficiently and quickly digest the
gelatin substrate. The proenzyme was activated with pꢀamino phenylmercuric acetate before
adsorption in order to provide appropriate conformation of the inhibitoryꢀbinding sites. Activated
MMPꢀ2, MMPꢀ2 or the same quantity of BSA protein control were adsorbed on a treated 96ꢀwell
plate in PBS (pH 7.4). Increasing amounts of biotinylated compound 5 and QDꢀstreptavidin
conjugate were subsequently incubated with the adsorbed MMPꢀ2. The QD fluorescence
intensity increased proportionally to the biotinylated inhibitor amount in the range from 0 to 100
ꢁg (Figure 14). Thus, the complex of MMPꢀ2 with the designed biotinylated compound 5 can be
quantitatively detected and visualized using compact and bright QDꢀstreptavidin conjugates.
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Figure 14. Quantitative detection of MMPꢀ2/biotinylated compound 5 complex with the
fluorescent streptavidinꢀQD800 nm conjugate in in vitro sandwichꢀtype biochemical assays. In a
control experiment, BSA was used instead of MMPꢀ2. Each analysis has been performed in
duplicate (p<0.001).
We performed additional experiments in order to explore the photostability of QDs enabling
longꢀterm accumulation of their fluorescence signal accompanied by orderꢀofꢀmagnitude
improvements of the signalꢀtoꢀnoise ratios in fluorogenic assay and, consequently, increasing the
sensitivity for application of QDꢀbased conjugates to MMPꢀ2 detection in vitro and to compare
the sensitivity of QDꢀbased assays with those based on organic dyes.
Figure 15 shows that accumulation of the signal from the fluorogenic assay employing
streptaꢀQD conjugate for visualization of MMPꢀ2ꢀinhibitor complexes leads to an increase in the
signalꢀtoꢀnoise ratio in the given detection scheme by more than two orders of magnitude and,
hence, the corresponding increase in the sensitivity of detection of the inhibitorꢀMMPꢀ2
complex. This increase may reach even higher values if longer signal accumulation times and
optimized detection schemes are employed. The result shown in Figure 15 was achieved due to
the rockꢀsolid photostability of QDs enabling longꢀterm QD fluorescent signal accumulation
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without significant photobleaching of the nanocrystals. Such a photostability is not characteristic
of the organic dyes used in currently available fluorogenic assays. Therefore, we have performed
additional comparative study of the model biochemical assays employing different organic dyes
in parallel with the assays employing streptaꢀQD conjugates in order to compare the changes in
their state during longꢀterm signal detection and accumulation.
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Figure 15. An increase of the fluorescent signal as a result of its accumulation during 2, 4, 6,
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, 10 or 20 min for different quantities of compound 5 complexed with equimolar quantities of
the fluorescent streptavidinꢀQD800 conjugate in an in vitro sandwichꢀtype biochemical assay.
The mean fluorescence intensities in the case of immediate signal recording (0 min of
accumulation) in the presence of 10, 50, and 100 ꢀg of compound 5 are 50, 220, and 420 a.u.,
respectively, which shows that the signalꢀtoꢀnoise ratios in assays employing QDs can be
increased by more than two orders of magnitude.
As seen from Figure 16, accumulation of the signal from fluorogenic assays employing
streptaꢀQD for revealing the (compound 5)ꢀMMPꢀ2 complex during 10ꢀ20 min may result in an
increase in the intensity of the signal by more than two orders of the magnitude, whereas the
signals of all typical organic dyes recorded under exactly the same conditions are photobleached.
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However, the most important fact is that the fluorescent signal from the assay employing streptaꢀ
QD obtained in the course of signal accumulation is more than two orders of magnitude stronger
than that for the maximal signals that may be obtained for typical fluorogenic assays employing
the best organic dyes under optimal conditions. This shows that the use of QDꢀbased labels in
typical fluorogenic assays is decisively advantageous.
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Figure 16. Comparative analysis of variations of fluorescent signals as a result of their
accumulation at permanent signalꢀtoꢀnoise ratios for typical in vitro biochemical sandwichꢀlike
fluorogenic assays employing two different concentrations of the fluorescent streptavidinꢀQD800
conjugate and the most popular organic dyes.
Panels (a) and (b) show variations of the fluorescence signals from the model assays during
signal accumulation for 20 min and 4 min, respectively. In Panel (a), the signals from organic
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dyes are too weak to be visible in comparison with the signals from streptaꢀQDs at the equimolar
concentration and even after a 1/100 dilution; in Panel (b), the signals from streptaꢀQD are
strong and visible only in the initial period of signal accumulation.
Panel (c) shows the same data as in Panel (a) but on a demiꢀlogarithmic scale. Here, the curves
corresponding to the evolutions of the fluorescence signals from streptaꢀQDs and from different
organic labels are visible in the same graph.
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CONCLUSION
Proteolytic enzymes of the MMP family, in particular, MMPꢀ2, are overexpressed during
cancer pathological processes. Activated MMPs have been detected in tissues, plasma, serum,
and urine of cancer patients at increased levels, they are also positively correlated with the
7
5ꢀ77
severity of metastatic tumours.
Thus, MMPs can serve as selective and specific tumour
markers for clinical applications.
Due to the unique potency to combine monitoring and therapy, nanoparticleꢀbased techniques
can provide an advantage over standard diagnostic and therapeutic tools with respect to disease
diagnosis and monitoring, drug delivery and release. Moreover, nanoparticleꢀbased diagnostic
tools can be easily modified, combined, and improved to ensure multiplexed detection and
advanced diagnostics sensitivity. Thus, nanoparticles (e.g., fluorescent nanocrystals or QDs) are
considered a useful and indispensable tool for advanced clinical diagnostics, imaging, and
78
therapeutic applications.
In this study, a series of new MMPꢀinhibitors, ilomastat derivatives, have been prepared and
subjected to preliminary biological evaluations in experiments with the main matrix
metalloproteinases (MMPꢀ1, MMPꢀ2, MMPꢀ9, MMPꢀ13, and MMPꢀ14). A systematic enzyme
inhibition / selectivity study was carried out, with carboxylic acid serving as a zincꢀbinding
group (ZBG), by introducing a phenyl group to the C2 carbon atom of the tryptophan indole ring
and by incorporating alkylidene chains of different lengths at the P’1 position of the succinic
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acid moiety. As a result of structureꢀactivity relationship (SAR) studies, we have identified the
most selective MMPꢀ2 inhibitor with convenient inhibition potency (IC50 = 80 nM) and
biotinylated it for the use in assays employing the QDꢀstreptavidin conjugate for visualisation.
The specific fluorescent complex of compound 5ꢀbiotin/streptavidinꢀQD was tested and
evaluated for MMPꢀ2 inhibition in model in vitro sandwichꢀtype analytic biochemical assays and
compared with similar model assays employing typical organic dyes. The data show that the
fluorogenic assays employing QDs allows the detection signal level to be increased by more than
two orders of magnitude compared to typical organic dyes, thus paving the way to a considerable
increase in the sensitivity of MMP detection in in vitro assays. The described ultrasensitive QDꢀ
based detection approach, exemplified here by MMPꢀ2 detection, is by no means limited to it.
Designing and using broadꢀspectrum inhibitors will allow this strategy to be efficiently modified
for detecting both other MMPs and other hydrolytic enzymes for different substrates.
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EXPERIMENTAL PROCEDURES
General synthesis methods and materials
Standard solvents were purchased from commercial sources and were dried by standard
2
procedures and redistilled under N prior to use. Reactions and products were routinely
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monitored by thin layer chromatography (TLC) on silica gel (KIESELGEL 60 PF254, Merck).
HBTU and 2,4,6ꢀcollidine were purchased from commercial sources. Pure products were
obtained by means of flash chromatography using Merck Geduran SI silica gel 60 (70ꢀ230 mesh
ASTM). The melting points were determined using a Reichert Thermovar hotꢀstage apparatus
and are uncorrected. The NIRꢀFT spectra (KBr or NaCl film) were measured using a Perkinꢀ
1
13
Elmer Spectrum BX FTIR instrument. The H NMR (300 MHz) and C NMR (75 MHz) spectra
were recorded by means of a Bruker AC 300 spectrometer using TMS as an internal standard;
the chemical shifts δ were expressed in ppm; the following abbreviations are used: singlet (s),
doublet (d), doublet of doublets (dd), triplet (t), multiplet (m). Coupling constants J were
expressed in hertz. Mass spectra were recorded by means of an MSQ ThermoFinnigan apparatus
using the chemical ionization (CI) method. Electrospray ionization mass spectrometry
experiments were carried out using a hybrid tandem quadrupole/timeꢀofꢀflight (QꢀTOF)
instrument equipped with a pneumatically assisted electrospray (Zꢀspray) ion source
(Micromass, Manchester, UK) operated in the positive mode. Optical rotations were measured
on a PerkinꢀElmer 241 polarimeter (Na lamp, λ = 589 nm).
Synthesis procedures
(S)ꢀethyl3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(2,2,2ꢀtrifluoroacetamido)propanoate 7
Lꢀtryptophan (5.0 g, 24.5 mmol) was dissolved in absolute ethanol (80 mL). Thionyl chloride
(3.6 mL, 2 equiv, 48.9 mmol) was added dropwise during 15 min. The mixture was refluxed for
3.5 h; then, the solvent was evaporated. The white solid was dissolved in EtOAc, the organic
phase was washed with an aqueous solution of NaHCO (5%) and then dried over MgSO . After
filtration and concentration of the solvent, a slightly yellow oil was obtained. Then, CH Cl (110
3
4
2
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mL), triethylamine (3.4 mL, 1 equiv, 24.5 mmol), and trifluoroacetic anhydride (8.2 mL, 2.4
equiv, 58.6 mmol) were added at 0°C. The mixture was stirred at room temperature overnight.
The solvent was evaporated; the crude product was purified by flash chromatography
(
Cyclohexane/CH
2
Cl
2
: 40/60) to afford the white solid of compound 7 (5.6 g, 72%). mp: 117ꢀ
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1
119°C. [α]
D
ꢀ21 (c 1.13, DMSO). H NMR (DMSOꢀd
6
, 300 MHz): δ 1.14 (t, 3H, J = 7.1 Hz),
3.20ꢀ3.35 (m, 2H), 4.12 (q, 2H, J = 7.1 Hz), 4.52ꢀ4.60 (m, 1H), 7.02ꢀ7.10 (m, 2H), 7.18 (s, 1H),
13
7.37 (d, 1H, J = 8.1 Hz), 7.55 (d, 1H, J = 7.8 Hz), 9.95 (d, 1H, J = 7.5 Hz), 10.92 (s, 1H). C
NMR (DMSOꢀd
6
, 75 MHz): δ 14.1, 26.2, 54.0, 61.3, 109.4, 110.6, 111.7, 114.4, 118.2, 118.4,
ꢀ1
1
18.7, 121.3, 122, 124.0, 127.1, 136.3, 155.9, 156.4, 156.9, 157.4, 170.5. IR (KBr): ν (cm )
3374, 3322, 1736, 1696, 1555, 1304, 1278, 1230, 1176, 1093, 1027, 873, 860, 741, 692. MS
+.
(CI): m/z 328.96 [M ] (100), 311 (14), 283 (15), 255 (25), 242 (23), 238 (59). Anal. Calcd for
: C 54.88, H 4.61, N, 8.53%. Found: C 54.64, H 4.74, N 8.38%.
15 15 2 3 3
C H N O F
(
S)ꢀethyl 3ꢀ(2ꢀphenylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(2,2,2ꢀtrifluoroacetamido)propanoate
Nꢀbromosuccinimide (54 mg, 0.3 mmol) was added to a suspension of
Lꢀtryptophan ethyl ester 7 (0.1 g, 0.3 mmol) in CCl
8
α
ꢀNꢀ(trifluoroacety1)ꢀ
4
(2 mL). The mixture was refluxed for 30
min under nitrogen, and the solvent was evaporated in vacuo. The filtrate was purified by flash
chromatography (Petroleum ether/EtOAc: 9/1) to afford a brominated compound (118 mg, 96%).
21
1
mp: 134ꢀ137°C. [α]
D
ꢀ14 (c 1.41, DMSO). H NMR (CDCl3, 300 MHz): δ 1.22 (t, 3H, J = 7.1
Hz), 3.29ꢀ3.35 (m, 2H), 4.08ꢀ4.24 (m, 2H), 4.91 (dd, 1H, J = 13.8 Hz, J = 5.8 Hz), 6.99 (d, 1H, J
13
=
7.1 Hz), 7.09ꢀ7.20 (m, 2H), 7.25 (d, 1H, J = 7.4 Hz), 7.46 (d, 1H, J = 7.7 Hz), 8.35 (s, 1H). C
NMR (CDCl , 75 MHz): δ 13.8, 27.3, 52.9, 62.5, 109.0, 109.6, 109.8, 110.7, 113.6, 117.4, 117.7,
3
ꢀ1
1
20.5, 121.2, 122.8, 127.5, 136.0, 156.0, 156.5, 157.0, 157.5, 170.2. IR (KBr): ν (cm ) 3339,
1727, 1701, 1555, 1450, 1419, 1278, 1194, 1164, 1027, 873, 741, 723, 648. Anal. Calcd for
C
15
H
14
N
2
O
3
3
BrF : C 44.25, H 3.47, N 6.88%. Found: C 44.41, H 3.34, N 6.78%.
Phenylboronic acid (52 mg, 1.5 equiv, 0.42 mmol) was dissolved in a mixture of toluene and
ethanol (1/1) (4 mL), the brominated compound (115 mg, 0.28 mmol), NaHCO (47 mg, 2 equiv,
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.56 mmol) dissolved in pure water (1 mL), and LiCl (36 mg, 3 equiv, 0.84 mmol); finally,
catalyst Pd(PPh
under reflux for 2 h. The red mixture was concentrated; then, CH
NaHCO (10%) were added. The aqueous phase was extracted twice with CH
phases were collected, dried over MgSO , filtered, and concentrated. The crude product was
purified by flash chromatography (petroleum ether/EtOAc: 9/1) to afford compound 8 (105 g,
3
)
4
(32 mg, 0.1 equiv, 0.028 mmol) was added. The yellow solution was stirred
Cl and an aqueous solution of
Cl . The organic
2
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2
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21
1
92%). mp: 172ꢀ174°C. [α]
D
ꢀ10.5 (c 0.84, DMSO). H NMR (CDCl3, 300 MHz): δ 1.02 (t, 3H,
J = 7.1 Hz), 3.56ꢀ3.62 (m, 3H), 3.91ꢀ3.97 (m, 1H), 4.81 (dd, 1H, J = 13.6 Hz, J = 5.8 Hz), 6.68
13
(d, 1H, J = 7.4 Hz), 7.12ꢀ7.57 (m, 9H), 8.22 (s, 1H). C NMR (CDCl
3
, 75 MHz): δ 13.7, 26.5,
53.4, 61.9, 105.6, 109.0, 111.0, 113.0, 117.0, 118.5, 120.2, 121.0, 122.7, 128.2, 128.3, 128.9,
ꢀ1
129.0, 129.1, 132.5, 135.6, 136.3, 155.1, 155.7, 156.3, 156.9, 170.2. IR (KBr): ν (cm ) 3374,
3
4
4
286, 1753, 1705, 1555, 1450, 1278, 1203, 1172, 1018, 877, 745, 697. MS (CI): m/z [M+1]
05.29 (100), 405 (60), 339 (15), 218 (20), 206 (15). Anal. Calcd for C21
.74, N 6.93%. Found: C 62.18, H 4.66, N 6.82%.
19 2 3 3
H N O F : C 62.37, H
(S)ꢀethyl3ꢀ(1ꢀ(3ꢀtertꢀbutoxyꢀ3ꢀoxopropyl)ꢀ2ꢀphenylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(2,2,2ꢀ
trifluoroacetamido)propanoate
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tBuOK (107 mg, 1.3 equiv, 0.95 mmol) was added to a solution of compound 8 (296 mg, 0.73
mmol) in THF (5 mL); the solution was stirred for 15 min under nitrogen. Then, tertꢀ
butylacrylate (1 mL) was added to the solution dropwise. The reaction mixture was incubated
4
upon heating to reflux for 3.5 h. The mixture was diluted with a solution of saturated NH Cl and
extracted with EtOAc. The organic phase was dried over MgSO and filtered, and the solvent
4
was evaporated. The crude product was purified by flash chromatography (petroleum
2
1
ether/EtOAc: 85/15) to afford compound 9 (351 mg, 90%). mp: 84ꢀ86°C. [α]
D
ꢀ2.4 (c 0.5,
1
MeOH). H NMR (CDCl3, 300 MHz): δ 1.08 (t, 3H, J = 7.1 Hz), 1.32 (s, 9H), 2.37ꢀ2.43 (m, 2H),
3
.33 (ddd, 2H, J = 5.7 Hz, J = 14.7 Hz, J = 20.4 Hz), 3.74ꢀ3.80 (m, 1H), 3.98ꢀ4.04 (m, 1H),
.25ꢀ4.30 (m, 2H), 4.71 (dd, 1H, J = 6.1 Hz, J = 13.9 Hz), 6.51 (d, 1H, J = 7.8 Hz), 7.15ꢀ7.61
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