Synthesis and binding monitoring of a new nanomolar PAMAM-based matrix metalloproteinases inhibitor (MMPIs)

Article abstract of DOI:10.1016/j.bmc.2016.11.028

Dendrimers are efficient drug delivery systems particularly useful in ocular diseases. In particular, low generation PAMAM dendrimers are non-toxic and non-immunogenic and they provide an enhancement of the residence time of drugs in the eyes. In this context, the synthesis of the PAMAM-based matrix metalloproteinases inhibitor 5, is reported. In particular, we demonstrated that 5 strongly binds (18.0?nM?±?2.5?nM) MMP-9, the most relevant MMP responsible of ocular surface damages in induced dry eyes syndrome (DES).

Full text of DOI:10.1016/j.bmc.2016.11.028

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and binding monitoring of a new nanomolar PAMAM-based
matrix metalloproteinases inhibitor (MMPIs)
Linda Cerofolini a,b,c, Veronica Baldoneschi a,c, Elisa Dragoni a, Andrea Storai a, Marianna Mamusa a,
Debora Berti a, Marco Fragai a,b, , Barbara Richichi a, , Cristina Nativi a,b
⇑
⇑
a Department of Chemistry ‘‘Ugo Schiff”, University of Florence, Via della Lastruccia 3-13, 50019 Sesto Fiorentino (FI), Italy
b CERM, University of Florence, Via Sacconi 6, 50019 Sesto Fiorentino (FI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Dendrimers are efficient drug delivery systems particularly useful in ocular diseases. In particular, low
generation PAMAM dendrimers are non-toxic and non-immunogenic and they provide an enhancement
of the residence time of drugs in the eyes. In this context, the synthesis of the PAMAM-based matrix
metalloproteinases inhibitor 5, is reported. In particular, we demonstrated that 5 strongly binds
(18.0 nM 2.5 nM) MMP-9, the most relevant MMP responsible of ocular surface damages in induced
dry eyes syndrome (DES).
Received 21 September 2016
Revised 10 November 2016
Accepted 11 November 2016
Available online xxxx
Keywords:
Inhibitors
Ó 2016 Elsevier Ltd. All rights reserved.
Metalloproteins
Dendrimers
Sulphonamidic scaffold
NMR spectroscopy
1. Introduction
called TIMPs.4 A loss of control of one of these steps results in an
unbalanced MMPs’ activity with the consequent onset of several
Matrix metalloproteinases (MMPs)1 also called matrixins, are a
large family of structurally related zinc-dependent endopeptidases
involved in the degradation of several extracellular matrix compo-
nents (ECMs) including collagens, glycoproteins and proteoglycans.
However, the enzymatic activity of MMPs is not only confined on
the degradation of ECMs because they also play a crucial role in
the proteolysis of other substrates, such as membrane receptors,
cytokines, growth factors and other proteases.2 Moreover, an intra-
cellular localization of some MMPs has been demonstrated, sug-
gesting a relevant role of these enzymes in the proteolysis of
intracellular substrates.3 Therefore, the functions of MMPs are
much more complex than straightforward breakdown of connec-
tive tissue and the complexity of their role in health, as well as
in diseases, is to date quite evident.2a Under physiological
conditions MMPs’ activity is the result of a complex balance
between several factors, like genes expression, proenzyme
activation and inhibition mediated by the endogenous inhibitors,
diseases, such as cancer, rheumatoid arthritis, heart failure and
inflammation.
MMPs are, thus, considered key targets and, thanks to the struc-
tural insights5 available on the catalytic domain of these proteases,
a large number of synthetic and natural MMP inhibitors (MMPIs),
targeting the aberrant activity of MMPs, have been identified.6 In
particular, the sulfonamide-based MMPIs, as NNGH and
CGS27023A, and the peptide-based inhibitors, as Batimastat7and
Marimastat,8 are potent MMPIs and bind these endopeptidases
with a low nanomolar affinity. Despite their high affinity toward
MMPs, MMPIs suffer from two main drawbacks, which limit their
clinical use: the low availability and the scarce selectivity towards
the members of MMPs’ family.
In the last twenty years, these MMPIs have been considered as
lead structures in the design of new compounds with improved
properties compared to the parent molecules.9 However, small
changes on the structure of the bioactive scaffolds can have a dra-
matic effect on the biological activity of the newly synthesized
compounds. For this reason, in designing new potential drugs,
the identification of the key pharmacophoric groups is essential.
In this regards, in 2005, the structure of the complex between
N-isobutyl-N-(4-methoxyphenylsulfonyl) glycil hydroxamic acid
(NNGH)10 (Fig. 1) and the catalytic domain of MMP-12 has been
solved by X-ray diffraction and NMR spectroscopy.5b
⇑
Corresponding authors at: Department of Chemistry ‘‘Ugo Schiff”, University of
Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino (FI), Italy (B. Richichi);
CERM, University of Florence, Via Sacconi 6, 50019 Sesto Fiorentino (FI), Italy
(M. Fragai).
E-mail addresses: fragai@cerm.unifi.it (M. Fragai), barbara.richichi@unifi.it
(B. Richichi).
c
L.C. and V.B. equally contributed to the work.
http://dx.doi.org/10.1016/j.bmc.2016.11.028
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cerofolini L., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.028

2
L. Cerofolini et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
HO
More recently, the leading concept in manipulating NNGH
skeleton was applied again to design compound 4 (Fig. 1), where
two residues of a -serine containing MMPI are conjugated to a
OH
OH
O
HO
HO
O
O
O
D
OH
O
O
O
HO
N
HO
N
O
divalent dendrimer. Dendrimers are intriguing systems for drug
delivery and they have been widely used for biomedical applica-
tions.15 In particular, they proved to be extremely efficient systems
in ocular drugs delivery.16 Indeed, dendrimers are nontoxic and
non-immunogenic molecules which are able to enhance the resi-
dence time of the drugs17 by avoiding the clearance mechanism
of the eyes. In this context, we recently reported18 on the synthesis
of the water-soluble PAMAM-based inhibitor 4 (Fig. 1). This com-
pound combines the unique bioadhesive properties of poly (ami-
doamine) (PAMAM) dendrimers and the high affinity of the
hydroxamate scaffold leading to significant biological effects
in vivo. Hence, it proved to be effective in rabbits affected by
induced dry eyes syndrome (DES), where the compound restores
the normal hydration degree on corneal surface, without the sys-
temic side-effects of conventional MMPIs.
N
H
N
H
S
S
O
O
O
HO
N
N
H
S
1
NNGH
2
O
O
O
O
O
HO
HO
O
O
OH
O
OH
O
S
N
H
O
N
O
4
N
4
O
H
HO
OH
OH
O
3
NH
HO
O
OH
O
S
H
N
O
N
Taking into account these in vivo encouraging results, we report
here on the synthesis of 5 (Fig. 2), and on its binding to MMP-9
(gelatinase) and MMP-12 (macrophage elastase).
OH
O
O
NH
In particular, 5 is a new PAMAM-based MMPI which maintains
the dendrimer-based scaffold of compound 4, but the bioactive sul-
phonamide is conjugated to the PAMAM dendrimer through a
longer aliphatic spacer to promote the interaction of the hydroxa-
mate moiety with the active site of the enzyme.
HN
NH
O
4
O
O
O
O
H
N
H
N
N
S
BocHN
N
N
H
O
O
O
2. Result and discussion
O
OH
NH
HO
2.1. Synthesis of the PAMAM-based inhibitor 5
Fig. 1. Structure of NNGH, the water-soluble derivatives 1 and 2, the ditopic MMPI
3, the divalent PAMAM-based inhibitor 4.
The precursor for the synthesis of the divalent PAMAM-based
MMPIs 5 is the activated sulfonamide 6, which was prepared fol-
lowing the efficient and few steps protocol reported in Scheme 1.
The D
-serine containing sulfonamide 712 was firstly protected as
The pharmacophoric enzyme-ligand interactions, like the bind-
ing of the aromatic ring with the hydrophobic S10 pocket and the
chelation of the catalytic Zn2+ ion by the hydroxamate moiety have
been confirmed. In addition, the X-ray structure disclosed relevant
insights about the role of the isobutyl group on the sulfonamide
nitrogen. Indeed, this residue did not directly participate in the
binding but rather it pointed away toward the solvent. This obser-
vation and the calculations in silico5a,11 prompted us to design new
MMPIs with improved properties by manipulating the scaffold of
NNGH9b,12 at the sulfonamide nitrogen substituent. In this
respect, in the last ten years, we reported the synthesis of several
NNGH derivatives, like 1–4 (Fig. 1), with specific
functionalization of the sulfonamide nitrogen, which exhibit a
remarkable affinity toward a panel of MMPs.
Thus, we demonstrated that, NNGH is a versatile scaffold to pre-
pare nanomolar inhibitors of MMPs with improved properties. In
particular, NNGH is insoluble in water, thus we replaced the isobu-
tyl group with several hydrophilic residues to generate a large fam-
ily of nanomolar water-soluble NNGH derivatives, such as
compounds 112 and 213 (Fig. 1).
Then, considering the structural similarity of the different
MMPs at the active site, and the well-known capability of these
enzymes of adapting the binding pocket to the inhibitor shape,
the development of selective MMPIs for each member of this pro-
tein family is still a demanding issue.
In this context, we decided to deal with the drawback related to
the lack of selectivity associated to MMPIs by exploiting a site-
specific delivery approach to target MMPs overexpressed at the
site of disease progression. In particular, the effective bifunctional
probe 3 (Fig. 1), targeting galectins for directing MMPIs to disease
site, has been successfully prepared.14
silylether, under standard conditions (tBuMe2SiOTf, NEt3, CH2Cl2,
0 °C), to afford, in high yield (97%), compound 8. Then, the sulfon-
amide nitrogen of 8 was alkylated, under Mitsunobu reaction
conditions (DIAD, PPh3, THF, 21 h, at room temperature, rt), using
the N-Cbz-5-amino-1-pentanol 9 to give the corresponding func-
tionalized derivative 10 in high yield (94%). Subsequent removal
of the silylether protective group in acidic conditions (AcOH:H2O
80:20, 11, 92%), and of the Cbz functional group (Pd(OH)2/C, H2,
AcOEt/MeOH) afforded compound 12, which in turn was reacted
with adipate ester 13 to afford the activated sulfonamide
derivative 6 (82% of yield over two steps).
Then, the sulfonamide 6 was firstly reacted at rt with the
divalent PAMAM-dendrimer 14 (Scheme 2) in dry DMF to give
the divalent compound 15 (88%), which was directly transformed
into the corresponding hydroxamic acid 5 (24%) by reaction with
O
O
S
N
O
OH
3
O
O
NHOH
NH
HN
5
O
O
NH
O
O
O
O
S
N
O
H
H
N
N
N
Boc
N
H
N
H
3
OH
NHOH
O
O
Fig. 2. Structure of the divalent PAMAM-based inhibitor 5.
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L. Cerofolini et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
NHCbz
3
H
N
H
O
O
O
COOCH3
OH
HO
NHCbz
9
N
COOCH3
N
COOCH3
S
S
S
3
O
tBuMe2SiOTf, NEt3
CH2Cl2
O
O
PPh3, DIAD THF
10
O
O
Si
Si
97%
86%
OCH3
OCH3
OCH3
7
8
AcOH/H2O
80/20
92%
NH2
NHCbz
3
3
O
O
H2
O
N
O
O
COOCH3
OH
N
COOCH3
OH
Pd(OH)2/C
AcOEt/MeOH
3/1
O
O
2
S
S
O
NO2
NO2
13
12
82%
over two steps
OCH3
OCH3
NMM
DMF
11
H
N
O
NO2
2
3
O
O
O
N
COOCH3
S
O
OH
6
OCH3
Scheme 1. Synthesis of the activated sulphonamide derivative 6.
O
O
O
3
S
N
OH
O
NH2
NH
O
O
O
NH
BocHN
HN
N
14
O
O
NH
NH
Boc
HN
O
NH2OH.HCl, KOH
MeOH
DMF
NH2
N
5
6
24%
88%
NH
O
O
HN
15
NH
O
O
O
S
3
N
O
O
OH
O
Scheme 2. Synthesis of the divalent PAMAM-based derivative 5.
hydroxylamine hydrochloride and potassium hydroxide, in MeOH
as solvent (Scheme 2).
particular, the assay provided a Ki value of 18.0 2.5 nM for com-
pound 5 (see Table S1 in Supporting information for the Ki values
toward other MMPs). These data indicate that the increased length
of the aliphatic spacer between the sulfonamide nitrogen and the
PAMAM scaffold, results in an improvement of the binding affinity
related to the divalent PAMAM-based inhibitor 5, toward MMP-9.
2.2. Fluorimetric assay on compound 5
To assess the binding properties of compound 5, the inhibition
constant toward MMP-9, the most relevant MMP responsible of
ocular surface damages in DES, was evaluated with a fluorimetric
assay by monitoring the hydrolysis of the fluorescence quenched
peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Enzo
Life Sciences, Inc.)19 (see Supporting information for details on
the protocol used). The Ki values, obtained by using the catalytic
domain of MMP-9, were compared with those previously obtained
from the divalent PAMAM-based inhibitor 4 (Ki = 289 54 nM). In
2.3. Structural information of the binding of compound 5 to MMP-12
The NMR binding studies were performed on the catalytic
domain of MMP-12 that is a widely studied and fully characterized
member of the MMPs’ family. Binding of compound 5 to the
15N-enriched catalytic domain of MMP-12 was monitored by 2D
1H-15N HSQC NMR spectroscopy during a slow stepwise titration
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4
L. Cerofolini et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
employing the widely studied MMP-12. These assays allow us to
fully characterize the binding by identifying the amino acids resi-
dues of the enzyme involved in the interaction and defining the
stoichiometry of the complex. The in vivo evaluation of the thera-
peutic potential of compound 5, in an experimental model of
induced DES, is underway.
4. Experimental
4.1. General and materials
Reagents were purchased from commercial suppliers and used
without purification. ESI-MS mass spectra were recorded on a
LCQ-Fleet Ion Trap equipped with a standard Ionspray interface
from Thermo Scientific. NMR spectra were recorded on Varian
Gemini 200, Gemini 300, Mercury Plus 400, Inova 400, Bruker
AVANCE 500 and 700 instruments. Elemental analyses were per-
formed with a PerkinElmer series II CHNS/O 2400 Elementary Ana-
Fig. 3. Surface representation of the catalytic domain of MMP-12. The residues
showing significant chemical shift perturbation upon the addition of the PAMAM-
based divalent inhibitor 5 (Y121, V162, H168, K177, G178, G179, I180, L181, A182,
H183, A195, H196, F197, D200, T215, A216, V217, H218, E219, S223, V235, M236,
F237, Y240, K241, Y242, V243, I245) are highlighted in yellow.
lyzer.
[a
]
D
values were measured using a JASCO DIP-370
instrument. Melting points were measured with a Melting Point
Büchi 510.
with a DMSO-d6 solution of the divalent inhibitor. The final con-
centration of compound 5 was 0.2 mM with 1 % of DMSO-d6. The
presence of compound 5 induced relevant changes in the HSQC
spectra, thus unequivocally revealing the interaction with the pro-
tein (see Fig. S1).
4.2. Synthesis of compound 6
To a stirred solution of 11 (184 mg, 0.36 mmol) in a mixture of
EtOAc/CH3OH (3/1, 4 mL), Pd(OH)2/C 20% wt. was added (92.0 mg).
The mixture was stirred at room temperature for 19 h under H2
atmosphere, then it was filtrated on a pad of CeliteÒ. Evaporation
of the solvent under reduced pressure gave 121 mg of the crude
product 14, which was used for the next step without further
purification. To a stirred solution of crude 12 (121 mg) in dry
The residues involved in the interaction were identified by
using the assignment of the protein5b and the 2D 1H-15N HSQC
correlation peaks. Binding of compound 5 affects amino acids
that form the active site of the enzyme, including some residues
located in the S10 pocket (Fig. 3), as usually observed for NNGH
derivatives MMPIs.5b
Then, in order to assess the stoichiometry of the interaction
between the MMP-12 binding site and compound 5, we character-
ized the complex in solution by means of dynamic light scattering
(DLS). The autocorrelation function was acquired at 90° and com-
pared with that obtained for the MMP-12/NNGH complex in the
same experimental conditions. As a result, both profiles showed
a single exponential decay. However, for MMP-12/NNGH the corre-
lation function showed a faster decay (see Supporting informa-
tion), in agreement with the presence of smaller object which
diffuse more rapidly in solution. By fitting the experimental data
DMF (3.0 mL), NMM (171 lL, 1.56 mmol) and 13 (1.51 g,
3.90 mmol) were added. The reaction mixture was left to stir for
18 h, then DMF was removed under reduced pressure. The crude
was dispersed in MeOH (15 mL), then the solid was filtered off
and the filtrate was concentrated and purified by a flash column
chromatography on silica gel (CHCl3 then CHCl3/CH3OH 15/1) to
25
afford 6 (183 mg, 82%) as a pale yellow oil. [
a
]
D
+37.6 (c 0.21,
CHCl3); HRMS m/z: calcd for C28H36O11N3S [MꢀH]ꢀ 622.20760
1
Found 622.20755; H NMR (500 MHz, CDCl3): d 8.28–8.25 (AA0 part
of an AA0MM0 system, JAM = 9.5 Hz, 2H, Ph), 7.78–7.75 (AA0 part of an
AA0MM0 system, JAM = 9.0 Hz, 2H, Ph), 7.30–7.26 (MM0 part of an
AA0MM0 system, JMA = 9.5 Hz, 2H, Ph), 6.98–6.95 (MM0 part of an
AA0MM0 system, JMA = 9.0 Hz, 2H, Ph), 5.83–5.81 (bs, 1H), 4.54–4.52
(X part of an ABX system, JXB = 6.5 MHz, JXA = 5.5 MHz, 1H,
CHCH2OH), 4.09–4.05 (A part of an ABX system, JAB = 11.5 MHz,
JAX = 5.5 MHz, 1H, CHCH2OH), 3.93–3.90 (B part of an ABX system,
JBA = 11.5 MHz, JBX = 6.5 MHz, 1H, CHCH2OH), 3.87 (s, 3H, PhOCH3),
3.58 (s, 3H, COOCH3), 3.34–3.29 (m, 2H, CH2NHCO), 3.26–3.17 (m,
1H, CH2NSO2), 3.17–3.09 (m, 1H, CH2NSO2), 2.64 (m, 2H, CH2-
COOPh-NO2), 2.25 (m, 2H, CH2CONH), 1.81–1.71 (m, 4H, CH2CH2-
CH2COOPh-NO2), 1.71–1.65 (m, 2H, CH2CH2NSO2), 1.53–1.49 (m,
2H, CH2CH2NHCO), 1.34–1.27 (m, 2H, CH2CH2CH2NSO2); 13C NMR
(125 MHz, CDCl3): d 172.7 (Cq), 171.0 (Cq), 170.1 (Cq), 155.4 (Cq),
145.3 (Cq), 131.3 (Cq), 129.5 (CH Ar), 125.1 (CH Ar), 122.5 (CH Ar),
114.0 (CH Ar), 61.8 (CH2OH), 61.4 (CHCH2OH), 55.6 (PhOCH3), 52.3
(COOCH3), 47.0 (CH2NHCO), 39.0 (CH2NSO2), 36.1 (CH2CONH), 33.9
(CH2COOPh-NO2), 29.7 (CH2CH2NSO2), 28.8 (CH2CH2NHCO),
24.9–24.1 (CH2CH2CH2COOPh-NO2), 23.5 (CH2CH2CH2NHCO).
with the cumulant method,
a hydrodynamic radius (RH) of
2.1 nm was calculated for the MMP-12/NNGH complex. The same
analysis performed on MMP-12/compound 5 complex provided a
RH of 3.0 nm, which is one and a half times larger than that calcu-
lated for MMP-12 in complex with the monovalent inhibitor
NNGH. The two different values of the hydrodynamic radius
strongly suggest that compound 5 behaves as a divalent inhibitor
capable to bind up to two molecules of MMP-12, simultaneously.
3. Conclusions
The unbalanced activity of MMPs is involved in the pathophys-
iology and pathogenesis of several diseases, thus the modulation of
the activity of these enzymes, by using high affinity MMPIs, is a
challenging task. Here we reported on the synthesis of the
PAMAM-based MMPI 5, a high affinity inhibitor of MMP-9. In par-
ticular, the dendrimer scaffold was conjugated to the sulphonami-
dic nitrogen of a NNGH – like scaffold, through the combination of
an aliphatic spacer and an adipate moiety. Thus, compound 5 com-
bines the unique features of the PAMAM constructs, as drug deliv-
ery systems in ocular diseases, with the bioactive hydroxamate
scaffold. Furthermore, the binding to the catalytic domain of the
4.3. Synthesis of compound 5
A suspension of KOH (60.0 mg, 1.06 mmol) in 350
and a suspension of NH2OHꢁHCl (49.0 mg, 0.71 mmol) in 590
CH3OH were refluxed for 150, then the solution of KOH was added
lL of CH3OH
metalloenzyme was monitored by 2D NMR experiment (1H-15
HSQC) and dynamic light scattering (DLS) of the complex, by
N
lL of
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L. Cerofolini et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
to the solution of NH2OHꢁHCl and the suspension was stirred at
40 °C for 100. This suspension was cooled at room temperature
and added to 15 (80.0 mg, 0.06 mmol). The reaction mixture was
stirred for 7 h at rt then it was filtered and concentrated to dryness.
The crude product which was purified by reversed-phase HPLC
tained in glass tubes, which were immersed in a thermostatic cell
filled with decahydronaphthalene to match the glass refractive
index. Data modelling with the cumulant method allowed to
derive the diffusion coefficient of the complexes in solution and,
by assuming a spherical shape, their average hydrodynamic radius
(RH) through the Stokes–Einstein law:
(Column Zorbax 300SB-C18, 9.4 ꢂ 250, 5
lm, from CH3OH/H2O
55/35–70/30) to afford 5 (18.0 mg, 24%) as a glassy white solid.
HRMS: calcd. for C59H97N12O20S2 1357.63890 [MꢀH]ꢀ. Found
1357.63882; 1H NMR (500 MHz, CD3OD): d 7.82–7.81 (m, 4H,
Ph), 7.09–7.07 (m, 4H, Ph), 4.34–4.31 (at, 2H, H-2), 3.89 (s, 6H,
PhOCH3), 3.88–3.84 (m, 2H, H-1), 3.62–3.59 (m, 2H, H-10), 3.47–
3.40 (m, 2H, H-3), 3.31–3.29 (m, 8H, H-12, H-13), 3.27–3.20 (m,
2H, H-30), 3.18–3.14 (m, 6H, H-7, H-17), 2.80–2.77 (m, 4H, H-15),
2.58–2.55 (m, 2H, H-16), 2.38–2.35 (m, 4H, H-14), 2.25–2.20 (m,
8H, H-8, H-11), 1.66–1.61 (m, 16H, H-9, H-10, H-4, H-6), 1.53–
1.48 (m, 4H, H-5), 1.44 (s, 9H, Boc); 13C NMR (125 MHz, CD3OD):
d 174.8 (Cq), 174.6 (Cq), 173.9 (Cq), 166.9 (Cq), 163.3 (Cq), 156.9
(Cq), 130.9 (Cq), 129.3 (Cq), 129.2 (CH Ar), 113.9 (CH Ar), 60.8
(C-1), 59.8 (C-2), 54.8 (PhOCH3), 52.1 (C-16), 49.5 (C-15), 45.1
(C-3), 38.9–38.8–38.7 (C-7, C-12, C-13, C-17), 35.4 (C-8, C-11),
33.1 (C-14), 30.0–29.9 (C-9, C-10), 28.5 (C-5), 27.4 (Boc),
25.2–25.1 (C-4, C-6).
RH ¼ kBT=6
pgDdiff
where kB is Boltzmann’s constant, T the absolute temperature, and
g
the medium’s viscosity.
Acknowledgments
Authors thank Prof Claudio Luchinat (CERM, University of Flor-
ence) for fruitful discussions. This work has been supported by
Ente Cassa di Risparmio di Firenze, MIUR PRIN 2012SK7ASN, and
EU ESFRI Instruct Core Centre CERM, Italy.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.11.028.
4.4. Protein expression and purification
References
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4.5. Fluorimetric assay
(c) Eble JA. Curr Pharm Des. 2009;15:1275–1276;
The inhibition constants for compound 5 were determined,
evaluating their ability to prevent the hydrolysis of the fluores-
cent-quenched peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-
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4.6. NMR binding to MMP-12
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4.7. Dynamic light scattering
DLS experiments were carried out on a BI-9000AT digital auto-
correlator (Brookhaven Instruments, USA) equipped with a green
laser (k = 532 nm; Torus, mpc3000, LaserQuantum, UK). The scat-
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Please cite this article in press as: Cerofolini L., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.028