New clicked thiirane derivatives as gelatinase inhibitors: The relevance of the P1′ segment

Article abstract of DOI:10.1039/c3ra46402d

Gelatinases (MMP-2 and MMP-9), a subfamily of Matrix Metalloproteinases (MMPs), are involved in several pathologies and especially in cancer. Thiirane is a latent-zinc binding group used for the design of potent inhibitors of gelatinases. Here we report a new family of thiirane inhibitors, obtained by click chemistry. Thus, an azide fragment containing the thiirane group was connected to several lipophilic alkynes, which were designed to interact with the S1′ pocket of the two gelatinases. Our hit compound (2f) displayed submicromolar inhibition of MMP-2 (IC₅₀ = 0.62 μM). Computational studies have been used to compare the binding mode of compound 2f in MMP-2 with the reference thiirane inhibitor (SB-3CT), allowing us to discuss the relevance of the P1′ segment in order to maximize potency. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ra46402d

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PAPER
New clicked thiirane derivatives as gelatinase
inhibitors: the relevance of the P10 segment†
Cite this: RSC Adv., 2014, 4, 17726
B. Fabre,a K. Filipiak,ab C. Coderch,a J. M. Zapico,a Rodrigo J. Carbajo,c
c
c
a
a
*
*
Anne K. Schott, Antonio Pineda-Lucena, B. de Pascual-Teresa and A. Ramos
Gelatinases (MMP-2 and MMP-9), a subfamily of Matrix Metalloproteinases (MMPs), are involved in several
pathologies and especially in cancer. Thiirane is a latent-zinc binding group used for the design of potent
inhibitors of gelatinases. Here we report a new family of thiirane inhibitors, obtained by click chemistry.
Thus, an azide fragment containing the thiirane group was connected to several lipophilic alkynes, which
were designed to interact with the S10 pocket of the two gelatinases. Our hit compound (2f) displayed
submicromolar inhibition of MMP-2 (IC50 ¼ 0.62 mM). Computational studies have been used to
compare the binding mode of compound 2f in MMP-2 with the reference thiirane inhibitor (SB-3CT),
allowing us to discuss the relevance of the P10 segment in order to maximize potency.
Received 4th November 2013
Accepted 1st April 2014
DOI: 10.1039/c3ra46402d
www.rsc.org/advances
pocket,8 using compounds including or not a group capable to
bind the catalytic zinc ion (zinc binding group or ZBG).9–11
Introduction
The hydroxamate moiety was largely used as ZBG for potent
inhibition of MMPs.11,12 However, hydroxamates suffer from
metabolic liability, toxicity and generally low selectivity. For this
reason, non-hydroxamate ZBGs have been developed, such as
carboxylates, thiolates, phosphonates, phosphinic derivatives,
hydroxypyrone derivatives and pyrimidinetriones, among
others.10 More than a decade ago, Brown et al.13 reported thiir-
ane SB-3CT as a new slow-binding inhibitor, that displayed
remarkable selective inhibition of gelatinases. Additionally, SB-
3CT efficiently inhibits cancer growth and metastasis in liver
Gelatinases (MMP-2 and MMP-9) belong to the matrix metal-
loprotease family (MMP), a group of zinc-dependent endopep-
tidases.1 In the 90 s, MMPs were considered as ideal targets,
mainly for cancer and inammation diseases, due to their
ability to degrade extracellular matrix (ECM) components such
as collagen.2 However, their role is more complicated than rst
thought, and MMPs have been demonstrated to participate also
in the activation, regulation or destruction of signalling mole-
cules, such as growth factors and cytokines.3,4 Furthermore,
related to their implication in cancer, some MMPs play
protective roles depending on the cancer localization or stage.5
This, in addition to the dose-limiting musculoskeletal
syndrome (MSS), is one of the reasons for the disappointing
results obtained in clinical trials with broad-spectrum MMP
inhibitors (MMPIs).6,7 Thus, medicinal chemists from the MMP
eld are now looking for more selective inhibitors within the
MMP family. This is mainly achieved by exploring differences in
the lipophilic S10 pocket of MMPs, also known as the selectivity
a
´
´
Departamento de Quımica y Bioquımica, Facultad de Farmacia, Universidad CEU San
´
Pablo, Urbanizacion Monteprincipe, 28668, Boadilla del Monte, Madrid, Spain.
E-mail: aramgon@ceu.es
bDepartment of Molecular Biology, Faculty of Biotechnology and Environment Sciences,
The John Paul II Catholic University of Lublin, 20-718, Lublin, Poland. E-mail:
bpaster@ceu.es
cStructural Biochemistry Laboratory, Advanced Therapies Program, Centro de
´
´
´
Investigacion Prıncipe Felipe, C/Eduardo Primo Yufera 3, 46012 Valencia, Spain
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of the compounds, competition waterLOGSY experiment for 2e, the method used
to obtain the 1CK7 structure with relaxed S10 pocket, as well as the starting and
nal structures of the MD simulations are found in ESI. See DOI:
10.1039/c3ra46402d
Fig. 1 Proposed mechanism for the slow inhibition of MMPs by SB-
3CT. The catalytic Glu404 removes a proton from the inhibitor, leading
to the opening of the thiirane ring. The catalytic zinc ion of the MMP is
represented in magenta and the S10 pocket is symbolized by orange
dots. Numbering of the full proMMP-2 is used in this work.26
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and bone murine models.14,15 The proposed inhibition mecha- click chemistry. The alkynes used here were chosen based on
nism (Fig. 1), based on experimental and computational our previous work in this eld.27
studies, implies the opening of SB-3CT thiirane ring within the
Selective S-alkylation of 4-aminothiophenol by allyl chloride
MMP catalytic cle, leading to the real active specie 1.16–19 As SB- provided compound 3. Less than 20% of the starting material
3CT displays high metabolism – in particular at the phenoxy- was converted into the corresponding N-alkylated by-product,
phenyl part – and poor aqueous solubility,20–24 Mobashery et al. which was easily separated from the S-alkylated product by
replaced the distal phenoxy group of SB-3CT by triazole frag- column chromatography. Compound 3 was treated with tert-
ments. However only weak gelatinases inhibitors were butyl nitrite to afford the diazonium salt, which was reacted in
obtained.25
situ with azidotrimethylsilane as azide source, to form azide 4.
We previously reported a series of highly potent triazole Oxidation with mCPBA led to a-sulfone epoxide 5. In this step,
based MMP-2 inhibitors with hydroxamate as ZBG obtained an excess of mCPBA was necessary, and the reaction was
following the click-chemistry approach,27,28 in which an azide stopped aer one week, before total completion. Other oxida-
containing segment was connected to a series of alkynes tion agents (peracetic acid and hydrogen peroxide) were tried,
(named P10 fragments according to Schechter and Berger as well as different temperatures of reaction, without any
nomenclature29) designed to interact with the lipophilic resi- improvement in the nal yield. Epoxide 5 was converted into
dues of the S10 pocket. This work led to inhibitors with high thiirane 6 by reaction with thiourea. From the azide 6, the tri-
selectivity for MMP-2 over MMP-9.30 Based on those encour- azole family 2a–f was obtained by mean of copper catalysed
aging results, we decided to replace the hydroxamate ZBG for azide–alkyne cycloadditions (CuAAC), using copper sulphate
the reasons stated above. Being interested in the inhibition of pentahydrate and sodium ascorbate as catalytic system. Three
MMP-2 because of its potential role in cancer,31 we chose the solvents (1 : 1H2O–tBuOH, MeCN and DMF) were screened for
thiirane latent ZBG as thiirane derivatives displayed good this transformation, DMF giving the best results.
selectivity for this enzyme.13,32 Here we report the discovery of a
The alkynes necessary for the synthesis of 2a–e are
hit compound (2f) that showed submicromolar activity against commercially available, whereas the alkyne necessary for the
MMP-2, thus improving the potency of the clicked thiirane synthesis of 2f was synthesized according to the literature
derivatives previously reported by Mobashery group in their procedure.34 In Scheme 1, we also show the results of the
attempt to improve the metabolic behaviour of SB-3CT.25 To opening of thiiranes 2a–f to form allyl thiolates 7a–f, expected to
design our compounds, we connected the azide core containing occur in the protein catalytic centre.
the latent-ZBG to the series of alkynes that provided the most
potent inhibitors in our previous work,27 as well as to two new
alkyne fragments (Fig. 2). Docking studies and molecular
Biology
dynamics (MD) experiments have been performed in order to Compounds activities against MMP-2 and MMP-9 were evalu-
rationalize the difference of inhibitory activity between 2f and ated in vitro, using a colorimetric assay.
the reference compound SB-3CT. This work allowed us to
The reaction was measured both aer 45 min and 2 h of
discuss the importance of the P10 fragment in the potency of incubation of the enzyme with the inhibitors (Table 1). Thiir-
this type of inhibitors.
anes are slow-binding inhibitors of gelatinases, and they need a
previous enzymatic transformation to the corresponding allyl
thiolate, which is the real inhibitor (see mechanism in Fig. 1).
The higher activity observed by us aer a longer incubation
period (2 h) is in accordance with the described mechanism of
action of thiirane inhibitors. The most active compound is 2f
(IC50 ¼ 0.62 mM against MMP-2 and IC50 ¼ 2.71 mM against
Results and discussion
Chemistry
Compounds 2a–f were synthesized as depicted in Scheme 1. As
the chirality of SB-3CT has no inuence on its biological action
on gelatinases,33 – the two enantiomers of SB-3CT and the MMP-9), with a 4-pyridyl substituent in the terminal aromatic
racemic mixture of both displayed similar kon and koff in vitro – ring. The replacement of the 4-phenoxy segment in SB-3CT by a
we synthesised the racemic mixtures of 2a–f. To obtain the triazole, using a CuAAC approach, is thus a useful strategy to
a-sulfone thiirane moiety, we used the methodology developed attain structural diversity in this type of inhibitors.
by Mobashery et al.23 An azide group was introduced to allow the
rapid growth of the molecule by coupling with fragments
designed to interact with gelatinases S10 pocket by means of
Substitution of the pyridyl ring present in 2f by phenyl (2c)
and m-F (2e) brought about a decrease in activity against MMP-2
and MMP-9. Finally, the introduction of N,N-dimethylamino,
n-pentyl or methoxy groups gave inactive compounds against
both metalloproteinases at 20 mM.
In a similar attempt to apply click chemistry to the synthesis
of thiirane-based gelatinase inhibitors by Mobashery's group,25
they obtained compounds 8 (Fig. 3) that showed to be weak
inhibitors of MMP-2 (in general, more than 50% of remaining
MMP-2 activity at 30 mM nal concentration and aer 3 h of
incubation). The authors suggested that the presence of a two
atoms link (–CH2O–) between the triazole and phenyl rings
Fig. 2 Click-chemistry approach for the synthesis of 2.
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Scheme 1 Synthesis of 2a–f. Reagents and conditions: (a) NaH at 0 ꢀC followed by allyl chloride, DMF, 68%; (b) tBuONO, TMSN3, CH3CN, 84%;
(c) mCPBA (excess), CH2Cl2, 37%; (d) thiourea, 1 : 1 MeOH–CH2Cl2, 81%; (e) CuSO4$5H2O (0.06 eq.), sodium ascorbate (0.20 eq.), alkyne.
Table 1 In vitro activities (IC50 values,a mM) for compounds 2a–f
ring (see Fig. 1); (3) binding of the resulting open compound
and chelation of the catalytic zinc ion by the thiolate moiety.
Classical SAR studies usually limit to step one, but here the
structure of the inhibitor may importantly inuence steps two
and three. We thus carried out computational studies to
examine the inuence of the P10 fragment on the inhibitor
potency.
MMP-9
MMP-2
R1
R2
45b min
2b h
2b h
Binding to MMP-2: NMR and computational studies
Cpd
Given the difference in potency of our inhibitors with the
reference thiirane SB-3CT, we veried if our compounds bind
the MMP-2 catalytic cle by NMR intermolecular-interaction
experiments. We have previously used the waterLOGSY experi-
ment35 in the characterization of novel inhibitors binding MMP-
2.27 Here we have applied a similar methodology to the study of
the interaction between MMP-2 and compounds 6, 2a–f. In
general, compounds 2a–f show very low solubility in water. As a
result, compounds 2a–d and 2f did not show recognizable
6
—
H
H
H
H
F
—
>50
>20
>50
>20
n.d.
2.36
87.5%d
10.9
>50
>20
n.d.
2a
2b
2c
2d
2e
N(CH3)2
(CH2)4CH3
C6H5
OCH3
H
>20
44.4%c
>20
12.04
>20
ꢁ20
79.9%d
2f
H
46.8%c
0.62
2.71
a
1
Determined by a colorimetric assay. Enzymatic data are mean values
resonances in the regular 1D H NMR spectra recorded in the
from three independent experiments. SD are within ꢂ10%.
b
c
d
protein buffer (see Materials & methods for further details) even
with the addition of supplementary DMSO (3%). Likewise, no
signicant information could be extracted from their water-
LOGSY experiments with MMP-2. However, compounds 6 and
2e were partially soluble under the NMR experimental condi-
tions, both showing positive intermolecular binding with MMP-
2 in the waterLOGSY spectra. To evaluate if such binding takes
place at the S10 pocket, waterLOGSY competition experiments
were repeated in the presence of 2R-[(4-biphenylsulfonyl)-
amino]-N-hydroxy-3-phenylpropinamide (BiPS), a commercially
available MMP2/9 inhibitor (IC50 (MMP-2): 17 nM; IC50 (MMP-
9): 30 nM). The addition of BiPS to the MMP-2/6 mixture yielded
positive interaction for both compounds in the waterLOGSY,
with no signicant reduction of signals from compound 6
compared to the MMP-2/6 waterLOGSY spectrum. The inverse
Incubation times. Remaining activity (%) at 10 mM. Remaining
activity (%) at 20 mM.
Fig. 3 Clicked-thiiranes synthesized by Mobashery et al.25
competition experiment (1st: waterLOGSY MMP-2/BiPS; 2nd
:
could be the cause of the lack of activity. The good inhibitory
activity observed for 2f, where the triazole is directly connected
to the phenyl is in complete accordance with this hypothesis.
Here it should be stressed that direct SAR analysis is rather
perilous. The binding of these thiirane derivatives could be
divided in three steps: (1) binding to the MMP; (2) hydrogen
abstraction by Glu404 and subsequent opening of the thiirane
addition of 6, waterLOGSY MMP-2/BiPS/6) gave analogous
results, suggesting that BiPS and compound 6 do not compete
for the same binding site of the protein. In the case of
compound 2e, the addition of BiPS to the MMP-2/2e sample
yielded positive binding for BiPS and no interaction for 2e,
indicating that BiPS displaces 2e from the binding pocket due
to its higher affinity for the protein (ESI gure†). This result was
17728 | RSC Adv., 2014, 4, 17726–17735
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conrmed by the inverse waterLOGSY competition experiment,
where the positive signals of the interaction BiPS – MMP-2 were
not affected by the addition of compound 2e. These results
indicate that compound 2e binds MMP-2 at the S10 pocket.
Docking studies
Docking simulations in MMP-2 catalytic centre were carried out
for 2a–f and SB-3CT for comparison.
As seen before, thiirane derivatives are slow binding inhibi-
tors due to the opening of the thiirane ring within the MMP-2
catalytic cle. For the docking studies, we considered both open
and closed forms to study the ability of the thiiranes, as well as
the corresponding allyl thiolates, to correctly bind to the MMP-2
catalytic centre. For each form (closed and open), each stereo-
isomer (S or R, Z or E) was considered. Indeed, using QM/MM
calculations, Mobashery et al. proposed that (R)-SB-3CT leads
preferentially to the allyl thiolate with E conguration,18
whereas (S)-SB-3CT leads to the Z isomer.36
According to the literature,18 three important interactions
could be expected for those compounds: (1) hydrophobic
interactions at the S10 pocket; (2) H-bond(s) between one
sulfone oxygen of the inhibitor and Leu191 and/or Ala192
backbone; (3) interaction between the sulphur atom and the
catalytic zinc ion, although such coordination was only
demonstrated experimentally for the allyl thiolate.
We carried out the docking studies as explained in the
Experimental section.
ꢃ Docking of thiirane derivatives 2a–f.
In a rst attempt, dockings were performed with 1CK7
structure, the same used to study the mechanism of SB-3CT.18 Fig. 4 Most energetically favoured binding mode for (R)-2f (A) and (S)-
2f (B) in MMP-2. The hydrogens from the ligand that are transferred to
While SB-3CT gave correct docking poses (that is to say with the
three interaction types listed above), 2a–f didn't. One possible
reason for this is that in 1CK7 structure no inhibitor is present,
leading to a tight S10 pocket, that cannot accommodate the long
P10 fragments used here (we previously observed false positive
Glu404 during the thiirane opening are depicted. H-bonds are rep-
resented by orange dots. Zn2+–sulphur distances (blue dots) are 2.62 A˚
(A) and 2.72 A˚ (B), respectively.
docking results with 1CK7 structure for inhibitors with long/
We have performed a comparative MD study of 2f and SB-
bulky P10 segment). Consequently, dockings were repeated using 3CT complexed to MMP-2 with the aim of exploring the ability
a structure extracted from a MD trajectory of the catalytic domain of these inhibitors to adopt conformations, within the MMP-2
of 1CK7 bound to a hydroxamate inhibitor with a similar P10 catalytic cle, that could allow their deprotonation by Glu404
fragment than those used here.28 Surprisingly, no correct binding and the subsequent opening of the thiirane ring (see below).
for SB-3CT was observed in this model. The curved biphenyl ether
moiety could not t into the S10 pocket together with a good
ꢃ Docking of thiolate derivatives 7a–f.
(E) and (Z) stereoisomers of 1 and 7a–f, resulting from the
coordination of the zinc ion by the sulphur atom. By contrast, in opening of respectively SB-3CT and 2a–f, were docked using
the most energetically favoured binding modes, 2a–f displayed the same MMP-2 structure coming from previous MD simu-
simultaneously the three interactions listed above. Fig. 4 shows lations.28 Both diastereoisomers of all compounds gave
the binding modes of (R)- and (S)-2f in this MMP-2 structure.
similar binding to MMP-2 with close and excellent binding
The binding modes of both enantiomers are similar in all scores. The most energetically favoured poses of those deriv-
compounds, what is in consonance with the results described atives (see Fig. 5 for the two isomers of 7f) showed the
for (S)- and (R)-SB-3CT.33 It is also important to note that the expected interactions: a sulfone oxygen establishes H-bonds
hydrogen that is taken by Glu404 in the mechanism reported in to Leu191 and Ala192 backbones, the P10 fragment goes deep
Fig. 1 does not point toward this residue and is relatively far into S10 pocket establishing various VdW contacts and the
from it (>4 A for all binding poses). To explain SB-3CT mecha- thiolate anion coordinates the catalytic Zn2+, forming a
˚
nism of inhibition,18 the authors faced the same problem and pseudo trigonal-based bipyramid, from which a ligand (water)
undertook MD simulations to explore the conformational is missing.
surface of MMP-2/SB-3CT complex and to select a conformation
in which the deprotonation of SB-3CT by Glu404 was feasible.
These results suggest that the opening of 2a–f would lead to
potent MMP-2 inhibitors.
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Fig. 6 Time evolution of the interaction distances measured (A˚) in the
complexes of MMP2 with (A) (R)-SB-3CT and (B) (R)-2f between the
sulfone moiety and Leu191 and Ala192 (black and grey lines respec-
tively), the reactive hydrogen and Glu404 (blue line) and the sulphur
atom of the thiirane ring and the catalytic zinc ion (red line).
Fig. 5 Most energetically favoured binding modes for (E)-7f (A) and
(Z)-7f (B) in MMP-2. H-bonds (orange dots) and alkene hydrogens are
depicted. Zn2+–sulphur distances are 2.20 A˚ (A) and 2.19 A˚ (B),
respectively.
MMP-2 with (R)-SB-3CT the distance and the bound confor-
mation remain very stable during the whole simulation, in the
complex with compound (R)-2f the interaction distances uc-
tuate and the interaction is lost at the end of the simulation
Molecular dynamics simulations
With the aim of rationalizing the difference of inhibitory activity time (Fig. S1†). In addition, we measured the distance between
experimentally observed between 2f and SB-3CT, we run MD the carboxylic group of Glu404 and the hydrogen that has to be
simulations for these two inhibitors. As it has been reported in abstracted in (R)-SB-3CT and (R)-2f in order to form thiolates 1
the literature, the inhibitory capacity of this type of compounds and 7f, respectively. We found that the distance uctuated
comes from their ability to react with Glu404. This is only considerably for (R)-2f even during the rst 16 ns of simulation
possible in an initial phase of the binding, in which the thiirane (in which the interactions with Leu191 and Ala192 were main-
sulphur atom is not coordinating the catalytic Zn ion.18,19,36 tained), while remained much more stable for (R)-SB-3CT. As a
Therefore 20 ns MD simulations were carried out in order to consequence, the probability for the proton abstraction and
assess the dynamic stability of the binding mode of the R- further ring opening reaction must be smaller for (R)-2f than for
enantiomer of SB-3CT in complex with MMP-2 and compare it (R)-SB-3CT.
to (R)-2f (Fig. 6A and B, respectively). We chose to study only the
The distance between the sulphur atom of the thiirane and
R enantiomer of both compounds as both enantiomers of SB- the catalytic zinc ion also uctuates much more in the MMP-2/
3CT showed similar inhibitory activity,33 and because more (R)-2f complex.
information about MD studies with (R)-SB-3CT can be found in
This different behaviour arises from the smaller size and the
the literature.18 The main stability parameter we focused on was exibility of (R)-SB-3CT compared to compound (R)-2f. The four
the H-bond established between the sulfone moiety of the two aromatic rings present on (R)-2f confer a rigid planar confor-
ligands and the backbone NH of both Leu191 and Ala192. Thus, mation to the molecule, which does not t in the S10 pocket as
we measured the hydrogen bonding distances between the good as the (R)-SB-3CT conformation. As a consequence, the
backbone NH of both residues and the nearest sulfone oxygen sulfone moiety of compound (R)-2f, although establishing
atom (black and grey line in Fig. 6). While in the complex of apparently stable interactions with the backbone NH of Leu191
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and Ala192, protrudes a little more out of the binding pocket (230–400 mesh) was used for ash chromatography. Analytical
than (R)-SB-3CT (Fig. S2†), inducing the observed uctuation of purity of tested compound was determined either by high-
the distance to Glu404. This increased mobility of the thiirane performance liquid chromatography coupled to mass spec-
end of compound (R)-2f, that goes along with a greater distance troscopy (HPLC-MS) or elemental analyses. HPLC-MS were done
uctuation between the hydrogen and the carbonyl of Glu404, in Synthelia Organics SL. 1100 HPLC (Xterra MS C18 5 mm
reduces the probability of a nucleophilic attack on the hydrogen reverse phase columns) coupled to 1946D MS detector, both
compared to that of compound (R)-SB-3CT and, therefore, also from Agilent, were used. Elemental analyses (C, H, N, S) were
reduces the reaction rate of the thiirane opening; all of which is performed on a LECO CHNS-932 apparatus at the Microanal-
in line with the experimental ndings.
yses Service of the Universidad Complutense de Madrid.
4-(Allylthio)aniline (3). To a suspension of sodium hydride
From these results, the P10 chain appears critical in the
potency of the inhibitors. P10 fragments that gave us excellent (1.53 g, 38.3 mmol) in dry DMF (15 mL) at 0 ꢀC, was slowly
results with the hydroxamate ZBG (such as in compounds 2a added a solution of p-aminothiophenol (4.0 g, 32.0 mmol) in dry
and 2d)27,28 are inadequate when using the thiirane latent ZBG. DMF. The resulting dark mixture was stirred 10 minutes at 0 ꢀC
Insufficient interactions at the S10 pocket – for instance in azide under argon and a solution of allyl chloride (2.7 g, 35.1 mmol) in
6 that misses a P10 fragment – or too rigid/bulky P10 groups may dry DMF was added dropwise. The reaction mixture was then
destabilize the inhibitor binding and thus affect the turn over of allowed to reach RT and stirred overnight under argon. The
the thiirane opening, as suggested by our comparative MD reaction was concentrated and the residue was partitioned
simulation studies. This hypothesis could explain the weak between water and EtOAc. The organic layer was washed with
potency of inhibitors 8 (previously reported25) and inhibitors 2a water (ꢄ3) and brine, dried over MgSO4 (anhydrous), ltered
and 2b that bear bulky P10 fragments.
and evaporated to afford 5.1 g of brown oil. The crude product
was puried on silica gel (9 : 1 to 7 : 3 hexane–EtOAc) to afford
N-alkylated by-product (860 mg, 16%) and 3 (3.6 g, 68%) as a
pale orange oil. H NMR (300 MHz, CDCl3) d 3.38 (dt, J ¼ 7.1,
Conclusions
1
We synthesized a series of clicked thiiranes as new gelatinases 1.1, 2H), 3.70 (br s, 2H), 4.92–5.00 (m, 2H), 5.77–5.90 (m, 1H),
inhibitors. Our hit (2f) displayed submicromolar IC50 against 6.60 (“d”, J ¼ 8.6 Hz, 2H), 7.23 (“d”, J ¼ 8.6 Hz, 2H); 13C NMR
MMP-2 in a colorimetric assay, strongly improving the potency (75.4 MHz, CDCl3) d 40.0 (CH2 in DEPT-135), 115.5 (CH), 117.1
of other clicked thiirane derivatives previously reported. To (CH2), 122.8, 134.4 (CH), 134.6 (CH), 146.2; MS (ESI) bad
understand the difference of potency between 2f and the ionization.
reference thiirane inhibitor SB-3CT, we carried out MD simu-
Allyl(4-azidophenyl)sulfane (4). To a solution of 3 (3.5 g, 21.2
lations for both inhibitors complexed to the MMP-2 catalytic mmol) in dry MeCN (30 mL) was added tert-butyl nitrite (2.4 g,
domain. We propose that the longer and more rigid P10 frag- 23.3 ꢀmmol) at 0 ꢀC under argon. The mixture was stirred 10 min
ment present in 2f is responsible for the lower stability of this at 0 C and TMSN3 (2.7 g, 23.3 mmol) was slowly added. Aer
compound in MMP-2, causing a reduction on the reaction rate stirring the mixture for 30 min at 0 ꢀC and 2 h at RT, the reaction
of the thiirane opening. These results put forward the relevance mixture was concentrated and the residue was partitioned
of the P10 fragment in the potency of this type of inhibitors. We between water and EtOAc. The organic layer was washed with
are now combining computational simulations with our click water and brine, dried over MgSO4 (anhydrous), ltered and
approach to better understand the effect of the P10 fragment on evaporated to afford 3.8 g of brown oil. The crude product was
the reaction rate of the thiirane opening within the MMP, and ltered through silica gel, eluting with 10% CH3Cl in hexane, to
with the nal aim of developing clicked thiirane derivatives with afford 4 (3.4 g, 84%) as a pale orange oil. IR (KBr): 2159 (N3
improved potency.
stretch) cmꢅ1; 1H NMR (300 MHz, CDCl3) d 3.50 (dt, J ¼ 7.2 1.1
Hz, 2H), 5.04–5.10 (m, 2H), 5.78–5.92 (m, 1H), 6.96 (“d”, J ¼ 8.7,
2H), 7.35 (“d”, J ¼ 8.7, 2H); 13C NMR (75.4 MHz, CDCl3) d 38.2,
117.9, 119.6, 132.0, 132.4, 133.6, 138.8; anal. calcd for C9H9N3S:
C, 56.52; H, 4.74; N, 21.97; S, 16.77. Found: C, 55.54; H, 5.30; N,
Experimental section
Chemistry
General procedures. Dry solvents were distilled before use 21.03; S, 16.68%.
and dried by standard methods; CH2Cl2 and MeCN, from CaH2.
2-(((4-Azidophenyl)sulfonyl)methyl)oxirane (5). To a solution of
All commercially available reagents were used without further 4 (3.0 g, 15.7 mmol) in CH2Cl2 (150 mL) was added mCPBA
ꢀ
purication. Melting points (uncorrected) were determined on a (16.2 g, 94.1 mmol, 6.00 eq.) at 0 C under argon. The mixture
Stuart Scientic SMP3 apparatus. Infrared (IR) spectra were was stirred at RT under argon. Aer 2 days, TLC showed a
recorded with a Perkin-Elmer 1330 infrared spectrophotometer, mixture of starting material, m-chlorobenzoic acid (mCBA) and
using KBr as solid matrix. 1H and 13C NMR data were recorded two intermediates. A sample was taken in CH2Cl2 and washed
on a Bruker DPX 300 MHz-BACS60 instrument. Chemical shis with 2% NaOH solution (ꢄ3), water and brine. Aer drying
(d) are expressed in parts per million relative to solvent reso- (MgSO4 anhydrous) and evaporating the organic layer, 1H NMR
nance as the internal standard; coupling constants (J) are in showed mCBA, starting material, ꢁ10% of epoxide
hertz. Mass spectra were run on a Bruker Esquire 3000 spec- compound(s), and a mixture of allyl derivatives. To remove
trometer (ESI-IT). Thin-layer chromatography (TLC) was run on mCBA, the reaction mixture was worked-up as above and plug-
Merck silica gel 60 F-254 plates and Merck silica gel 60 ged on silica pad (eluting with CH2Cl2) and dried. The yellow oil
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obtained was set back to react using the same conditions 1.8 Hz, 1H), 2.57 (dd, J ¼ 6.2 1.8 Hz, 1H), 2.66 (t, J ¼ 7.9 Hz, 2H),
described above. TLC showed progress. Aer 2 days this 3.07–3.15 (m, 1H), 3.33 (dd, J ¼ 14.4 7.2 Hz; 1H), 3.52 (dd, J ¼
manipulation was repeated. Aer 3 more days, the reaction was 14.4 6.2 Hz, 1H), 7.30 (d, J ¼ 8.2 Hz, 2H), 7.83 (d, J ¼ 8.2 Hz, 2H),
stopped and worked-up as previously described to give 2.3 g of 8.08 (d, J ¼ 8.9 Hz, 2H), 8.14 (d, J ¼ 8.9 Hz, 2H), 8.27 (s, 1H); 13
C
yellow oil. The crude product was puried on silica gel (9 : 1 to NMR (75.4 MHz, CDCl3) d 14.1, 22.7, 24.1, 26.0, 31.2, 31.6, 35.9,
7 : 3 hexane–EtOAc) to afford 5 (1.4 g, 37%) as a yellow oil. IR 62.7, 116.9, 120.0, 120.7, 126.1, 129.2, 130.7, 138.5, 141.2, 144.2,
(KBr): 2140 (N3 stretch) cmꢅ1; 1H NMR (300 MHz, CDCl3) d 2.45 149.5; MS (ESI) 428.0 [M + H]+. Purity by HPLC-MS: 87%.
(dd, J ¼ 4.8, 2.0 Hz, 1H), 2.79–2.82 (m, 1H), 3.24–3.37 (m, 3H),
General procedure for the preparation of triazoles 2c–f. A
7.20 (d, J ¼ 8.8 Hz, 2H), 7.93 (d, J ¼ 8.8 Hz, 2H); 13C NMR (75.4 solution of azide 6 (100 mg, 0.39 mmol, 1.00 eq.), the corre-
MHz, CDCl3) d 46.0, 59.8, 119.8, 130.4, 135.3, 146.5.
sponding alkyne (0.43 mmol, 1.10 eq.), 0.1M CuSO4$5H2O
2-(((4-Azidophenyl)sulfonyl)methyl)thiirane (6). A solution of 5 solution (390 mL, 0.039 mmol, 0.10 eq.), 0.5 M sodium ascorbate
(1.3 g, 5.4 mmol) and thiourea (827 mg, 10.9 mmol) in 1 : 1 solution (312 mL, 0.16 mmol, 0.40 eq.) in DMF (2–3 mL) was
MeOH–CH2Cl2 (30 mL) was stirred overnight at RT under argon. stirred overnight at RT under argon. Water was added and the
The reaction mixture was concentrated and the residue was precipitate was washed with water (ꢄ3), EtOAc, Et2O and dried
partitioned between water and EtOAc. The organic layer was to dryness. The resulting solid was recrystallized or washed in
washed with water, dried over MgSO4 (anhydrous), ltered and hot MeCN in case recrystallization failed.
evaporated to afford 1.2 g of a white solid. The crude product
4-([1,10-Biphenyl]-4-yl)-1-(4-((thiiran-2-ylmethyl)sulfonyl)-
was puried on silica gel, eluting with 8 : 2 hexane–EtOAc to phenyl)-1H-1,2,3-triazole (2c). Following the general procedure,
afford 6 (1.1 g, 81%) as a white solid. IR (KBr): 2119 (N3 stretch) starting from 4-ethynyl-1,10-biphenyl, triazole 2c (70 mg, 41%)
cmꢅ1; 1H NMR (300 MHz, CDCl3) d 2.13 (dd, J ¼ 5.1 1.7 Hz, 1H), was obtained as a beige solid, mp 236–237 ꢀC (from MeCN). 1H
2.53 (dd, J ¼ 6.1 1.5 Hz, 1H), 3.02–3.10 (m, 1H), 3.23 (dd, J ¼ 14.3 NMR (300 MHz, CDCl3) d 2.23 (d, J ¼ 4.8 Hz, 1H), 2.58 (d, J ¼ 5.3
7.4 Hz, 1H), 3.48 (dd, J ¼ 14.3 5.9 Hz, 1H), 7.20 (d, J ¼ 8.7 Hz, Hz, 1H), 3.03–3.11 (m, 1H), 3.72–3.86 (m, 2H), 7.40 (t, J ¼ 7.3 Hz,
2H), 7.91 (d, J ¼ 8.7 Hz, 2H); 13C NMR (75.4 MHz, CDCl3) d 24.2 1H), 7.50 (t, J ¼ 7.3 Hz, 2H), 7.76 (d, J ¼ 7.3 Hz, 2H), 7.85 (d, J ¼
(CH2 in DEPT-135), 26.1 (CH), 62.7 (CH2), 119.8 (CH), 130.6 (CH), 8.4 Hz, 2H), 8.06 (d, J ¼ 8.4 Hz, 2H), 8.20 (d, J ¼ 8.8 Hz, 2H), 8.31
134.8, 146.6.
(d, J ¼ 8.8 Hz, 2H), 9.58 (s, 1H); 13C NMR (75.4 MHz, CDCl3) d
N,N-Dimethyl-4-(1-(4-((thiiran-2-ylmethyl)sulfonyl)phenyl)-1H- 23.9, 26.8, 60.4, 120.0, 120.3, 126.0, 126.6, 127.3, 127.7, 128.9,
1,2,3-triazole-4-yl)aniline (2a). A mixture of 6 (150 mg, 0.59 129.0, 130.3, 138.2, 139.4, 140.1, 140.3, 147.4; MS (ESI) 434.1 [M
mmol), 4-ethynyl-N,N-dimethylaniline (102 mg, 0.70 mmol), + H]+. Anal. calcd for C23H19N3O2S2: C, 63.72; H, 4.41; N, 9.69; S,
freshly made aqueous 0.1 M CuSO4$5H2O solution (350 mL, 0.03 14.79. Found: C, 63.64; H, 4.50; N, 9.80; S, 13.79%.
mmol, 0.06 eq.) and 0.5 M sodium ascorbate solution (234 mL,
4-(4-Methoxyphenyl)-1-(4-((thiiran-2-ylmethyl)sulfonyl)phenyl)-
0.12 mmol, 0.20 eq.) in 1 : 1 H2O–tBuOH (3 mL) was stirred at 1H-1,2,3-triazole (2d). Following the general procedure, starting
RT under argon for 24 h. The reaction mixture was partitioned from 1-ethynyl-4-methoxybenzene, triazole 2d (55 mg, 36%) was
1
ꢀ
between EtOAc and water. A brown precipitate appeared in the obtained as a white solid, mp 207–208 C. H NMR (300 MHz,
organic layer. The aqueous part was discarded and the solvent CDCl3) d 2.23 (d, J ¼ 4.5 Hz, 1H), 2.57 (d, J ¼ 5.6 Hz, 1H), 3.02–
was evaporated. The resulting brown solid was triturated in 3.10 (m, 1H), 3.71–3.81 (m, 2H), 3.82 (s, 3H), 7.09 (d, J ¼ 8.8 Hz,
Et2O and recrystallized from EtOAc to afford 2a (53 mg, 22%) as 2H), 7.89 (d, J ¼ 8.8 Hz, 2H), 8.17 (d, J ¼ 8.8 Hz, 2H), 8.28 (d, J ¼
1
an orange solid, mp 226–227 C. H NMR (300 MHz, CDCl3) d 8.8 Hz, 2H), 9.40 (s, 1H); 13C NMR (75.4 MHz, CDCl3) d 23.9,
ꢀ
2.23 (dd, J ¼ 5.3, 1.0 Hz, 1H), 2.58 (dd, J ¼ 5.3, 1.0 Hz, 1H), 2.97 26.8, 55.2, 60.4, 114.5, 118.8, 120.2, 122.3, 126.8, 130.3, 138.1,
(s, 6H), 3.02–3.11 (m, 1H), 3.71–3.85 (m, 2H), 6.84 (d, J ¼ 8.9 Hz, 140.3, 147.7, 159.5; MS (ESI) 388.1 [M + H]+. Purity by HPLC-MS:
2H), 7.77 (d, J ¼ 8.9 Hz, 2H), 8.16 (d, J ¼ 8.9 Hz, 2H), 8.27 (d, J ¼ 89%.
8.9 Hz, 2H), 9.30 (s, 1H); 13C NMR (75.4 MHz, CDCl3) d 23.9,
4-(3-Fluorophenyl)-1-(4-((thiiran-2-ylmethyl)sulfonyl)phenyl)-
26.8, 39.9, 60.4, 112.3, 117.4, 117.6, 120.0, 126.3, 130.3, 137.8, 1H-1,2,3-triazole (2e). Following the general procedure, starting
140.4, 148.4, 150.4; MS (ESI) 401.0 [M + H]+; anal. calcd for from 1-ethynyl-3-uorobenzene, triazole 2e (51 mg, 34%) was
1
ꢀ
C
19H20N4O2S2: C, 56.98; H, 5.03; N, 13.99; S, 16.01. Found: C, obtained as a beige solid, mp 189–190 C. H NMR (300 MHz,
57.03; H, 5.07; N, 13.91; S, 15.72%.
DMSO-d6) d 2.23 (d, J ¼ 5.3 Hz, 1H), 2.58 (d, J ¼ 5.3 Hz, 1H),
4-(4-Pentylphenyl)-1-(4-((thiiran-2-ylmethyl)sulfonyl)phenyl)- 3.05–3.09 (m, 1H), 3.72–3.86 (m, 2H), 7.26 (td, J ¼ 8.9, 2.4 Hz,
1H-1,2,3-triazole (2b). A mixture of 6 (100 mg, 0.39 mmol), 1- 1H), 7.55–7.62 (m, 1H), 7.76 (d, J ¼ 10.1 Hz, 1H), 7.82 (d, J ¼ 7.7
ethynyl-4-pentylbenzene (81 mg, 0.470 mmol), freshly made Hz, 1H), 8.20 (d, J ¼ 8.8 Hz, 2H), 8.27 (d, J ¼ 8.8 Hz, 2H), 9.59 (s,
aqueous 0.1 M CuSO4$5H2O solution (235 mL, 0.02 mmol, 0.06 1H); 13C NMR (75.4 MHz, DMSO-d6) d 23.9, 26.8, 60.4, 112.0 (d,
eq.) and 0.5 M sodium ascorbate solution (157 mL, 0.08 mmol, 2JCF ¼ 23.0 Hz), 115.2 (d, JCF ¼ 21.2 Hz), 120.3, 120.7, 121.5,
2
3
3
0.20 eq.) in MeCN (2 mL) was stirred at RT under argon for 24 h. 130.4, 131.3 (d, JCF ¼ 8.7 Hz), 132.2 (d, JCF ¼ 8.6 Hz), 138.3,
Water was added and the precipitate was ltered, washed with 140.2, 146.6, 162.6 (d, JCF ¼ 243 Hz); MS (ESI) 375.9 [M + H]+.
1
water and CH2Cl2 to give 104 mg of a yellow solid. The crude Purity by HPLC-MS: 86%.
product was consequently puried by column chromatography
4-(4-(1-(4-((Thiiran-2-ylmethyl)sulfonyl)phenyl)-1H-1,2,3-tri-
on silica gel (1–5% MeOH–CH2Cl2) to give 2b (25 mg, 15%) as a azole-4-yl)phenyl)pyridine (2f). Following the general procedure,
1
white solid, mp 187–188 C. H NMR (300 MHz, CDCl3) d 0.90 starting from 4-(4-ethynylphenyl)pyridine,28 triazole 2f (42 mg,
ꢀ
(“t”, 3H), 1.32–1.37 (m, 4H), 1.61–1.71 (m, 2H), 2.18 (dd, J ¼ 5.1 25%) was obtained as a white solid, mp 213–214 ꢀC (from
17732 | RSC Adv., 2014, 4, 17726–17735
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MeCN). 1H NMR (300 MHz, DMSO-d6) d 2.24 (d, J ¼ 5.2 Hz, 1H), DMSO-d6. For each sample, 1D 1H and waterLOGSY (direct
2.58 (d, J ¼ 5.7 Hz, 1H), 3.03–3.1 (m, 1H), 3.72–3.87 (m, 2H), 7.83 interaction and competition) experiments were recorded. 8k
(br s, 2H), 7.99 (d, J ¼ 8.3 Hz, 2H), 8.12 (d, J ¼ 8.3 Hz, 2H), 8.20 points were used for a sweep width of 9600 Hz and a total of 512
(d, J ¼ 8.7 Hz, 2H), 8.30 (d, J ¼ 8.7 Hz, 2H), 8.76 (br s, 1–2H), 9.62 scans were accumulated for the waterLOGSY experiment. In
(s, 1H); 13C NMR (75.4 MHz, DMSO-d6) d 22.9, 25.8, 59.4, 119.3, these experiments, the large bulk water magnetization is
119.4, 120.0, 125.1, 126.5, 129.4, 129.7, 135.9, 137.3, 139.3, partially transferred via the protein–ligand complex to the free
145.2, 146.1, 149.3; MS (ESI) 435.0 [M + H]+. Anal. calcd for ligand in a selective manner. A non-interacting compound
C
22H18N4O2S2: C, 60.81; H, 4.18; N, 12.89; S, 14.76. Found: C, results in negative resonances, whereas protein–ligand inter-
60.55; H, 4.24; N, 12.84; S, 14.01%.
actions are characterized by positive signals or by a reduction in
the negative signals obtained in the absence of the protein.
MMPs inhibition assays
MMPs activity measurements were performed using MMP
Inhibitor Proling Kit purchased from Enzo Life Science Inter-
national, Inc., and following the manufacturer's protocol with
slight modications. Proteolytic activity was measured using a
thiopeptide substrate (Ac-PLG-[2-mercapto-4-methylpentanoyl]-
LG-OC2H5) where the MMP cleavage site peptide bond has been
replaced by a thioester bond.37,38 Hydrolysis of this bond by
MMP produces a sulydryl group that reacts with DTNB to
form 2-nitro-5-thiobenzoic acid, which was detected by its
absorbance at 414 nm (microplate photometer Thermo Scien-
tic Multiscan FC). Enzyme reactions were carried out at 37 ꢀC
in a 100 mL nal volume of solutions, where the catalytic
domains of the corresponding MMP were incubated 45 min or
2 h in triplicate with at least seven concentrations of inhibitors.
The compounds were dissolved in DMSO, with 2% nal
concentration in each well. The negative control (enzyme +
substrate contained the same amount of DMSO solvent). The
assay buffer contained the following components: 50 mM
HEPES, 10 mM CaCl2, 0.05% Brij-35 and 1 mM DTNB at pH 7.5.
Aer addition of substrate, the increase of absorbance was
recorded in 1 min time intervals for 30 min. Data were plotted
as OD versus time for each sample, in order to obtain the
reaction velocity (V) in OD per min. The percentage of residual
activity for each compound was calculated using the following
formula: % of remaining activity ¼ (V in the presence of
inhibitor/Vcontrol) ꢄ 100. An inhibitor, NNGH, was included as a
prototypic control inhibitor.39 The concentration of compound
that provided 50% inhibition of enzymatic activity (IC50) was
determined by semi-logarithmic dose–response plots (Graph
Pad Prism 5.0 for Windows, Graph Pad Soware Inc., San Diego,
California, USA, 2007).
Computational methods
Preparation of the protein structures. For the preparation of
1CK7 structure, the starting coordinates for the MMP-2 catalytic
domain were taken from the crystal structure, which corre-
sponds to the Ala404Glu mutant of the full-length pro-MMP-2
enzyme.26 The propeptide (Pro31-Asn109) and the C-Term
hemopexin-domain (Leu461-Cys660) were deleted from the
initial crystal structure, and the three bronectin-type domains
(Glu217-Gln393) were replaced by a short peptide segment (Lys-
Gly-Val) as in the 1QIB crystal structure40 of the MMP-2 catalytic
domain.
To relax and ensure a correct opening of S10 in 1CK7, we used
a structure extracted from a previously reported MD simulation
of a MMP-2 hydroxamate inhibitor bearing the same P10 frag-
ment as 2a.28
Docking setting. Flexible docking calculations were carried
out with the Glide program,41–43 using default parameters and
requiring a maximum of ten output poses. We selected the XP
(Extra-Precision) method allowing a more extensive sampling
method as well as a more exigent scoring function.43 We
removed all water molecules and counterions (Cl) but kept zinc
and calcium ions in the experimental structures. The proteins
were prepared with the protein preparation wizard44 provided in
¨
Schrodinger suite and the grid was constructed with the grid
receptor generation module included in Glide. During the
docking calculations, all the protein residues were xed and
only the inhibitor atoms were allowed to move. The catalytic
zinc ion was assigned a +2 charge. For the docking of thiirane
SB-3CT and 2a–f the Glu404 side chain was considered depro-
tonated (ꢅ1 charge) and the ligand neutral, whereas for the
docking of thiolate 1 and 7a–f, Glu404 side chain was in its
protonated form (neutral) and the ligand with a ꢅ1 charge. The
ligand was prepared using LigPrep45 application also provided
NMR studies
¨
All spectra were recorded at 300 K with a Bruker Avance in the Schrodinger suite.
600 MHz spectrometer equipped with a 5 mm TCI cryoprobe. A
MD simulations. The complexes of MMP-2 bound to (R)-SB-
typical NMR sample contained a concentration of 5 mM of MMP- 3CT and (R)-2f issued from the docking experiments were used
2 and 100 mM of ligand (from a 50 mM stock in DMSO-d6), in an as initial structures for the MD simulations. The water molecule
approximate protein–ligand ratio of 1 : 20, optimal for the coordination sphere of the catalytic zinc ion was taken from the
˚
waterLOGSY experiments. To 450 mL of sample (buffer: 10 mM 2.00 A resolution crystal structure of the complex between
deuterated-Tris/HCl pH 7.4 with 50 mM NaCl, 0.02% NaN3, MMP-8 and a non-zinc chelating inhibitor (PDB code: 3DNG).
100 mM CaCl2 and 100 mM ZnCl2) 25 mL of D2O were added for Addition of the missing hydrogen atoms and computation of
locking purposes, and 15 mL of DMSO-d6 to increase the solu- the protonation state of tritable groups were carried out using
bility of the compounds. BiPS (2R-[(4-biphenylsulfonyl)amino]- the H++ web server,46 which relies on the AMBER force eld
N-hydroxy-3-phenylpropinamide; VWR, Barcelona, Spain) was parameters47 and nite difference solutions to the Poisson–
used for the competition experiments from a 50 mM stock in Boltzmann equation. The charge distribution for the ligands
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´
studied was obtained by tting the quantum mechanically 11 P. Serra, M. Bruczko, J. Zapico, A. Puckowska, M. Garcıa,
´
´
calculated (RHF/6-31G**//RHF/3-21G*) molecular electrostatic
potential (MEP) of the geometry-optimized molecules to a point
S. Martın-Santamarıa, A. Ramos and B. de Pascual-Teresa,
Curr. Med. Chem., 2012, 19, 1036–1064.
charge model, as implemented in Gaussian 03.48 Consistent 12 R. Yadav, S. Gupta, P. Sharma and V. Patil, Curr. Med. Chem.,
bonded and non-bonded AMBER parameters for both mole-
2011, 18, 1704–1722.
cules were assigned by analogy or through interpolation from 13 S. Brown, M. M. Bernardo, Z. H. Li, L. P. Kotra, Y. Tanaka,
those already present in the AMBER database (ff03). Each
R. Fridman and S. Mobashery, J. Am. Chem. Soc., 2000, 122,
molecular system was immersed in a truncated octahedron
6799–6800.
+
containing ꢁ4600 TIP3P water molecules49 and 23 Na ions50 to 14 A. Kruger, M. J. E. Arlt, M. Gerg, C. Kopitz, M. M. Bernardo,
¨
achieve system electroneutrality. The sander and pmemd
modules from the AMBER10 suite51 were used for the restrained
M. Chang, S. Mobashery and R. Fridman, Cancer Res., 2005,
65, 3523–3526.
and unrestrained MD simulations, respectively. Periodic 15 R. D. Bonl, A. Sabbota, S. Nabha, M. M. Bernardo, Z. Dong,
boundary conditions were applied and electrostatic interactions
H. Meng, H. Yamamoto, S. R. Chinni, I. T. Lim, M. Chang,
S. Mobashery and R. Fridman, Int. J. Cancer, 2006, 118,
2721–2726.
were treated using the smooth particle mesh Ewald method52
˚
with a grid spacing of 1 A. The cutoff distance for the non-
53
˚
bonded interactions was 9 A, the SHAKE algorithm was 16 O. Kleifeld, L. P. Kotra, D. C. Gervasi, S. Brown,
applied to all bonds, and an integration step of 2.0 fs was used
M. M. Bernardo, R. Fridman, S. Mobashery and I. Sagi,
throughout. Aer an initial energy minimization of the water
J. Biol. Chem., 2001, 276, 17125–17131.
molecules and counterions, the system was heated to 300 K in 17 C. Forbes, Q. Shi, J. F. Fisher, M. Lee, D. Hesek, L. I. Llarrull,
25 ps, aer which the solvent was allowed to redistribute
around the positionally restrained solute for 220 ps. Then, the
M. Toth, M. Gossing, R. Fridman and S. Mobashery, Chem.
Biol. Drug Des., 2009, 74, 527–534.
system was allowed to move freely so as to explore the mutual 18 P. Tao, J. F. Fisher, Q. Shi, T. Vreven, S. Mobashery and
adaptation between ligand and protein. Snapshots from each H. B. Schlegel, Biochemistry, 2009, 48, 9839–9847.
20 ns MD trajectory were collected every 20 ps for further 19 P. Tao, J. F. Fisher, Q. Shi, S. Mobashery and H. B. Schlegel,
analysis. The resulting trajectories were processed with the ptraj J. Phys. Chem. B, 2009, 114, 1030–1037.
module of AMBER to monitor the hydrogen bonding distances 20 M. Lee, A. Villegas-Estrada, G. Celenza, B. Boggess, M. Toth,
between the ligands and the protein.
G. Kreitinger, C. Forbes, R. Fridman, S. Mobashery and
M. Chang, Chem. Biol. Drug Des., 2007, 70, 371–382.
21 G. Celenza, A. Villegas-Estrada, M. Lee, B. Boggess,
C. Forbes, W. R. Wolter, M. A. Suckow, S. Mobashery and
M. Chang, Chem. Biol. Drug Des., 2008, 71, 187–
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22 M. Gooyit, M. Lee, D. Hesek, B. Boggess, A. G. Oliver,
R. Fridman, S. Mobashery and M. Chang, Chem. Biol. Drug
Des., 2009, 74, 535–546.
23 S. A. Testero, M. Lee, R. T. Staran, M. Espahbodi,
L. I. Llarrull, M. Toth, S. Mobashery and M. Chang, ACS
Med. Chem. Lett., 2011, 2, 177–181.
24 M. Gooyit, M. Lee, V. A. Schroeder, M. Ikejiri, M. A. Suckow,
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Acknowledgements
This work was supported by the Spanish Ministry of Science and
Innovation (SAF2008-00945, CTQ2011-24741, SAF2011-28350).
Grants to B. F. and K. F. from Fundacion Universitaria San
Pablo CEU are also acknowledged. We thank Synthelia Organics
SL. for HPLC-MS experiments.
´
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