Discovery of new heterocycles with activity against human neutrophile elastase based on a boron promoted one-pot assembly reaction

Article abstract of DOI:10.1039/c3ob40614h

Herein we demonstrate for the first time that a boron promoted one-pot assembly reaction may be used to discover novel enzyme inhibitors. Inhibitors for HNE were simply assembled in excellent yields, high diastereoselectivities and IC₅₀ up to 1

Full text of DOI:10.1039/c3ob40614h

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Discovery of new heterocycles with activity against
human neutrophile elastase based on a boron
promoted one-pot assembly reaction†
Cite this: DOI: 10.1039/c3ob40614h
Francesco Montalbano,a Pedro M. S. D. Cal,a Marta A. B. R. Carvalho,a
Lídia M. Gonçalves,a Susana D. Lucas,a Rita C. Guedes,a Luís F. Veiros,b Rui Moreiraa
and Pedro M. P. Gois*a
Herein we demonstrate for the first time that a boron promoted one-pot assembly reaction may be used
to discover novel enzyme inhibitors. Inhibitors for HNE were simply assembled in excellent yields, high
diastereoselectivities and IC50 up to 1.10 μM, based on components like salicylaldehyde, aryl boronic
acids and amino acids. The combination of synthetic, biochemical, analytical and theoretical studies
allowed the identification of the 4-methoxy or the 4-diethyl amino substituent of the salicylaldehyde as
the most important recognition moiety and the imine alkylation, lactone ring opening as key events in
the mechanism of inhibition.
Received 27th March 2013,
Accepted 10th May 2013
DOI: 10.1039/c3ob40614h
www.rsc.org/obc
collections is emerging as a powerful method for identifying
chemical probes that may more efficiently interact with bio-
Introduction
Medicinal chemists incessantly face the daunting task of dis- logical macromolecules.7,8
covering new drugs with potency, specificity, and low toxicity
In this sense, it is now accepted that small molecules dis-
for human diseases.1 To unravel new therapeutically useful playing more complex architectures enriched with oxygen and
molecules, medicinal chemists frequently use natural pro- with hydrogen bond donors and acceptors embedded in cyclic
ducts’ architecture and biological properties as inspiration.2–4 systems comprising multiple sp3-hybridized carbons and
Alternatively, ligand- and structure-based drug designs poten- stereogenic centres are privileged candidates for these
tiated by modern in silico tools are invaluable strategies in campaigns.9–11 To prepare such compounds, medicinal che-
current hit discovery and lead optimization processes.1,5 Never- mists rely on labor intensive synthetic strategies that combine
theless, when the drug target or its biological mechanism of a plethora of complex methodologies frequently used in a
action is unknown, the high-throughput screening of a large multi-step sequence, which often yield the target compounds
number of compounds becomes the most practical strategy to in low yields determining the need for extensive purification
identify new hits.1,6 This approach has benefited tremendously steps. Therefore, based on the recent interest in medicinal
from the expansion of synthetic methodologies that enabled chemistry for boron containing compounds,12–15 we conceived
the simple manipulation of traditional cores such as benzene, that small drug-like molecules could be easily created by
pyridine and imidazole rings.7,8 Nevertheless, the extensive assemblage of simple building blocks promoted by a boron
exploitation of these scaffolds limited the medicinal chemistry tether.16–18 This innovative strategy would enable the straight-
chemical space and introduced deficiencies in many collec- forward generation of discrete molecular scaffolds with dis-
tions of compounds currently available for screening.7,8 Con- tinct pharmacophores that may be readily tuned for optimal
sequently, the quest for functionally diverse compound interaction with a biological target (Scheme 1).
aResearch Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of
Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
Discussion
E-mail: pedrogois@ff.ul.pt
bCentro de Química Estrutural, Departamento de Engenharia Química, Instituto
To test this hypothesis we selected Human Neutrophile
Elastase (HNE) which is an important serine protease involved
Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa, Portugal
†Electronic supplementary information (ESI) available: Experimental pro-
in the degradation of tissue matrix proteins such as elastin
cedures, Fig. S1: energy profile calculated for methanol addition to 3 with the
and for which there are still no efficient inhibitors marketed.19
first attack on the lactone ring, atomic coordinates for the optimized species
and spectral data for all compounds. See DOI: 10.1039/c3ob40614h
The imbalance between HNE and its endogenous inhibitors
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Scheme 1 Synthesis of drug-like molecules based on the assembly of simple
building blocks promoted by a boron tether.
Scheme 3 Evaluation of compounds 1–3 prepared from L-alanine, L-leucine
and L-phenylalanine amino acids, phenyl boronic acid and salicylaldehyde
against HNE.
Scheme 2 One-pot assembly of inhibitors for HNE with different reactive
centres and multiple possible recognition moieties (PRM).
synthesized with L-phenylalanine, displayed a very promising
IC50 of 20.27 μM, at the same time that the individual com-
ponents, phenyl boronic acid and salicylaldehyde, had no
activity. These results clearly demonstrated our initial hypo-
thesis and the usefulness of this approach to create assembled
structures with activity against this target.
leads to excessive elastin proteolysis and the destruction of
connective tissues in several inflammatory diseases such as
the chronic obstructive pulmonary disease (COPD).19 Typically,
HNE inhibitors are acylating agents that react with the catalytic
serine residue to form a stable acyl-enzyme. This reaction
involves the formation of a tetrahedral intermediate en route to
the acyl-enzyme, which is stabilized at the so-called oxyanion
hole of the active site.20 HNE inhibitors spread over many
different families of compounds with very distinct chemo-
types, among these, fused cyclic lactones have been described
for the inhibition of HNE with IC50’s ranging from 0.024 to
1.603 μM.21 Based on this, we envisioned that lactone mimics
could be readily assembled by combining a chelating alde-
hyde, a boronic acid and an amino acid.22 These constructs
could be simply tuned to promote a selective enzyme inhi-
bition via formation of an acyl–enzyme complex as shown in
Scheme 2. Importantly, the boron construct depicted in
Scheme 2 can provide two major structural requirements for
enzyme inhibition: (i) a substitution pattern appropriate for
molecular recognition and (ii) an increased reactivity of the
5-membered ring towards the catalytic serine.
In addition to the catalytic triad formed by Ser195, His57
and Asp102, the topology of the HNE active site comprises
extended molecular recognition binding sites, of which the S1
is the primary specificity binding pocket with affinity for small
hydrophobic groups.23 Based on this, we selected amino acids
with hydrophobic side chains like L-alanine, L-leucine and
L-phenylalanine amino acids as partners together with phenyl
boronic acid and salicylaldehyde to generate the fused lactone
mimic. The components were reacted in water for 20 h at
90 °C. This simple approach afforded the expected com-
pounds, which were collected by filtration in good to excellent
yields and diastereoselectivities.12 Once prepared, compounds
Motivated by the identification of compound 3 as an HNE
inhibitor, we embarked on a hit optimization program empow-
ered by the chemical versatility offered by this synthetic
strategy. Therefore, heterocycles 4–6 were successfully prepared
using para substituted aromatic boronic acids, L-phenylalanine
and salicylaldehyde though, with these compounds, no signifi-
cant improvement in the inhibition of HNE was observed.
Based on this, the modification was extended to the aromatic
moiety of the aldehyde. The inclusion of a methyl group only
slightly improved the activity of 7, though a methoxy at the
same position had a much more pronounced effect and
reduced the IC50 up to 9.3 μM. Therefore, compound 8 was
selected as the starting point for another round of optimi-
zation in which different boronic acids were explored. Dis-
appointingly compounds 9–10 displayed a poor inhibitory
activity when compared with 8, though the introduction of a
bromide atom either in the para or ortho positions of the
boronic acid aromatic moiety greatly improved the potency up
to 2 μM. The same trend was observed in compounds 14
(IC50 = 2 μM) and 15 which feature a chlorinated aromatic
boronic acid ortho and para substituted and displayed an
IC50 of 1.9 μM. Based on all modifications operated in this
scaffold the methoxy substituent introduced into the aldehyde
aromatic moiety was shown instrumental for potency. For this
reason, compound 16 was prepared with 4-diethylamino-
salicylaldehyde with the objective of improving the effect pre-
viously observed. Therefore, L-phenylalanine, 4-diethylamino-
salicylaldehyde and 2,4-dichlorophenyl boronic acid were
reacted in ethanol for 20 h at 70 °C to yield 16 in 89% yield
and 98% dr. Very gratifyingly the operated modification
resulted in the improvement of the activity against HNE and
1–3 were readily evaluated against HNE and
1 and 2,
were shown almost inactive (Scheme 3). Very differently, 3,
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Scheme 5 Biological evaluation of compounds 17 and 18 featuring a reduced
imine and lactone respectively. NPA atomic charges in parentheses.
the lactone was reduced. As expected, the evaluation of com-
pound 17 against HNE revealed the loss of activity when com-
pared with the reference compound 3. Encouraged by this
result, compound 18 was synthesized aiming to demonstrate
that the lactone was indeed the reactive centre and not the
imine moiety, but to our surprise, compound 18 was also
shown to be inactive (Scheme 5).
Puzzled by these results, we considered that the saturation
of the imine bond would significantly alter the electrophilicity
of the lactone carbon, disfavouring by this way, the nucleophi-
lic addition to this position. Nevertheless, the 11B NMR of 3,
17 and 18 did not expose any substantial differences between
these molecules neither the atomic charges (NPA) calculated
for 3, 17 and 18 by means of DFT calculations,24 corroborated
this assumption.
Based on these results, we performed the evaluation of the
stability of 16 in the presence of sodium methoxide, a strategy
often used to mimic the action of the serine hydroxyl group at
the active centre of the enzyme.25 The reaction was performed
in methanol at room temperature, followed by mass spec-
trometry. Surprisingly, the product (ESI+ m/z = 385.26) formed
in this transformation indicates that two molecules of meth-
oxide reacted with 16 and that the boronic acid component
was released in the process. Interestingly, no product corres-
ponding to the addition of just one molecule of methoxide to
16 was detected in the reaction mixture (ESI†). This un-
expected result indicated that the mechanism of action would
probably involve the addition to both the imine and the
lactone moieties. Therefore, the mechanism of the reaction of
O-nucleophilic attack on the heterocycles tested as HNE inhi-
bitors was studied by means of DFT calculations.
The system chosen was compound 3 as a substrate and
methanol as a nucleophile, for computational convenience.
The energy profile obtained is represented in Fig. 1. The mech-
anism starts with substrate 3 plus two molecules of methanol,
represented by A in Fig. 1. In the first step there is a nucleophi-
lic attack of one methanol molecule on the imine C-atom,
while at the same time the other MeOH molecule assists in
the proton transfer from the OH bond in the nucleophile to
the oxygen of the carbonyl group of the lactone. In the corres-
ponding transition state, TSAB, formation of the new C–O bond
is almost accomplished, as shown by a distance of 1.58 Å,
while the proton transfer is underway with the four relevant
O–H bond lengths ranging from 1.07 to 1.40 Å. Then, there is
proton transfer from the oxygen, in B, to the nitrogen, in C,
where there is a formally positive N-atom and the lactone
CvO group is regenerated. Interestingly, this N-protonation
Scheme 4 Biological evaluation and structure–activity relationship of com-
pounds 4–16 against HNE. SA = 4-methoxy salicylaldehyde, BA = 4-chlorophenyl
boronic acid.
16, with an IC50 = 1.1 μM, becomes the most efficient inhibitor
prepared in this work. After this, we addressed the selectivity
of 16 in the presence of other proteases like porcine pancreatic
elastase (PPE), proteinase 3 (PR3) and cathepsin G. As shown
in Scheme 4, compound 16, at 50 μM concentration, displayed
an IC50 of 12 μM against cathepsin G and was inactive towards
PPE and PR3. Finally, the inhibitory activity of compound 16
towards HNE was also determined using the progress curve
method (ESI†). Significantly, a time-dependent inhibitory
profile was observed and the corresponding pseudo-first-order
rate constant values, kobs, varied linearly with concentration of
16, yielding a second-order rate constant of inactivation,
kinact/Ki, of 1.2 × 102 M−1 s−1. This value compares well with
those reported for other potent HNE inhibitors.23b Overall, our
results suggest that 16 inhibits HNE irreversibly, and contrasts
sharply with compound 3, for which reversible inhibition kine-
tics were observed (ESI†).
Once demonstrated the potential of this methodology to
prepare new HNE inhibitors, we initiated a study to unravel
their mechanism of action. Considering the general structure
of this family of compounds we envisioned that the nucleophi-
lic attack of the Ser195 hydroxyl oxygen atom on the lactone
moiety would be the most likely mechanism of action. Based
on this assumption, we synthesized compound 17 in which
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Fig. 1 Energy profile (kcal mol−1) calculated for the reaction of 3 with methanol. Relevant distances are indicated (Å).
step produces a significant stabilization of the intermediate, C
being more stable than B by 21 kcal mol−1. A third molecule of
methanol is added to the model, from C to D, as it will be
required to assist the second nucleophilic attack. This attack
occurs in the following step, from D to E, and the extra MeOH
molecule assists in the protonation of the O in the carbonyl
group of the lactone, while, at the same time, there is for-
mation of the new C–O bond between the oxygen atom of
methanol and the carbonyl C-atom. In the transition state,
TSDE, formation of the new C–O bond is well advanced as indi-
cated by a distance of 1.65 Å, while the process of proton trans-
fer, from the methanol OH bond to the carbonyl O-atom, is
half way through, as shown by the relevant O–H distances
(1.11–1.32 Å). From E to F there is a rearrangement in the
H-bonds between the intermediate and the neighbour MeOH
Fig. 2 Energy profile (kcal mol−1) calculated for nucleophilic attack of metha-
nol on the lactone CvO group of 3. The energy values are referred to A and
relevant distances are indicated (Å).
molecule. Finally, in the last step there is proton transfer from methanol additions and, thus, the fact that the reaction
the carbonyl O-atom to the oxygen connected to the boron cannot be stopped after the first attack, on the imine C-atom.
atom, with the corresponding hydrolysis of this bond and the
Since there are two O-nucleophilic attacks in the reaction
opening of the former lactone ring. In the transition state, described above it is important to study the sequence of those
TSFG, scission of the C–O bond is just starting, with a distance attacks in order to gain insight into the mechanism of HNE
(1.59 Å) that is only 0.19 Å longer than the corresponding one inhibition. In other words, what occurs first, addition of the
in intermediate F. Once again, assistance of the adjacent nucleophile to the carbonyl C-atom or attack on the imine
MeOH molecule is crucial to the H-transfer process that occurs C-atom? In order to answer this question the alternative path
along that step and the relevant O–H distances indicate that, was explored by means of DFT calculations, i.e., a mechanism
in fact, such process is underway in TSFG (1.08–1.39 Å).
where the first step corresponds to methanol nucleophilic
It is important to notice that the barriers calculated for the attack on the CvO of the lactone. The energy profile obtained
mechanism represented in Fig. 1 are rather high, presenting for the first step is represented in Fig. 2, while the entire path
values of 24 and 30 kcal mol−1 for the two steps corresponding is presented as ESI (Fig. S1†).
to nucleophilic attack. This is due to the use of methanol as a
The O-nucleophilic attack of methanol on the lactone CvO
nucleophile in the model employed in the calculations, a group of substrate 3 occurs similarly to the equivalent steps in
much poorer nucleophile than methoxide, the nucleophile the first mechanism addressed. Thus, there is methanol
used in the experiment described above. Nevertheless, the bar- assisted proton transfer, from the incoming methanol mole-
riers obtained indicate a feasible reaction, especially taking cule to the O-atom in the carbonyl group of the substrate,
into account an expected large excess of methanol if this simultaneously leading to the formation of the new C–O
species is used as a solvent in the reaction medium. In between the substrate and the nucleophile. Formation of that
addition, the similarity between the values calculated for the bond is well advanced in the transition state, TSHI, with a C–O
two energy barriers justifies the existence of two consecutive distance of 1.70 Å. Also, the process of H-transfer is evident in
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Fig. 3 Representation of the LUMO of heterocycle 3.
TSHI with the four relevant O–H distances between 1.13 and
1.29 Å. Most interestingly, the energy barrier calculated for this
step is 3 kcal mol−1 higher than the one obtained for the
attack on the imine C-atom, as the first step of the reaction
mechanism (TSAB in Fig. 1). This indicates the imine as the
more favourable location on the substrate for the first
O-nucleophilic attack, being a rather surprising result, taking
into account the atomic charges calculated for 3 (Scheme 5),
which show the carbonyl C-atom as more electrophilic than
the imine one. However, the LUMO of the substrate (Fig. 3)
has a significant participation in the imine CvN bond, repre-
senting a π* interaction between those two atoms and present-
ing 51% of the electron density of that orbital. The results
above indicate that the reaction is under orbital control, rather
than being ruled by charge.
Fig. 4 Modeling the interaction of compounds 1, 3, 15 and 16 in the HNE
active site (A, B, C and D, respectively), using GOLD 5.1 software.
Scheme 6 Biological evaluation of compound 19 aiming to demonstrate the
importance of the aldehyde aromatic substituent in the compound recognition
and potency.
Aiming to explain the observed biological activity of the
various heterocycles (Scheme 4) and to get insight into their
mechanisms of action at the molecular level, we also per-
formed in silico molecular docking studies in the active site of
HNE, using GOLD 5.1 software.26 The coordinates of the
enzyme structure were obtained from Protein Data Bank select-
ing the structure with accession code 1HNE (X-ray coordinates
at 1.84 Å resolution, previously validated for docking pro-
cedures23). Modelling the interaction of compounds 1, 3, 15
and 16 in the HNE active site (Fig. 4A, B, C and D, respect-
ively), the most striking feature evidenced by the docking of all
4 compounds is their distinct recognition by the enzyme’s
primary specificity binding pocket (S1). As shown in Fig. 4A,
the inactive compound 1 does not occupy S1, contrasting with
the moderately active compound 3 (IC50 of 20 μM), prepared
with L-phenylalanine, which fits the benzyl side chain well
within the S1 pocket (Fig. 4B). Interestingly, in this optimized
conformation, the imine and lactone possible reactive sites are
still quite distant from any HNE nucleophile. Regarding the
most stable conformations obtained for the most potent
inhibitors 15 and 16, inspection of Fig. 4C and D indicates
that the L-phenylalanine side chain is no longer occupying the
S1 pocket, being replaced by the 4-methoxy or the 4-diethyl
amino substituent of the aldehyde aromatic moiety. Interest-
ingly, this arrangement visibly favours the proximity of the
imine reactive site to the Ser195 (4.2 Å (15); 4.1 Å (16)) and
His57 (2.8 Å (15); 2.9 Å (16)).
To validate this model and confirm the importance of the
aldehyde aromatic substituent for the recognition and
potency, compound 19 was prepared from glycine, 4-methoxy-
salicylaldehyde and 2,4-dichlorophenylboronic acid. Very grati-
fyingly, compound 19 that lacks the benzyl group displayed a
3.17 μM IC50 which is comparable with the activity exhibited
by compound 15 prepared from L-phenylalanine (Scheme 6).
Regarding the mechanism of inhibition, the lack of activity
of compounds 17 and 18 indicates that reaction of the imine
and lactone ring opening are both key events in the inhibitory
process. Although the exact sequence of events of the mech-
anism is uncertain, we envision that the Ser195 hydroxyl group
firstly adds to the nearby imine carbon centre to form an
amino ketal (Fig. 5A), in line with the accepted first step of
serine proteases mode of action,20 and with the mechanism of
methanol addition to 3, obtained by DFT calculations (see
above).
The O-nucleophilic attack on the imine neutralizes the
nitrogen atom and generates a negatively charged intermedi-
ate, which may be readily protonated in the N-atom by the
near His57, re-establishing the N–B ylide. This parallels the
intermediate step in the mechanism of Fig. 1, from B to C in
which there is a gain of 21 kcal mol−1. Subsequently, the proxi-
mity of the His57 to the amino ketal carbon centre (2.9 Å) may
assist in the dissociation of the hydroxyl group of Ser195
(Fig. 5B), liberating this fragment for the following lactone
ring opening that promotes the release of the boronic acid
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us to conclude that the boron tether can provide the structural
requirements necessary for the enzyme inhibition. Moreover,
we were able to identify the 4-methoxy or the 4-diethyl amino
substituent of the salicylaldehyde as the most important recog-
nition moiety in the molecule, and the imine alkylation,
lactone ring opening and release of the boronic acid as key
events in the mechanism of inhibition. Further studies are
conducted to optimize this scaffold and to apply this strategy
in the design of inhibitors for other important enzymes.
Experimental section
Fig. 5 Proposed mechanism of inhibition against HNE for heterocycle 16.
General procedure for preparation of boron heterocycles using
water as a solvent
A round bottom flask equipped with a magnetic stirrer was
charged with amino acid (2.0 equiv.), aldehyde (1.5 equiv.) and
distilled water (2.0 mL). This suspension was stirred at 90 °C
for 1 h after which the boronic acid (0.41 mmol) was added,
the mixture was then stirred at 90 °C for 20 h. The reaction
mixture, which appears as a biphasic composition of the pre-
cipitate and a supernatant liquid, was filtered and the solid
retained in the filter was then washed with water followed by
hexane. The desired compound was recovered with dichloro-
methane, which was subsequently removed under reduced
pressure. Diastereomeric ratios were determined based on the
1H NMR.
Scheme 7 LC-ESI-MS analysis of the reaction between inhibitor 16 and HNE.
moiety as suggested by the reaction of 16 with sodium meth-
oxide (Fig. 5C). In agreement with this proposal, the mini-
mized structure generated from the reaction of 16 with His57
and Ser195 in the HNE pocket (performed with MOE 2011.10
software27) shows the maintenance of the 4-diethyl amino
counterpart of the aldehyde aromatic moiety well fitted to the
S1 pocket as shown in Fig. 5.
Based on this proposal, we perform the LC-ESI-MS analysis
of the reaction between inhibitor 16 and HNE. Very gratify-
ingly, we observed that 16 is completely consumed in the pres-
ence of HNE, at the same time that compound 20 is formed in
the reaction mixture (Scheme 7 and ESI†). The in situ for-
mation of 20 is a particularly important observation as it is in
good agreement with the aforementioned mechanism, in
which imine 20 may be generated via hydrolysis of the inter-
mediate depicted in Fig. 5A. Nevertheless, these results should
be taken cautiously as the appearance of 20 as a result of a
spontaneous reaction of phenylalanine and salicylaldehyde
formed via alternative pathways cannot be ruled out.
Human neutrophil elastase activity with a fluorogenic peptide
substrate
Fluorometric assays for the Human neutrophil elastase (HNE)
(Merck, Germany) inhibition activity were carried out in 200 μL
assay buffer (0.1 M HEPES pH 7.5 at 25 °C) containing 20 μL of
0.17 μM HNE in assay buffer (stock solution 1.7 μM in 0.05 M
acetate buffer, pH 5.5), and 5 μL of each of tested inhibitors.
The reaction was initiated by the addition of 175 μL of a fluoro-
genic substrate to a final concentration of 200 μM (MeO-Suc-
Ala-Ala-Pro-Val-AMC, Merck, Germany) and the activity was
monitored (excitation 380 nm; emission 460 nm) for 30 min,
at 25 °C on a Fluorescence Microplate Reader Tecan infinite
M200 (Tecan, Switzerland). The Km of this substrate of HNE
was previously determined to be 185 μM (data not shown). For
all assays, saturated substrate concentration was used,
Conclusion
In summary, as far as our knowledge goes, herein we have throughout, in order to obtain linear fluorescence curves.
demonstrated for the first time that a boron promoted one-pot Inhibitors stock solutions were prepared in DMSO, and serial
assembly reaction can be used to design novel bioactive dilutions were made in DMSO. Controls were performed using
scaffolds. This strategy was applied to the discovery of new enzyme alone, substrate alone, enzyme with DMSO and a posi-
HNE inhibitors generated from components like salicylalde- tive control (MeOSuc-Ala-Ala-Pro-Ala-CMK, Calbiochem,
hyde, aryl boronic acids and amino acids. The versatility of Germany). By computing the log of inhibitors concentrations
this assembly strategy allowed the synthesis of new HNE versus the percentage of activity and using the GraphPad
inhibitors in excellent yields, high diastereoselectivities and program the IC50 values were determined by non-linear
IC50 up to 1.1 μM (compound 16). The combination of syn- regression analysis. Assays were performed in triplicate and
thetic, biochemical, analytical and theoretical studies allowed data presented as the mean and the standard deviation.
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Docking studies
2 D. J. Newman and G. M. Cragg, J. Nat. Prod., 2012, 75, 311–
335.
The 3D structure coordinates of HNE were obtained from the
Protein Data Bank, PDB code 1HNE with a 1.84 Å resolution.
To prepare the enzyme for the docking studies, the co-crystal-
lized inhibitor as well as crystallographic water molecules,
included in the PDB structure, were removed. Hydrogen atoms
were added and the protonation states were correctly assigned
using the Protonate-3D tool within the Molecular Operating
Environment (MOE) 2011.10 software package,27 energy was
minimized using MMFF94x force field. Molecular docking
studies were then performed using the GoldScore scoring func-
tion from the GOLD 5.1 software package and each ligand was
subjected to 1000 docking runs. Minimization of the structure
generated from the reaction of 16 with His57 and the Ser195
in the HNE pocket was performed with MOE software using
MMFF94x force field.
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DFT calculations
Calculations were performed using the Gaussian 09 software
package,28 and the M06-2X functional, without symmetry con-
straints. That is a hybrid meta-GGA functional developed by
Truhlar and Zhao,29 and it was shown to perform very well for
main-group kinetics, providing a good description of long range
effects such as van der Waals interactions or π–π stacking.30,31
The optimized geometries were obtained using the 6-31G(d,p)
basis set.32 Transition state optimizations were performed with
the Synchronous Transit-Guided Quasi-Newton Method (STQN)
developed by Schlegel et al.,33 following extensive searches of
the potential energy surface. Frequency calculations were per-
formed to confirm the nature of the stationary points, yielding
one imaginary frequency for the transition states and none for
the minima. Each transition state was further confirmed by fol-
lowing its vibrational mode downhill on both sides and obtain-
ing the minima presented on the energy profile. The atomic
charges presented were obtained by means of a Natural Popu-
lation Analysis (NPA),34 performed with the NBO 5.0 program.35
Single point energy calculations were performed using the
6-311++G(d,p)36 basis set, with solvent effects (MeOH) calcu-
lated by means of the Polarizable Continuum Model (PCM)
initially devised by Tomasi and coworkers,37 with radii and
non-electrostatic terms of the SMD solvation model, developed
by Truhler et al.38
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Acknowledgements
Fundação para a Ciência e Tecnologia (PEst-OE/SAU/UI4013/
2011; PEst-OE/QUI/UI0100/2011; PTDC/QUI-QUI/118315/2010;
PTDC/CTM-NAN/115110/2009;
SFRH/BD/61419/2009
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