Clickable 4-Oxo-β-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes

Article abstract of DOI:10.1002/cmdc.201600258

Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper-catalyzed alkyne–azide 1,3-dipolar cycloaddition (CuAAC, or ′click′ chemistry) approach based on the 4-oxo-β-lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure–activity relationships set the basis to develop fluorescent and biotinylated activity-based probes as tools for molecular functional analysis. Attaching the tags to the 4-oxo-β-lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC₅₀values in the nanomolar range (56–118 nm) displayed by the probes. The nitrobenzoxadiazole (NBD)-based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity-based probes with promising applications in target validation and identification, as well as diagnostic tools.

Full text of DOI:10.1002/cmdc.201600258

DOI: 10.1002/cmdc.201600258
Full Papers
Clickable 4-Oxo-b-lactam-Based Selective Probing for
Human Neutrophil Elastase Related Proteomes
Eduardo F. P. Ruivo,[a] Lꢀdia M. GonÅalves,[a] Luꢀs A. R. Carvalho,[a] Rita C. Guedes,[a]
Stefan Hofbauer,[b, c] Josꢁ A. Brito,[b] Margarida Archer,[b] Rui Moreira,*[a] and
Susana D. Lucas*[a]
Human neutrophil elastase (HNE) is a serine protease associat-
ed with several inflammatory processes such as chronic ob-
structive pulmonary disease (COPD). The precise involvement
of HNE in COPD and other inflammatory disease mechanisms
has yet to be clarified. Herein we report a copper-catalyzed
alkyne–azide 1,3-dipolar cycloaddition (CuAAC, or ’click’
chemistry) approach based on the 4-oxo-b-lactam warhead
that yielded potent HNE inhibitors containing a triazole moiety.
The resulting structure–activity relationships set the basis to
develop fluorescent and biotinylated activity-based probes as
tools for molecular functional analysis. Attaching the tags to
the 4-oxo-b-lactam scaffold did not affect HNE inhibitory activi-
ty, as revealed by the IC50 values in the nanomolar range (56–
118 nm) displayed by the probes. The nitrobenzoxadiazole
(NBD)-based probe presented the best binding properties
(ligand efficiency (LE)=0.31) combined with an excellent lipo-
philic ligand efficiency (LLE=4.7). Moreover, the probes
showed adequate fluorescence properties, internalization in
human neutrophils, and suitable detection of HNE in the pres-
ence of a large excess of cell lysate proteins. This allows the
development of activity-based probes with promising applica-
tions in target validation and identification, as well as diagnos-
tic tools.
Introduction
Serine hydrolases are one of the largest, most diverse and bio-
logically important enzyme classes in eukaryotic and prokary-
otic proteomes.[1] Not surprisingly, an increasing number of
clinically approved drugs target several members of this
enzyme class such as lipases, esterases, amidases, and proteas-
es. Human neutrophil elastase (HNE) is a member of the chy-
motrypsin family of serine proteases that degrades tissue
matrix proteins such as elastin when released from the azuro-
philic granules in neutrophils due to inflammatory stimuli and
mediators.[2] Several pulmonary dysfunctions result from the
massive migration of neutrophils to the lungs during inflam-
mation and the subsequent release of proteolytic enzymes.[2]
The imbalance between HNE and its endogenous inhibitors
leads to severe tissue injuries resulting in a variety of diseases
such as chronic obstructive pulmonary disease (COPD), rheu-
matoid arthritis, pulmonary emphysema, and psoriasis.[3] More-
over, it has been postulated that HNE can contribute to the
progression of non-small-cell lung cancer.[4]
The need for understanding essential molecular recognition
events in biological systems has directed considerable efforts
toward the development of chemical probes.[5] The design of
small-molecule activity-based probes (ABPs) has proven to be
a successful approach to track the spatial–temporal location
and activity of proteases.[6] Hence, the use of ABPs that target
HNE may lead to an easy methodology for the quantification
of HNE activity in lung disorders, and may ultimately lead to
new diagnostic tools that function through non-invasive proto-
cols.[7,8]
The design of new HNE inhibitors has been intensively pur-
sued, and exploiting the reactivity and molecular recognition
properties of scaffolds used as enzyme inhibitors has led to
breakthrough advances in the development of ABP war-
heads.[6] G-quadruplex-based complexes were reported as the
first efficient probes targeting HNE,[9] but only recently have
non-natural peptidic substrates of HNE been assembled as sen-
sitive ABPs.[7] This approach can be applied to HNE activity
profiling as a promising tool to perform molecular function
analyses of HNE-related disease proteomes. In addition, it has
already been shown that the use of ABPs targeting HNE for
the analysis of human specimens from patients with chronic
lung diseases might lead to an efficient methodology for the
validation of HNE as a diagnostic tool via a non-invasive proto-
col.[10]
[a] E. F. P. Ruivo, Dr. L. M. GonÅalves, L. A. R. Carvalho, Dr. R. C. Guedes,
Prof. R. Moreira, Dr. S. D. Lucas
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Uni-
versidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon (Portugal)
E-mail: sdlucas@ff.ulisboa.pt
rmoreira@ff.ulisboa.pt
[b] Dr. S. Hofbauer, Dr. J. A. Brito, Dr. M. Archer
Instituto de Tecnologia Quꢀmica e Biolꢁgica—Antꢁnio Xavier, Universidade
Nova de Lisboa, Avenida da Repfflblica, 2780-157 Oeiras (Portugal)
[c] Dr. S. Hofbauer
Present address: Department for Structural and Computational Biology,
Max F. Perutz Laboratories, University of Vienna, 1030 Vienna (Austria)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600258.
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Kasperkiewicz et al.[7] reported non-natural peptidic sub-
strates of HNE as sensitive ABPs, revealing that the level of
active HNE during the process of neutrophil extracellular trap
formation was not as high as anticipated, highlighting the utili-
ty of this approach in dissecting biological events. As the use
of peptidic substrates can raise some challenges, herein we
present the use of small-molecular scaffolds as ABPs that pro-
vide both ease of handling and high specificity toward the
target protein. A 1,2,3-triazole-based oxo-b-lactam library was
synthesized to rapidly explore the impact of the triazole
moiety on HNE inhibitory potency. This information was then
extended to the design of ABPs that were validated for HNE
activity. Neutrophil internalization and selectivity in the pres-
ence of a complex proteome was observed for an ABP contain-
ing a nitrobenzoxadiazole (NBD) tag. Moreover, a bio-orthogo-
nal probing methodology is envisaged, as the starting oxo-b-
lactam alkyne was also shown to inhibit HNE.
of LE as result of fragment growth, all compounds 2 displayed
LE values >0.3, thus meeting the requirements of an early
lead. Furthermore, LLE values remained in the range of 5–6, re-
flecting the fact that lipophilicity was kept fairly constant
during fragment growth, and suggesting that that the 1,2,3-tri-
azole moiety may lead to additional interactions with the
enzyme active site.
To gain molecular insight on the preferred interactions of
the synthesized compounds and the active site of HNE, molec-
ular docking studies were performed using HNE coordinates
from PDB ID: 3Q77.[18] All the compounds showed a correct
pose for the oxo-b-lactam in the S1 pocket, promoting Ser195
nucleophilic attack in agreement with high activities obtained
for the synthesized library (further details and molecular poses
are given in the Supporting Information). We were unable to
co-crystallize a triazole-based oxo-b-lactam either with HNE or
porcine pancreatic elastase (PPE), but we have crystallized[19]
and determined the X-ray structure of PPE in complex with the
closely related diethyl N-(methyl)pyridinyl-substituted oxo-b-
lactam, at 1.8 ꢃ resolution (Figure 2A, details of structure de-
termination are given in the Supporting Information, and the
atomic coordinates and structure factor amplitudes have been
deposited in the RCSB Protein Data Bank (PDB) as PDB ID:
4YM9). This structure confirmed the expected covalent mecha-
nism of action, involving ring opening and the suitable accom-
modation of the diethyl moiety in the S1 pocket. Additional p–
p stacking with His57 is likely to contribute to the stability of
the complex, decreasing the rate of hydrolysis and promoting
strong inhibition.
Results and Discussion
1,2,3-Triazole-based 4-oxo-b-lactams are potent HNE
inhibitors
Previous 4-oxo-b-lactam structure–activity relationship (SAR)
studies against HNE showed that diethyl substitution at C3 is
preferred for optimal S1 subsite recognition, leading to im-
proved inhibitory activity and
target selectivity. Furthermore, the
N-substitution pattern can be ex-
plored as an anchor to probe
the interaction with Sn subsites
(Figure 1).[11,12]
Design and synthesis of oxo-b-lactam-based probes
Click chemistry is as a powerful
The encouraging results obtained with 1,2,3-triazole-substitut-
ed 4-oxo-b-lactams as HNE inhibitors led us to design a group
of ABPs in which NBD, fluorescein, and biotin moieties were
used as reporter tags and attached to the 4-oxo-b-lactam war-
head via a 1,2,3-triazole linker (3–5, Figure 3). Docking studies
revealed that the tag did not affect the positioning of the oxo-
b-lactam ring inside the active site of HNE when compared
with smaller triazole analogues (Figure S2, Supporting Informa-
tion). Moreover, covalent docking performed using the coordi-
nates from the PPE–oxo-b-lactam complex showed that the
acyl-enzyme formed after Ser195 attack presents additional p–
p stacking between the triazole moiety and His57 and cation–
p stacking with the PPE backbone Arg61 residue (Figure 2B,
ABP 3 example), that may increase the complex lifetime
toward feasible ABP profiling.
and versatile synthetic tool for
drug development and chemical
biology applications.[13,14] More
Figure 1. Schematic represen-
tation of the spatial arrange-
ment of oxo-b-lactam sub-
stituents in the active site of
HNE.
specifically,
copper-catalyzed
alkyne–azide 1,3-dipolar cycloaddi-
tion (CuAAC) has been explored as
an efficient approach for the gen-
eration of compound libraries,
where the formed 1,2,3-triazole moiety is more than a passive
linker, as it presents favorable physicochemical properties.[15]
We first prepared the 4-oxo-b-lactam alkyne 1 (Table 1) as the
starting material for CuAAC chemistry. Remarkably, compound
1 showed to be a potent inhibitor of HNE, with an IC50 value
of 82 nm (Table 1), while displaying a fragment-like nature
[Mr =179.22 Da, ligand efficiency (LE)=0.81, lipophilic ligand
efficiency (LLE)=5.53].[16,17]
The synthetic strategy was first to bind a tag moiety with
a linker containing an appropriate handle for further reaction
with the oxo-b-lactam alkyne 1 using CuAAC, yielding the
NBD-, fluorescein-, and biotin-tagged probes 3–5 (Figure 3).
To validate compounds 3–5 as potential ABPs, their activity
was measured against HNE, showing conserved inhibitory ac-
tivity as anticipated, with remarkable IC50 values in the nano-
molar range (56–118 nm), deeming the structures as potent
HNE inhibitors and suitable candidates for an ABP. The NBD-
based probe 3 displayed the best binding properties (LE=
With this encouraging result in hand, we then synthesized
a small set of 1,2,3-triazole-based 4-oxo-b-lactam derivatives,
2a–l (Table 1) by reacting alkyne 1 with the appropriate azides.
Compounds 2a–l revealed to be very potent HNE inhibitors,
with IC50 values ranging from 14 to 103 nm (Table 1). These
values compare favorably with that determined for ONO-5046
(15 nm), the only HNE inhibitor in clinical use and included as
a positive control in our assay. Although there was some loss
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Table 1. Oxo-b-lactams synthesized by Huisgen 1,3-dipolar cycloaddition.
Compd
R
–
Yield [%]
IC50 [nm][a]
LE [kcalmolÀ1
]
LLE
1
21
18
25
28
41
35
62
47
74
31
58
82Æ15
21Æ6
18Æ3
48Æ1
19Æ9
25Æ7
103Æ10
14Æ4
17Æ1
20Æ5
31Æ6
0.81
0.50
0.48
0.40
0.46
0.47
0.44
0.43
0.46
0.44
0.41
5.53
5.63
5.34
4.36
5.71
4.82
4.78
5.94
5.01
5.41
5.62
2a
2b
2c
2d
2e
2 f
2g
2h
2i
Ph
C6H4-4-Me
C6H4-4-CF3
C6H4-4-OMe
C6H4-4-Br
CH2Ph
CH2C6H4-4-NO2
CH2C6H4-4-Cl
CH2C6H4-4-OMe
C6H4-2-NO2
2j
2k
44
16Æ6
0.33
4.68
2l
94
–
30Æ5
15Æ1
0.28
–
4.91
–
ONO-5046
–
[a] Against HNE; values are the meanÆSD of n=3 experiments performed in triplicate.
Figure 2. A) View of the active site of PPE complexed with a diethyl N-(methyl)pyridinyl-substituted oxo-b-lactam (JM102, PDB ID: 4YM9). The simulated an-
nealing omit map is shown as a blue mesh contouring JM102; surrounding residues are shown in stick format: O: red, N: blue, S: gold, C: yellow (protein)
and green (inhibitor). B) Covalent docking of compound 3 using the coordinates of the PPE–oxo-b-lactam complex (PDB ID: 4YM9).
0.31) combined with excellent lipophilic ligand efficiency
(LLE=4.7).
can be used for a variety of assays under different conditions
(Figure S4).
ABP 3 was characterized for its spectroscopic properties in
solvents with various polarities. It was observed that the ab-
sorption and fluorescence of ABP 3 did not change with sol-
vents of different polarities, which indicates that these probes
Fluorescent ABPs 3 and 4 were incubated for 30 min with
human neutrophils in order to test neutrophil internalization,
where HNE is localized in biological systems. By comparing
ABPs 3 and 4 (Figure 4A) with the DAPI-stained nuclei (Fig-
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Table 2. Enzyme selectivity assay.
Enzyme
IC50 [mm][a]
1
2g
3
HNE
PR3
Chymotrypsin
Urokinase
Trypsin
0.082
8Æ1
0.014
>100
>100
>100
>100
0.056
3Æ1
23Æ1
12Æ1
>100
>100
>100
>100
Thrombin
ND[b]
41Æ2
1Æ1
[a] Values are the meanÆSD of n=3 experiments performed in triplicate.
[b] Not determined.
related proteases, displaying selectivity indices (SI) of >2900.
The oxo-b-lactam alkyne 1 exhibited a good selectivity profile,
with SI>100 toward all proteases studied, thus being a good
candidate for bioorthogonal chemistry. The ABP 3 (IC50 HNE=
56 nm) was able to maintain an excellent selectivity profile,
a remarkable achievement usually observed only with peptido-
mimetic probes.[7,20] Overall, the results show suitable HNE se-
lectivity for activity-based protein profiling.
Figure 3. Oxo-b-lactam-based probes 3–5.
ure 4B), it was observed that these were co-localized with the
stained nuclei, indicating that both NBD- and fluorescein-
tagged ABPs 3 and 4 were internalized in the neutrophils (Fig-
ure 4C).
Moreover, depending on the target proteomes, further
assays are needed to evaluate experimental specificity. Hence,
ABPs 3 and 4 were applied to label HNE in the presence of
a complex proteome (HEK293T cell line was chosen as being
a non-carcinogenic human cell line that does not express elas-
tase) and subjected to SDS-PAGE for analysis by protein stain-
ing and fluorescence imaging (Figure 5). Remarkably, this assay
revealed selective labelling of HNE in the presence of a large
excess of lysate proteins without nonspecific interactions of
either NBD or fluorescein ABPs 3 and 4. SDS-PAGE showed
unique HNE–ABP fluorescent bands (lanes 8 and 9, Figure 5B)
clearly detected at the HNE region (for HNE control, lane 5,
Figure 5), that was not observed in the absence of HNE
(lanes 3 and 4, Figure 5B), revealing good selectivity of the flu-
orescent oxo-b-lactam ABPs for HNE.
The capacity of 3 and 4 to be internalized by neutrophils
highlights the advantages of our small-warhead-based ABPs
over the less permeable peptidic probes and expands the
scope of biological assays that can be performed, allowing
HNE visualization not only in neutrophil extracellular traps, but
also in its intracellular localization. Moreover, the nanomolar
biotinylated ABP will allow quantification and identification of
targets by affinity isolation.
Selectivity for HNE was evaluated for the most potent inhibi-
tor 2g, ABP 3, and the oxo-b-lactam containing the alkyne
handle 1 against five related proteases (Table 2). Inhibitor 2g
(IC50 HNE=14 nm) showed outstanding selectivity for HNE over
Figure 4. A) Human neutrophils after 30 min contact with ABPs 3 (top row) and 4 (bottom row). B) Nuclei stained with DAPI, incubation with ABP. C) Merged
images.
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Figure 5. Imaging human leukocyte elastase with the fluorescent probes ABP 3 and 4 by SDS-PAGE. Laser image scanned at excitation wavelength 532 nm
and 580 nm bandpass emission filter (Typhoon Trio) before total protein staining (B) and SDS-PAGE Coomassie blue labeling (A). Lanes 1: pre-stained protein
standard markers, 2: 5 mg HEK293T protein extracts, 3: 5 mg HEK293T protein extracts and 1 mm ABP 4, 4: 5 mg HEK293T protein extracts and 1 mm ABP 3,
5: 40 ng HNE, 6: 40 ng HNE and 1 mm ABP 4, 7: 40 ng HNE and 1 mm ABP 3, 8: 5 mg human neutrophil protein extracts and 1 mm ABP 4, 9: 5 mg human neutro-
phil protein extracts and 1 mm ABP 3, 10: 5 mg human neutrophil protein extracts.
The amount of HNE for labeling experiments was decreased
down to 5 ng (lanes 2–4, Figure 6), and a clear band was ob-
served for ABP 3 detection in each lane, which strongly sup-
ports the suitability of the synthesized probes for low-nano-
gram-level detection of HNE.
tory activity against HNE together with adequate fluorescence
properties, internalization in human neutrophils, and suitable
detection of HNE in the presence of a large excess of cell
lysate proteins. The biotinylated probe is envisaged for further
affinity isolation and quantification of HNE in inflammation-re-
lated proteomes. Moreover, a bio-orthogonal probing method-
ology can be foreseen by the use of the oxo-b-lactam alkyne
1 that may help to overcome possible cell permeability pitfalls.
Notably, 1 exhibited a remarkable IC50 value of 82 nm (Table 1),
a result consistent with the observation that the HNE inhibitory
potency of 4-oxo-b-lactams is determined largely by the intrin-
sic reactivity of the 4-oxo-b-lactam warhead combined with
the diethyl group at C3, responsible for molecular recognition.
The ABPs presented herein will be further studied as promising
tools for molecular mechanism studies of HNE-related diseases,
namely COPD. Taken together, the results presented in this
work highlight not only the versatility of the oxo-b-lactam scaf-
fold for the generation of protease inhibitor libraries, but also
the possibility to develop ABPs with promising applications in
target validation and identification as well as in the develop-
ment of diagnostic tools.
Figure 6. Imaging human leukocyte elastase with the fluorescent probe ABP
3 by SDS-PAGE. Laser image scanned at excitation wavelength 532 nm and
580 nm bandpass emission filter (Typhoon Trio) before total protein staining
(B) and SDS-PAGE Coomassie blue labeling (A). Lanes 1: pre-stained protein
standard markers, 2: 40 ng HNE and 1 mm ABP 3, 3: 20 ng HNE and 1 mm
ABP 3, 4: 5 ng HNE and 1 mm ABP 3, 5: 40 ng HNE.
Experimental Section
3,3-Diethyl-1-(prop-2-yn-1-yl)azetidine-2,4-dione (1): To a stirring
solution of diethylmalonyl dichloride (36 mmol, 6.3 mL) in dry diox-
ane (21 mL) at room temperature under nitrogen atmosphere, a so-
lution of propargylamine (1 equiv, 2.3 mL) in the same solvent
(21 mL) was added dropwise. A solution of triethylamine (18 mL) in
dry dioxane (21 mL) was then also added dropwise, and the reac-
tion was stirred at room temperature overnight. The solid residues
were filtered and washed with hexanes. The filtered solution was
concentrated, and the resulting residue was purified by flash chro-
matography, using a gradient of hexanes/EtOAc (0–100% EtOAc)
to yield the desired alkyne 1 as a colorless waxy solid (1.35 g,
Conclusions
From the results presented in this work, we can conclude that
clickable 4-oxo-b-lactams give access to potent and selective
inhibitors against HNE and are valuable scaffolds for emerging
molecular profiling approaches. The click chemistry approach
proved to be an efficient tool for the development of new
potent HNE inhibitors in which the 1,2,3-triazole moiety pro-
vides additional interactions in the enzyme active site. Proof-
of-concept was performed with NBD-, fluorescein-, and biotin-
tagged ABPs 3–5 that show conservation of the potent inhibi-
1
21%). H NMR (400 MHz, CDCl3): d=4.07 (d, J=1.6 Hz, 2H), 2.31 (s,
1H), 1.76 (q, J=7.5 Hz, 4H), 1.00 ppm (t, J=7.5 Hz, 6H).
Synthesis of azide building blocks: Azide building blocks were
synthesized from the corresponding bromides, chlorides, or tri-
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fluoromethanesulfonates by reaction with sodium azide and used
without further purification. For the azide precursor of compound
2j, a synthetic procedure via diazonium salt using 2-nitroaniline as
starting material was used. Further details are given in the Sup-
porting Information.
thank Dr. Maria Elisa Alves for neutrophil collection and isolation
from healthy donor samples, Prof. Isabel Sµ-Correia (IST-ULisboa)
for access to the Multimager system Thyphon Trio 6300583, Dr.
Miguel Pessanha and Dr. Przemyslaw Nogly for help with X-ray
structure characterization, and Dr. Paula Gomes for MS data ac-
quisition at Faculdade de CiÞncias da Universidade do Porto.
Copper-catalyzed alkyne–azide 1,3-dipolar cycloaddition; Typi-
cal example for the synthesis of ABP 3: To a 0.1m solution of
alkyne 1 (0.32 mmol, 57 mg) in DMF, N-(3-azidopropyl)-7-nitroben-
zoxadiazol-4-amine (1.1 equiv, 91.8 mg), aqueous sodium ascorbate
(0.1m, 10% mmol) and aqueous copper sulfate (0.1m, 10% mmol)
were added. The reaction was stirred overnight, and the reaction
mixture was diluted with water (four volumes) and then extracted
with EtOAc (3ꢄ1 volume). The organic layers were combined,
dried with anhydrous Na2SO4, filtered and concentrated. The ob-
tained residue was purified by flash chromatography (elution with
hexanes/EtOAc gradient) followed by recrystallization from CH2Cl2/
n-hexane to yield the target compound as an orange solid
Keywords: 4-oxo-b-lactams
· activity-based probes · click
chemistry · fluorescent probes · human neutrophil elastase
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1
(94.5 mg, 67%); mp: 199–2018C; H NMR (300 MHz, (CD3)2SO): d=
9.59 (brs, 1H), 8.56 (d, J=8.9 Hz, 1H), 8.21 (s, 1H), 6.39 (d, J=
8.8 Hz, 1H), 4.63 (s, 2H), 4.56 (t, J=6.9 Hz, 2H), 3.30 (m, 2H), 2.29
(quint, 2H), 1.72 (q, J=7.5 Hz, 4H), 0.91 ppm (t, J=7.5 Hz, 6H);
ESIMS(+) m/z: 443 [M+H]+; Anal. calcd (C19H22N8O5): C 51.58, H
5.01, N 25.33%, found: C 51.79, H 5.21, N 25.34%.
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a diethyl N-(methyl)pyridinyl-substituted oxo-b-
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CCD detector at 100 K. Further details are given in the Supporting
Information.
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were used at 1.85 ꢃ resolution. To prepare the enzymes for dock-
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formed with the GoldScore scoring function from the GOLD soft-
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in the Supporting Information.
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Acknowledgements
This work was funded by Fundażo para a CiÞncia e a Tecnologia
(FCT, Portugal): PTDC/BBB-BEP/2463/2014, Pest-OE/SAU/UI4013/
2014, PTDC/BIA-PRO/118535/2010, PEst-OE/EQB/LA0004/2011,
SFRH/BPD/79224/2011 (J.A.B.), SFRH/BD/100400/2014 (L.A.R.C.),
and IF/00472/2014 (S.D.L.). E.F.P.R. acknowledges a research grant
awarded by Fundażo AstraZeneca Innovate Competition (Portu-
gal). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-
007344), which is co-funded by FCT/MCES through national
funds and by FEDER under PT2020, is acknowledged. The authors
[21] GOLD ver. 5.1: https://www.ccdc.cam.ac.uk/solutions/csd-discovery/
components/gold/ (accessed July 19, 2016).
Received: May 19, 2016
Revised: June 28, 2016
Published online on && &&, 0000
&
ChemMedChem 2016, 11, 1 – 7
www.chemmedchem.org
6
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
E. F. P. Ruivo, L. M. GonÅalves,
L. A. R. Carvalho, R. C. Guedes,
S. Hofbauer, J. A. Brito, M. Archer,
R. Moreira,* S. D. Lucas*
&& – &&
Clickable 4-Oxo-b-lactam-Based
Selective Probing for Human
Neutrophil Elastase Related
Proteomes
Click here! Human neutrophil elastase
(HNE) is a serine protease implicated in
several inflammatory processes. A click
chemistry approach is reported based
on the 4-oxo-b-lactam warhead toward
the development of fluorescent and
biotinylated activity-based probes as
tools for molecular functional analysis.
The proof-of-concept study revealed se-
lective detection of HNE activity in
human neutrophils and in the presence
of a large excess of cell lysate proteins.
ChemMedChem 2016, 11, 1 – 7
www.chemmedchem.org
7
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ