Target-activated prodrugs (TAPs) for the autoregulated inhibition of MMP12

Article abstract of DOI:10.1021/ml3001193

We describe a prodrug concept in which the target enzyme MMP12 produces its own inhibitor in a two-step activation procedure. By using an MMP12-specific peptide sequence and a known sulfonamide drug integrated in the backbone, the active inhibitor is released upon enzyme cleavage. In in vitro experiments, we present proof of concept that the activation proceeds with useful kinetics. The approach is highly selective over the closely related MMP8. If applied in vivo in the future, these prodrugs might release the active entity in a highly specific manner only at such sites where enzyme activity resides.

Full text of DOI:10.1021/ml3001193

Letter
pubs.acs.org/acsmedchemlett
Target-Activated Prodrugs (TAPs) for the Autoregulated Inhibition of
MMP12
Amanda Cobos-Correa,†,‡ Frank Stein,† and Carsten Schultz*,†,‡,§
†Cell Biology and Biophysics Unit, EMBL, Meyerhofstraße 1, 69117 Heidelberg, Germany
‡Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and EMBL, Otto-Meyerhof-Centre, Im Neuenheimer
Feld, 69124 Heidelberg, Germany
§Translational Lung Research Center Heidelberg (TLRC), Member of the German Center for Lung Research
S
* Supporting Information
ABSTRACT: We describe a prodrug concept in which the
target enzyme MMP12 produces its own inhibitor in a two-
step activation procedure. By using an MMP12-specific
peptide sequence and a known sulfonamide drug integrated
in the backbone, the active inhibitor is released upon enzyme
cleavage. In in vitro experiments, we present proof of concept
that the activation proceeds with useful kinetics. The approach
is highly selective over the closely related MMP8. If applied in vivo in the future, these prodrugs might release the active entity in
a highly specific manner only at such sites where enzyme activity resides.
KEYWORDS: prodrug, MMP, inhibitors, inflammation, proteases
roteases are potential drug targets for a broad range of
diseases such as cancer or inflammation, and some
disease would produce useful treatments in the future.8 As a
result, a large number of low nanomolar MMP inhibitors with
improved selectivity were synthesized, but their applicability in
the clinic has not yet been proven.9 Therefore, there is still a
need for new strategies to develop MMP inhibitors that avoid
side effects and make MMPs druggable targets.10 A particularly
desirable feature would be the exclusive targeting of MMP12 at
the site of inflammation rather than the global inhibition of
enzyme activity.
P
inhibitors have been successfully used in the clinic.1 However,
developing new drugs in the last years has been challenging,
partially due to the difficulty in achieving selectivity and the risk
of hitting antitargets, leading to serious side effects. To open
doors to new treatments for diseases where proteases have
become validated targets, we propose a novel prodrug approach
involving target-activated prodrugs (TAPs), to inhibit proteo-
lytic activity selectively and in an autoregulated fashion. Here,
we focus on matrix metalloproteinase-12 (MMP12), which is a
proteolytic enzyme mainly secreted by macrophages. MMP12 is
able to degrade components of the extracellular matrix such as
elastin and therefore plays an important role in macrophage
migration and extracellular matrix homeostasis.2 However,
under certain pathological conditions, especially when linked to
inflammation, its proteolytic activity might result in excessive
tissue destruction and lung emphysema. For this reason,
MMP12 has been considered a drug target for inflammatory
lung diseases for a long time.3 Consequently, significant effort
has been spent on the development of specific MMP inhibitors,
and several compound libraries have been created and
screened.4
Some of the hits obtained were evaluated in mouse models
and later in clinical trials against cancer and vascular and
inflammatory diseases.5,6 However, these molecules revealed
side effects such as musculoskeletal pain as well as insufficient
clinical benefit.7 The ineffectiveness of synthetic MMP
inhibitors was most probably due to the lack of selectivity
but also a lack of knowledge about MMP biology. For this
reason, it was speculated that an improvement in the selectivity
toward a specific MMP and a better understanding of the
Received: May 18, 2012
Accepted: July 14, 2012
Published: July 14, 2012
© 2012 American Chemical Society
653
dx.doi.org/10.1021/ml3001193 | ACS Med. Chem. Lett. 2012, 3, 653−657

ACS Medicinal Chemistry Letters
Letter
Figure 1. HPLC traces showing hMMP12-mediated drug release from TAP. (a) Prodrug 3 (10 μM) was incubated with hMMP12 (12 nM) in TCN
buffer at 37 °C for different times. After 3 h, the prodrug had been significantly cleaved by hMMP12 to generate predrug 5. Over time, 5 hydrolyzed
spontaneously to the potent inhibitor 1. Uncleaved prodrug 3 hydrolyzed to compound 7. (b) Compound 5 (10 μM) was incubated without enzyme
to study the spontaneous hydrolysis of the molecule. After 20 h, a substantial amount of 5 had converted to the free inhibitor (1). (c) In the absence
of hMMP12, prodrug 3 degraded slowly to the noninhibitory compound 7. (d) When prodrug 3 (10 μM) was incubated with hMMP8 (20 nM), the
enzyme did not cleave the prodrug substantially, and thus, inhibitor release was poor. Instead, most of the prodrug degraded to compound 7 over
time.
a
Scheme 1. Mechanism of TAP Activation by hMMP12 and Inhibitor Release
a
An MMP12 inhibitor is inserted into a peptidic sequence (3), which is specifically cleaved by hMMP12. Upon proteolytic cleavage, the released
molecule (5) shows a moderate IC50 towards hMMP12 but degrades spontaneously, generating the more potent hMMP12 inhibitor 1. In the
absence of enzyme, the prodrug eventually degrades to the poor hMMP12 inhibitor (7).
Our aim was to develop a prodrug that is selectively activated
by its target, MMP12, to release its own inhibitor. This
approach of TAPs will ensure that the release of the inhibitor
will be closely linked to the localization and activity of the
target enzyme. Unlike other protease-activated prodrugs,11
TAPs are unique in that the released inhibitor is not designed
to act on a different target than the activating protein.
Additionally, the prodrug requires proteolytic activation by
the target to become an inhibitor, quite contrary to approaches
where the prodrug itself is already a good inhibitor of the
target.12 Areas of enzyme activity will therefore produce local
inhibitor release at the site of inflammation.
As a starting point for the development of a TAP for
hMMP12, we chose as a starting point two MMP inhibitors
containing an aryl-sulfonamide scaffold (1 and 2). This class of
compounds has been extensively investigated13 and presents
various positions that are easily functionalized.14 The molecules
are able to block MMP12 activity by chelating the catalytic zinc
ion in the active site of the enzyme via their carboxylic acid
group.15 Although hydroxamic acid derivatives would have a
higher affinity,16 the carboxylic acid provides higher stability
and bioavailability17,18 and is synthetically more accessible.
Our first aim was to mask the inhibitory potency of
compounds 1 and 2 and at the same time generate a specific
substrate for MMP12. We therefore incorporated the inhibitors
into a peptidic sequence cleavable by the target protease. We
chose the sequence PLGLEEA, previously shown to be specific
for hMMP12 over other hMMPs, where the cleavage site is
located between glycine and leucine and the specificity relies on
two glutamates located at the P′ site.19 The inhibitor was
incorporated between the two leucines as an N-substituted
glycine generating compounds 3 and 4. The P′ site should
destroy the inhibitory activity from these compounds as it
masks the zinc binding group (ZBG). On the other hand, the
ZPL sequence located on the P site is known to be crucial for
enzyme recognition.20,21 The combination should provide
specificity toward hMMP12 over other hMMPs and make the
inhibitory effect sensitive to hMMP12. All peptides were
prepared by solid-phase peptide synthesis (see the Supporting
Information).
To test the modified peptide 3 as a substrate for recombinant
hMMP12, we incubated the prodrug with the enzyme and
monitored its integrity by HPLC. Only in the presence of
hMMP12 (Figure 1a), we observed the conversion of the
starting material into compound 5 (Scheme 1), which is the
product resulting from the predicted proteolytic cleavage at the
N terminus of leucine. This demonstrated the unaltered
substrate behavior of the prodrugs against hMMP12 whose
catalytic efficiency [Kcat/Km = (3.7
0.1) 103 M−1 s] was
determined by HPLC (see Figure S1a in the Supporting
Information). Prodrug 3 showed a substrate behavior toward
five other MMPs (see Figure S1b in the Supporting
654
dx.doi.org/10.1021/ml3001193 | ACS Med. Chem. Lett. 2012, 3, 653−657

ACS Medicinal Chemistry Letters
Letter
Information) very similar to what was previously published for
the unmodified peptide.19 Only MMP13 showed significant
cleavage of 3, albeit much slower than MMP12.
weak IC50 of 48 μM, most probably due to its inability to
chelate zinc. This indicates that in the absence of hMMP12,
nonproductive degradation will occur, whereas in the presence
of hMMP12, a weak inhibitor will be formed followed by
productive degradation to the potent hMMP12 inhibitor 1 over
time (Scheme 1).
Furthermore, 5 hydrolyzed over time to the more hydro-
philic free MMP12 inhibitor 1. Hence, the successful TAP
design produced inhibitor 1 in a completely MMP12-
dependent fashion through a two-step process: the initial
enzymatic cleavage of prodrug 3 to release predrug 5 followed
by its spontaneous conversion into the final drug (1) (Scheme
1). However, there was a significant delay between enzyme
activity and production of its own inhibitor. This is desirable to
produce a burst of predrug 5 that upon hydrolysis to 1
effectively inhibits MMP12. Together with the MMP12
inhibitor (1), we found an additional peak corresponding to
compound 7 (Scheme 1), a result of the sulfonamide hydrolysis
of 3. N-acylated sulfonamides hydrolyze spontaneously to
secondary sulfonamides as we showed by incubating predrug 5
and prodrug 3 in TCN buffer pH 7.5 at 37 °C in the absence of
enzyme (Figure 1b,c). In both cases, the starting materials
converted over time into compounds 1 and 7, respectively, at a
different rate (Table 1). A similar degree of stability was
Following this mechanism, the limiting step for the
conversion to the inhibitor is the spontaneous degradation of
acylated sulfonamides; therefore, the concentration of inhibitor
released will be proportional to the concentration of active
hMMP12 since predrug 5 does not strongly inhibit MMP12
(see Figure S4a,b in the Supporting Information). To expand
this concept to similar inhibitors, the same set of experiments
was performed with prodrug 4. Again, the generation of the
inhibitor, in this case compound 2, happened exclusively in the
presence of hMMP12 (see Figure S5 in the Supporting
Information). Next, we studied the molecular factors affecting
the spontaneous hydrolysis of the prodrugs and intermediates.
It was previously demonstrated that the hydrolysis of N-
acylated sulfonamides highly depends on the pH.24 We
examined how different substitutions affect the stability of
these model compounds. When leucine at the P1 site in 4 was
substituted by glycine (10, Table 1), the stability in buffer pH
7.5 decreased. However, the terminal free carboxylic acid as in 5
or 6 increased the stability of the molecule as compared to the
amide (3 or 4), suggesting that the conversion occurs via a
typical nucleophilic attack by water on the sulfonamide. The
biphenyl derivatives degraded generally faster than those
containing the methoxyphenyl group (Table 1). These data
indicate that by modifying either the peptidic sequence or the
aryl-sulfonamide scaffold, it is possible to control the timing of
drug release and therefore the window between the enzymatic
activation and the end of drug delivery. In this example, the
spontaneous conversion of 3 into 7 is slow as compared to its
proteolytic cleavage in the presence of hMMP12 (Figure 1a).
Therefore, enzymatic cleavage will dominate. Once converted
into predrug 5, irreversible release of the potent drug 1 will
occur (see Figure S4c in the Supporting Information).
Additionally, we used an activity-based assay to demonstrate
the TAP concept and confirm the HPLC results (Figure 2).
The longer the incubation time of prodrug 3 with hMMP12,
the more inhibitor was released, and the lower was the
enzymatic activity (Figure 2a). The same result was obtained
when compound 5 was incubated without hMMP12, since 5
spontaneously degraded to generate the potent MMP12
inhibitor 1, as observed by HPLC (Figure 2b). On the other
hand, the incubation of 3 without hMMP12 did not lead to any
reduced enzymatic activity, showing once more that the
spontaneous degradation of the prodrug will not have an effect
on the target enzyme (Figure 2c). As a control, the stability of
hMMP12 activity over time was monitored (see Figure S6a in
the Supporting Information). One of the key features of the
TAP approach is that a broad-spectrum inhibitor can be
converted to a specific inhibitor. To prove this, we tested the
prodrug with hMMP8, another protease that is inhibited by 1
(see Figure S7 in the Supporting Information). Unlike
hMMP12, hMMP8 fails to significantly cleave the prodrug
due to the specificity of the peptidic sequence toward
hMMP12; thus, inhibitor 1 is only sparsely released as
compared to hMMP12. Instead, the prodrug mainly degrades
over time to compound 7, which has no effect on the enzymatic
activity of MMPs (Figures 1d and 2d). To support the
importance of the peptidic sequence as supplier of drug
Table 1. Compounds Containing the Arylsulfonamide
Scaffold Tested as Substrates of hMMP12 and Half Lifetimes
a
in TCN Buffer pH 7.5
t50 pH 7.5
compd
R1
R2
R3
(h)
3
Z-Pro-Leu(CO)− −HN-Leu-Glu-Glu-
−Ph
5.1 0.5
Ala(NH2)
4
Z-Pro-Leu(CO)− −HN-Leu-Glu-Glu-
−OMe 28
4
Ala(NH2)
5
6
7
Z-Pro-Leu(CO)− −OH
Z-Pro-Leu(CO)− −OH
−Ph
−OMe 77
−Ph
27 0.9
9
−H
−HN-Leu-Glu-Glu-
NH
Ala(NH2)
8
9
−H
−HN-Leu-Glu-Glu-
−OMe NH
−OMe 27
Ala(NH2)
Z-Pro-Leu-
Gly(CO)−
−OH
1
10
Z-Pro-Gly(CO)− −HN-Leu-Glu-Glu-
−OMe 6.4 0.6
Ala(NH2)
a
NH, no hydrolysis.
observed in fetal calf serum (FCS) and heat inactivated FCS,
suggesting a lack of enzymatic activity in these media that could
accelerate hydrolysis or promote undesirable degradation
pathways (see Figure S2 in the Supporting Information). The
hydrolysis of N-acylated sulfonamides leading to secondary
sulfonamides has been observed in vivo.22 On the other hand,
the lability of prodrug 3 indicates that the present TAP design
leads to byproduct that might affect the target enzyme.
Therefore, we investigated the inhibitory potency of all
molecules generated upon incubation of compound 3 with
and without hMMP12. Inhibition constants were determined
using the MMP12 FRET reporter LaRee5, previously
developed in our lab23 (see Figure S3 in the Supporting
Information). Compound 1 was by far the most potent
inhibitor with an IC50 = 0.29 μM. Compound 5, which also has
a free carboxylic acid group, inhibited hMMP12 with an IC50
=
13 μM. Byproduct 7 was only able to inhibit hMMP12 with a
655
dx.doi.org/10.1021/ml3001193 | ACS Med. Chem. Lett. 2012, 3, 653−657

ACS Medicinal Chemistry Letters
Letter
Figure 2. Activity-based assay showing the selective inhibition of hMMP12 by TAP. (a) Prodrug 3 (10 μM) was preincubated with hMMP12 (12
nM) in TCN buffer at 37 °C for different times. After that, the activity of the enzyme was measured with the fluorogenic substrate LaRee5. With
longer preincubation times, the enzyme was significantly inhibited due to the presence of compound 1. (b) Compound 5 (10 μM) was preincubated
in buffer without hMMP12. After that, we added hMMP12 (12 nM) and measured its activity. Strong inhibition occurred after long preincubation
times due to the spontaneous conversion of 5 into 1. (c) Incubation of prodrug 3 in the absence of hMMP12 did not generate any inhibitory
molecule over time; thus, hMMP12 activity remained high. (d) When prodrug 3 was incubated with hMMP8 (20 nM), the enzyme activity also
remained high over time, as measured with a commercial fluorogenic substrate, since compound 1, which also inhibits hMMP8, is not significantly
formed.
specificity, we synthesized a modified substrate (10) where
leucine at the P1 site of 4 was substituted by glycine. hMMP12
failed to cleave this substrate and release the inhibitor (see
Figure S8 in the Supporting Information).
effects. Especially for the cancer antitarget MMP12 and its
dramatic role in lung emphysema formation, the TAP concept
may lead to successful MMP inhibitors in the future.
In conclusion, we have described a novel concept to inhibit
proteolytic activity using a prodrug approach that relies on the
local catalytic activity of the target enzyme itself. Only in the
presence of the target enzyme, in this case hMMP12, the
prodrug gets activated and releases its inhibitor (Scheme 1).
This mechanism of drug release has the following advantages
over the direct application of an inhibitor or other known
prodrug concepts: (i) Only the active target protease is able to
promote the release of the inhibitor. (ii) An unspecific inhibitor
(here 1) is employed to produce a specific inhibitory effect. (iii)
For a given reaction and inhibitor, the release of the drug will
be proportional to the concentration of enzyme and substrate
in the system. If there is little enzyme to be inhibited, only a
small portion of the prodrug will be converted. (iv) The
window between enzymatic activation and inhibitor release is
tunable by hydrolytic stability rather than drug−enzyme
interaction. (v) The release of the drug will occur locally at
the sites of enzymatic hydrolysis and a gradient of inhibitor
concentration will be generated locally only where (damaging)
proteolysis occurs. It needs to be stressed that so far only the
release of the predrug is local and that the lack of its
spontaneous hydrolysis still hampers spatially restricted activity.
One solution would be much faster hydrolysis. Alternatively,
the flexibility of the TAP design would allow the insertion of
chemical moieties, that is, lipidation, to modulate its residence
time. If applied in vivo, the combined advantages would likely
avoid that off-targets are hit in areas unaffected by the
inflammation. This might largely reduce side effects and
opens the opportunity to use known potent inhibitors with
already studied pharmacological properties. Of course,
pharmacokinetic aspects of the prodrugs need to be considered
to go beyond the proof of concept presented in this study.
Expanding the TAP concept to other proteases by using
different small-molecule inhibitors and other peptidic sequen-
ces will open new doors to personalized medicine, where the
individual equipment of enzyme activities will take care of
releasing as much inhibitor as needed. This will avoid excessive
and unspecific inhibition and, thus, the main cause of side
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplementary figures, experimental protocols for synthesis,
and biochemical assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Schultz@embl.de.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
The work was financially supported by the EU 7FP
(LIVIMODE) and Transregio83 of the DFG.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Turk, B. Targeting proteases: Successes, failures and future
prospects. Nat. Rev. Drug Discovery 2006, 5 (9), 785−799.
(2) Belaaouaj, A.; Shipley, J. M.; Kobayashi, D. K.; Zimonjic, D. B.;
Popescu, N.; Silverman, G. A.; Shapiro, S. D. Human macrophage
metalloelastase. Genomic organization, chromosomal location, gene
linkage, and tissue-specific expression. J. Biol. Chem. 1995, 270 (24),
14568−14575.
(3) Lagente, V. L.; Le Quement, C.; Boichot, E. Macrophage
metalloelastase (MMP-12) as a target for inflammatory respiratory
diseases. Expert Opin. Ther. Targets 2009, 13 (3), 287−295.
(4) Uttamchandani, M. W.; Wang, J.; Li, J.; Hu, M.; Sun, H.; Chen,
K. Y.; Liu, K.; Yao, S. Q. Inhibitor fingerprinting of matrix
metalloproteases using a combinatorial peptide hydroxamate library.
J. Am. Chem. Soc. 2007, 129 (25), 7848−7858.
(5) Hu, J.; Van den Steen, P. E.; Sang, Q. X.; Opdenakker, G. Matrix
metalloproteinase inhibitors as therapy for inflammatory and vascular
diseases. Nat. Rev. Drug Discovery 2007, 6 (6), 480−498.
656
dx.doi.org/10.1021/ml3001193 | ACS Med. Chem. Lett. 2012, 3, 653−657

ACS Medicinal Chemistry Letters
Letter
(6) Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Matrix
metalloproteinase inhibitors and cancer: Trials and tribulations. Science
2002, 295 (5564), 2387−2392.
(7) Tu, G.; Xu, W.; Huang, H.; Li, S. Progress in the development of
matrix metalloproteinase inhibitors. Curr. Med. Chem. 2008, 15 (14),
1388−1395.
(8) Skiles, J. W.; Gonnella, N. C.; Jeng, A. Y. The design, structure,
and therapeutic application of matrix metalloproteinase inhibitors.
Curr. Med. Chem. 2001, 8 (4), 425−474.
(9) Li, X.; Wu, J. F. Recent developments in patent anti-cancer agents
targeting the matrix metalloproteinases (MMPs). Recent Pat.
Anticancer Drug Discovery 2010, 5 (2), 109−141.
(10) Overall, C. M.; Kleifeld, O. Towards third generation matrix
metalloproteinase inhibitors for cancer therapy. Br. J. Cancer 2006, 94
(7), 941−946.
(11) Kline, T.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.;
Senter, P. D. Novel antitumor prodrugs designed for activation by
matrix metalloproteinases-2 and -9. Mol. Pharmaceutics 2004, 1 (1), 9−
22.
(12) Milo, L. J. J.; Lai, J. H.; Wu, W.; Liu, Y.; Maw, H.; Li, Y.; Jin, Z.;
Shu, Y.; Poplawski, S. E.; Wu, Y.; Sanford, D. G.; Sudmeier, J. L.;
Bachovchin, W. W. Chemical and biological evaluation of dipeptidyl
boronic acid proteasome inhibitors for use in prodrugs and pro-soft
drugs targeting solid tumors. J. Med. Chem. 2011, 54 (13), 4365−4377.
(13) Pickett, S. D.; Green, D. V. S.; Hunt, D. L.; Pardoe, D. A.;
Hughes, I. Automated Lead Optimization of MMP-12 Inhibitors Using
a Genetic Algorithm. ACS Med. Chem. Lett. 2011, 2 (1), 28−33.
(14) Rouffet, M.; Denhez, C.; Bourguet, E.; Bohr, F.; Guillaume, D.
In silico study of MMP inhibition. Org. Biomol. Chem. 2009, 7, 3817−
3825.
(15) Dragoni, E.; Calderone, V.; Fragai, M.; Jaiswal, R.; Luchinat, C.;
Nativi, C. Biotin-Tagged Probes for MMP Expression and Activation:
Design, Synthesis, and Binding Properties. Bioconjugate Chem. 2009,
20 (4), 719−727.
(16) Agrawal, A.; Romero-Perez, D.; Jacobsen, J. A.; Villarreal, F. J.;
Cohen, S. M. Zinc-binding groups modulate selective inhibition of
MMPs. ChemMedChem 2008, 3 (5), 812−20.
(17) Summers, J. B.; Gunn, B. P.; Mazdiyasni, H.; Goetze, A. M.;
Young, P. R.; Bouska, J. B.; Dyer, R. D.; Brooks, D. W.; Carter, G. W.
In vivo characterization of hydroxamic acid inhibitors of 5-
lipoxygenase. J. Med. Chem. 1987, 30 (11), 2121−2126.
(18) Puerta, D. T.; Cohen, S. M. A bioinorganic perspective on
matrix metalloproteinase inhibition. Curr. Top. Med. Chem. 2004, 4
(15), 1551−1573.
(19) Devel, L.; Rogakos, V.; David, A.; Makaritis, A.; Beau, F.;
Cuniasse, P.; Yiotakis, A.; Dive, V. Development of selective inhibitors
and substrate of matrix metalloproteinase-12. J. Biol. Chem. 2006, 281
(16), 11152−11160.
(20) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. Design
and therapeutic application of matrix metalloproteinase inhibitors.
Chem. Rev. 1999, 99 (9), 2735−2776.
(21) Verma, R. P.; Hansch, C. Matrix metalloproteinases (MMPs):
Chemical-biological functions and (Q)SARs. Bioorg. Med. Chem. 2007,
15 (6), 2223−2268.
(22) Vaccarino, A. L.; Paul, D.; Mukherjee, P. K.; Rodríguez de
Turco, E. B.; Marcheselli, V. L.; Xu, L.; Trudell, M. L.; Minguez, J. M.;
Matía, M. P.; Sunkel, C.; Alvarez-Builla, J.; Bazan, N. G. Synthesis and
in vivo evaluation of non-hepatotoxic acetaminophen analogs. Bioorg.
Med. Chem. 2007, 15 (5), 2206.
(23) Cobos-Correa, A.; Trojanek, J. B.; Diemer, S.; Mall, M. A.;
Schultz, C. Membrane-bound FRET probe visualizes MMP12 activity
in pulmonary inflammation. Nat. Chem. Biol. 2009, 5 (9), 628−630.
(24) Larsen, J. D.; Bundgaard, H.; Lee, V. H. L. Prodrug forms for the
sulfonamide group. II. Water-soluble amino acid derivatives of N-
methylsulfonamides as possible prodrugs. Int. J. Pharm. 1988, 41 (1−
3), 103−110.
657
dx.doi.org/10.1021/ml3001193 | ACS Med. Chem. Lett. 2012, 3, 653−657