3-hydroxy-1-alkyl-2-methylpyridine-4(1 H)-thiones: Inhibition of the pseudomonas aeruginosa virulence factor LasB

Article abstract of DOI:10.1021/ml300128f

Bacterial resistance coupled to our current arsenal of antibiotics presents us with a growing threat to public health, thus warranting the exploration of alternative antibacterial strategies. In particular, the targeting of virulence factors has been regarded as a second generation antibiotic approach. In Pseudomonas aeruginosa, a Zn²⁺ metalloprotease virulence factor, LasB or P. aeruginosa elastase, has been implicated in the development of P. aeruginosa-related keratitis, pneumonia, and burn infection. Moreover, the enzyme also plays a critical role in swarming and biofilm formation, both of which are processes that have been linked to antibiotic resistance. To further validate the importance of LasB in P. aeruginosa infection, we describe our efforts toward the discovery of nonpeptidic small molecule inhibitors of LasB. Using identified compounds, we have confirmed the role that LasB plays in P. aeruginosa swarming and demonstrate the potential for LasB-targeted small molecules in studying antimicrobial-resistant P. aeruginosa phenotypes.

Full text of DOI:10.1021/ml300128f

Letter
pubs.acs.org/acsmedchemlett
3-Hydroxy-1-alkyl-2-methylpyridine-4(1H)-thiones: Inhibition of the
Pseudomonas aeruginosa Virulence Factor LasB
Amanda L. Garner,† Anjali K. Struss,† Jessica L. Fullagar,‡ Arpita Agrawal,‡ Amira Y. Moreno,†
Seth M. Cohen,*,‡ and Kim D. Janda*,†
†Departments of Chemistry and Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, The Worm Institute
for Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, United States
‡Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California, United
States
S
* Supporting Information
ABSTRACT: Bacterial resistance coupled to our current
arsenal of antibiotics presents us with a growing threat to
public health, thus warranting the exploration of alternative
antibacterial strategies. In particular, the targeting of virulence
factors has been regarded as a “second generation” antibiotic
approach. In Pseudomonas aeruginosa, a Zn2+ metalloprotease
virulence factor, LasB or P. aeruginosa elastase, has been
implicated in the development of P. aeruginosa-related keratitis,
pneumonia, and burn infection. Moreover, the enzyme also
plays a critical role in swarming and biofilm formation, both of which are processes that have been linked to antibiotic resistance.
To further validate the importance of LasB in P. aeruginosa infection, we describe our efforts toward the discovery of nonpeptidic
small molecule inhibitors of LasB. Using identified compounds, we have confirmed the role that LasB plays in P. aeruginosa
swarming and demonstrate the potential for LasB-targeted small molecules in studying antimicrobial-resistant P. aeruginosa
phenotypes.
KEYWORDS: antibiotics, metalloprotease inhibitors, virulence factors, hydroxypyridinthiones
he emergence of antibiotic resistance to nearly all
antimicrobial agents on the market today is a great threat
number of surface and secreted virulence factors that facilitate
bacterial attachment, colonization and invasion, and tissue
damage and cytokine production, respectively.4,5 Cell-associ-
ated virulence factors include flagella, pili, lectins, alginate, and
lipopolysaccharide, while extracellular virulence factors include
proteases, hemolysins, cytotoxin, pyocyanin, siderophores, and
exotoxin A.5 This arsenal of virulence factors ensures that P.
aeruginosa infections are both invasive and toxigenic.5 Thus,
because of growing antibiotic resistance in P. aeruginosa
including the identification of many multidrug resistant strains,4
new approaches for tackling such infections are warranted.
One P. aeruginosa virulence factor that has been shown to be
highly toxic to the host6 is a Zn2+ metalloprotease, LasB or P.
aeruginosa elastase,7 which is secreted by the bacteria in a
quorum sensing-dependent fashion.8 In particular, LasB has
been demonstrated to cause tissue damage and persistent
inflammation, degrade plasma proteins, and promote invasion
and colonization among other pathologies.6 To do so, the
enzyme degrades a wide variety of substrates including elastin,
fibrin, immunoglobulins, complement factors, and cytokines.6
Importantly, infection with a P. aeruginosa strain producing a
T
to public health, requiring the development of alternative
antibacterial strategies. Of these tactics, the targeting of
virulence factors has been regarded as a “second generation”
antibiotic approach.1−3 Pathogenic bacteria produce virulence
factors such as adhesion molecules, secretion systems, and
other toxic factors, including proteases to enhance their ability
to cause disease and damage the host's tissues.2,3 Importantly,
because virulence factors do not alter viability, agents targeting
such factors should impose weaker selective pressure for the
development of resistance.2,3 Moreover, as many virulence
factors are unique to a specific organism, virulence-targeting
drugs are unlikely to impact the host's commensal flora.2,3 The
efficacy of exploring such targets remains to be seen; however,
inhibitors targeting toxin function and delivery, the regulation
of virulence expression, and adhesion have been reported with
promising in vivo anti-infective properties.2,3
Pseudomonas aeruginosa is a nosocomial, opportunistic Gram-
negative bacteria that is the predominant cause of pneumonia
in ventilated patients and lung infection in patients with cystic
fibrosis.4 In addition to respiratory tract infections, the
pathogen also causes blood, ear, skin, eye, urinary, and
gastrointestinal tract infections among others and is responsible
for 10−15% of hospital-acquired infections worldwide.4,5 To
promote these clinical manifestations, P. aeruginosa produces a
Received: May 25, 2012
Accepted: June 19, 2012
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
mutant, inactive elastase was found to be less virulent than the
parent strain in a chronic lung infection model in rats.9
Additionally, with respect to the bacteria itself, LasB has been
linked to biofilm formation10 and swarming,11 further aiding in
its elusion from the host's immune system. Accordingly, LasB
has been shown to play a role in the development of P.
aeruginosa-related keratitis, pneumonia, and burn infection.6
Our laboratories have invested much effort in exploring the
modulation of metalloproteases for human health, such as
identifying small molecule inhibitors of the botulinum neuro-
toxins,12−18 anthrax lethal factor,19−22 and matrix metal-
loproteinases.21,22 To do so, we have designed and developed
nonpeptidic small molecules containing a variety of metal-
chelating warheads as active site inhibitors. Because only
peptide-based LasB inhibitors have been previously re-
ported23−26 coupled with the potential therapeutic significance
of this P. aeruginosa virulence factor, we were interested in
expanding the scope of our metalloprotease-targeting design
strategies toward the goal of identifying potent small molecule
LasB antagonists. Herein, we describe our efforts to discover
the first nonpeptidic small molecule inhibitors of LasB and
validate the role of this virulence factor in swarming, a
multicellular bacterial behavior recently linked to antimicrobial
resistant phenotypes.27
To initiate our screening efforts, we examined a 323-member
small molecule hydroxamic acid library, which was constructed
using solid-phase organic synthesis and originally used to
identify inhibitors of the botulinum neurotoxin A protease.14
All compounds were analyzed for LasB inhibition using a
previously reported fluorescence assay based on a LasB-
cleavable fluorescence resonance energy transfer (FRET)
peptide substrate (see the Supporting Information for assay
details).23 Library members were initially examined at 50 μM,
and from our screening efforts, eight hits showing 100%
inhibition at this concentration were identified following
triplicate experiments (see the Supporting Information). Of
these hit compounds, only two demonstrated dose-dependent
LasB antagonism, hydroxamic acids 1 and 2 (Figure 1), with
Members of this fragment library were screened at 1 mM for
inhibition of LasB, and 11 fragment hits were identified (see the
Supporting Information). Of these compounds, four showed
dose-dependent inhibition of LasB with IC50 values ranging
from 80 to 400 μM (compounds 3−6, Figure 2). Interestingly,
all hits contained an O,S atom donor set and have previously
been identified from this library as inhibitors of other Zn2+
metalloproteases.19−22
Figure 2. Hit compounds from CFL-1.1 with IC50 values for LasB
inhibition.
To identify 3,4-HOPTO [3-hydroxy-1-alkyl-2-methylpyri-
dine-4(1H)-thione; 3−5] derivatives with improved in vitro
potency against LasB, a sublibrary of compounds based on
fragments 3 and 4 (see the Supporting Information for
structures), was screened.21 3,4-HOPTO analogues 8 were
constructed via a one-step condensation of a primary amine
with thiomaltol 7 (Scheme 1)21 and initially tested for
Scheme 1. Synthesis of 3,4-HOPTO Analogues
inhibition at 100 μM. From this sublibrary, two compounds
were discovered with ∼30-fold improvement in potency:
analogues 8a and 8b exhibited measured IC50 values of 3.34
and 2.73 μM, respectively (Table 1; see the Supporting
Information for the activity of other sublibrary members).
Structure−activity relationship studies were then conducted on
8b by varying the substituents of the biphenyl ring; however, no
compounds with enhanced LasB inhibition were identified
(Table 1). On the basis of initial docking studies, we
hypothesize that the diminished activity of compounds 8c−g
may be due to the restrictive geometry of the 3,4-HOPTO−
Zn2+ complex, which forces the biphenyl side chain into solvent
space as the active site of LasB is quite open similar to that of
thermolysin (data not shown).23
With the in vitro activity of 3,4-HOPTO analogues 8a and
8b confirmed, we were then anxious to examine their activity in
a bacterial phenotypic assay, particularly since hydroxamates 1
and 2 displayed no secreted LasB inhibition. In P. aeruginosa,
LasB has been demonstrated to be required for both swarming
and biofilm formation, and lasB mutants were shown to exhibit
strong swarming defects and a ∼50% decrease in biofilm
formation.11 Swarming and biofilm formation are both social
phenomena that require differentiated cells and are regulated by
cell-to-cell communication, in particular, quorum sensing.27 Of
significance, both bacterial cell states have been found to be
highly resistant to antibiotics, and it has recently been
hypothesized that antimicrobial resistance is a general feature
Figure 1. Hydroxamic acid LasB inhibitor hits with IC50 values.
measured IC50 values of 13.6 and 16.4 μM, respectively. Despite
the in vitro activity of these compounds, unfortunately, no
antagonism in bacterial LasB assays was observed. One possible
explanation for the lack of bacterial cell activity is enzymatic
discharging of the hydroxamate moiety, as this chelating motif
is known to be unstable.28
Because of the potential instability and off-target effects of
the hydroxamate group,28 we were eager to explore additional
Zn2+ chelating motifs that may be more stable under biological
conditions. Recently, a library of metal chelating fragments
(CFL-1.1) was designed by the Cohen laboratory.21,22 The
library consists of 96 fragment chelators, each containing 2−4
donor atoms for metal binding. Some representative library
members include picolinic acids, hydroxyquinolones, pyrimi-
dines, and hydroxypyrones in addition to other well-known
metal-binding units such as sulfonamides and hydroxamic acids.
B
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ACS Medicinal Chemistry Letters
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DMSO-treated control sample that exhibited a dendritic
swarming phenotype typical of P. aeruginosa. We highlight
this result as 8a and 8b are the first targeted compounds to
show swarming antagonism. Previously, only general virulence
factor expression29,30 and bacterial adhesion inhibitors31 have
been examined in swarming assays. Similar to biofilm
formation, swarming is also of significance pathologically, as
the environment required for swarming motility is similar to
that covering epithelial surfaces and the hyperabundant mucous
found the in lungs of cystic fibrosis patients.11 Thus, we are
hopeful that these compounds will be useful in further
understanding the role that LasB plays not only in swarming
but also the development of antibiotic resistance in P.
aeruginosa swarm cells and invasion and colonization of tissues
by P. aeruginosa.6 Moreover, upon improvement in potency, we
are also interested in exploring the applicability of our
nonpeptidic small molecule LasB inhibitors in animal models
of P. aeruginosa infection to study the impact of this
metalloprotease in promoting bacterial virulence. Importantly,
3,4-HOPTO derivatives have been previously demonstrated to
be nearly nontoxic at concentrations up to 100 μM in
mammalian cells.32,33 We envision that a combination of
small molecule LasB inhibitors with traditional antibiotics may
represent a new therapy for targeting multidrug resistant P.
aeruginosa.
Table 1. Structures and IC50 Values for 3,4-HOPTO
Analogue Hits
Lastly, we wish to bring to light that although hydroxamic
acids 1 and 2 were only ∼5-fold less potent than 3,4-HOPTO
analogues 8a and 8b in the in vitro LasB assay, these
compounds showed no swarming inhibition (data not
shown). As indicated, it is quite possible that instability and
off-target effects may be at play as highlighted by others;28
however, it remains to be seen if other factors are involved such
as bacterial cell metabolism. Initial stability studies in bacterial
culture medium indicate the hydrolytic instability of 1 and 2 in
comparison to 8a and 8b, which showed no detectable S-
oxidation or decomposition over a 48 h period (see the
Supporting Information). Regardless, compounds 1 and 2 will
serve as useful scaffolds for future warhead “hopping”
experiments, and these studies will be reported in due course.
In conclusion, we have identified small molecule, Zn2+-
chelating inhibitors of LasB with antiswarming activity. These
inhibitors were identified via initial screening of a chelator
fragment library, further validating this approach for the
identification of metalloprotein inhibitors. Although com-
pounds 8a and 8b are not the most potent LasB inhibitors to
date, they are the first nonpeptidic small molecule antagonists
discovered.26 Moreover, they are the first targeted compounds
to exhibit swarming inhibition. On the basis of our preliminary
findings described here, LasB-targeted chemical probes should
aid in elucidating the role of LasB in P. aeruginosa-related
infections and its ability to resist antibiotics. Additionally, the
discovery and study of such small molecule virulence factor
inhibitors will provide additional evidence to discern the impact
of targeting bacterial virulence factors as a “second generation”
antibiotic approach.
of bacterial multicellularity.27 As the study of swarming motility
is believed to be a useful model to investigate antibiotic
resistance in biofilms,27 particularly due to reproducibility issues
that often go unreported in biofilm assays, we chose to
investigate swarming inhibition in the presence of compounds
8a and 8b.
To examine the effect of compounds 8a and 8b on swarming,
P. aeruginosa strain PA14 was grown on swarm agar plates
containing 25 μM 8a or 8b (∼95% viability at this
concentration; see the Supporting Information) for 16 h at
30 °C. As Figure 3 shows, both 8a and 8b almost completely
abolished swarming at this concentration in comparison to a
ASSOCIATED CONTENT
* Supporting Information
■
S
Representative synthetic procedure, characterization of com-
pounds 8a and 8c−8g, LasB fluorescence assay protocol,
supplemental screening results, stability and bacterial toxicity of
8a and 8b, and swarming details. This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 3. Swarming of P. aeruginosa strain PA14 in the presence of 25
μM (a) DMSO (control), (b) 8a (left), or 8b (right).
C
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ACS Medicinal Chemistry Letters
Letter
(12) Boldt, G. E.; Eubanks, L. M.; Janda, K. D. Identification of a
botulinum neurotoxin A protease inhibitor displaying efficacy in a
cellular model. Chem. Commun. 2006, 3063−3065.
(13) Boldt, G. E.; Kennedy, J. P.; Hixon, M. S.; McAllister, L. A.;
Barbieri, J. T.; Tzipori, S.; Janda, K. D. Synthesis, characterization and
development of a high-throughput methodology for the discovery of
botulinum neurotoxin A inhibitors. J. Comb. Chem. 2006, 8, 513−521.
(14) Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Identification of a
potent botulinum neurotoxin A protease inhibitor using in situ lead
identification chemistry. Org. Lett. 2006, 8, 1729−1732.
(15) Capkova, K.; Yoneda, Y.; Dickerson, T. J.; Janda, K. D. Synthesis
and structure-activity relationships of second-generation hydroxamate
botulinum neurotoxin A protease inhibitors. Bioorg. Med. Chem. Lett.
2007, 17, 6463−6466.
(16) Capkova, K.; Salzameda, N. T.; Janda, K. D. Investigations into
small molecule non-peptidic inhibitors of the botulinum neurotoxins.
Toxicon 2009, 54, 575−582.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 858-822-5596. E-mail: scohen@ucsd.edu (S.M.C.). Tel:
858-784-2515. Fax: 858-784-2595. E-mail: kdjanda@scripps.
edu (K.D.J.).
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was funded by the NIH (Grants R01 AI077644 to
K.D.J. and R01 GM098435 to S.M.C.).
Notes
The authors declare no competing financial interest.
(17) Capkova, K.; Hixon, M. S.; Pellett, S.; Barbieri, J. T.; Johnson, E.
A.; Janda, K. D. Benzylidene cyclopentenediones: First irreversible
inhibitors against botulinum neurotoxin A's zinc endopeptidase. Bioorg.
Med. Chem. Lett. 2010, 20, 206−208.
(18) Stowe, G. N.; Silhar, P.; Hixon, M. S.; Silvaggi, N. R.; Allen, K.
N.; Moe, S. T.; Jacobson, A. R.; Barbieri, J. T.; Janda, K. D. Chirality
holds the key for potent inhibition of the botulinum neurotoxin
serotype A protease. Org. Lett. 2010, 12, 756−759.
(19) Lewis, J. A.; Mongan, J.; McCammon, J. A.; Cohen, S. M.
Evaluation and binding-mode prediction of thiopyrone-based inhib-
itors of anthrax lethal factor. ChemMedChem 2006, 1, 694−697.
(20) Agrawal, A.; de Oliveira, C. A. F.; Cheng, Y.; Jacobsen, J. A.;
McCammon, J. A.; Cohen, S. M. Thioamide hydroxypyrothiones
supersede amide hydroxypyrothiones in potency against anthrax lethal
factor. J. Med. Chem. 2009, 52, 1063−1074.
(21) Agrawal, A.; Johnson, S. L.; Jacobsen, J. A.; Miller, M. T.; Chen,
L.; Pellecchia, M.; Cohen, S. M. Chelator fragment libraries for
targeting metalloproteinases. ChemMedChem 2010, 5, 195−199.
(22) Jacobsen, J. A.; Fullager, J. L.; Miller, M. T.; Cohen, S. M.
Identifying chelators for metalloprotein inhibitors using a fragment-
based approach. J. Med. Chem. 2011, 54, 590−602.
(23) Nishino, N.; Powers, J. C. Pseudomonas aeruginosa Elastase:
Development of a new substrate, inhibitors, and an affinity ligand. J.
Biol. Chem. 1980, 255, 3482−3486.
(24) Kessler, E.; Israel, M.; Landshman, N.; Chechick, A.; Blumberg,
S. In vitro inhibition of Pseudomonas aeruginosa elastase by metal-
chelating peptide derivatives. Infect. Immun. 1982, 38, 716−723.
(25) Cathcart, G. R.; Gilmore, B. F.; Greer, B.; Harriott, P.; Walker,
B. Inhibitor profiling of the Pseudomonas aeruginosa virulence factor
LasB using N-alpha mercaptoamide template-based inhibitors. Bioorg.
Med. Chem. Lett. 2009, 19, 6230−6232.
ACKNOWLEDGMENTS
■
We thank Greg McElhaney for the preparation of compounds
8a and 8e−g.
ABBREVIATIONS
■
P. aeruginosa, Pseudomonas aeruginosa; LasB, P. aeruginosa
elastase; FRET, fluorescence resonance energy transfer; 3,4-
HOPTO, 3-hydroxy-1-alkyl-2-methylpyridine-4(1H)-thione
REFERENCES
■
(1) Travis, J.; Potempa, J. Bacterial proteinases as targets for the
development of second-generation antibiotics. Biochim. Biophys. Acta
2000, 1477, 35−50.
(2) Clatworthy, A. E.; Pierson, E.; Hung, D. T. Targeting virulence: A
new paradigm for antimicrobial therapy. Nat. Chem. Biol. 2007, 3,
541−548.
(3) Barczak, A. K.; Hung, D. T. Productive steps toward an
antimicrobial targeting virulence. Curr. Opin. Microbiol. 2009, 12, 490−
496.
(4) Mesaros, N.; Nordmann, P.; Plesiat, P.; Roussel-Delvallez, M.;
Van Eldere, J.; Glupczynski, Y.; Van Laethem, Y.; Lebecque, P.;
Malfroot, A.; Tulkens, P. M.; Van Bambeke, F. Pseudomonas
aeruginosa: Resistance and therapeutic options at the turn of the
millennium. Clin. Microbiol. Infect. 2007, 13, 560−578.
(5) Strateva, T.; Mitov, I. Contribution of an arsenal of virulence
factors to pathogenesis of Pseudomonas aeruginosa infections. Ann.
Microbiol. 2011, 61, 717−732.
(6) Wretlind, B.; Pavlovskis, O. R. Pseudomonas aeruginosa elastase
and its role in pseudomonas infections. Rev. Infect. Dis. 1983, 5, S998−
S1004.
(7) Morihara, K.; Tsuzuki, H.; Oka, T.; Inoue, H.; Ebata, M.
Pseudomonas aeruginosa Elastase: Isolation, crystallization, and
preliminary characterization. J. Biol. Chem. 1965, 240, 3295−3304.
(8) Pearson, J. P.; Gray, K. M.; Passador, L.; Tucker, K. D.; Eberhard,
A.; Iglewski, B. H.; Greenberg, E. P. Structure of the autoinducer
required for expression of Pseudomonas aeruginosa virulence genes.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 197−201.
(9) Woods, D. E.; Cryz, S. J.; Friedman, R. L.; Iglewski, B. H.
Contribution of toxin A and elastase to virulence of Pseudomonas
aeruginosa in chronic lung infections in rats. Infect. Immun. 1982, 36,
1223−1228.
(10) Kamath, S.; Kapatral, V.; Chakrabarty, A. M. Cellular function of
elastase in Pseudomonas aeruginosa: Role in the cleavage of nucleotide
diphosphate kinase and in alginate synthesis. Mol. Microbiol. 1998, 30,
933−941.
(11) Overhage, J.; Bains, M.; Brazas, M. D.; Hancock, R. E. W.
Swarming of Pseudomonas aeruginosa is a complex adaptation leading
to increased production of virulence factors and antibiotic resistance. J.
Bacteriol. 2008, 190, 2671−2679.
(26) Cathcart, G. R.; Quinn, D.; Greer, B.; Harriott, P.; Lynas, J. F.;
Gilmore, B. F.; Walker, B. Novel inhibitors of the Pseudomonas
aeruginosa virulence factor LasB: A potential therapeutic approach for
the attenuation of virulence mechanisms in pseudomonal infection.
Antimicrob. Agents Chemother. 2011, 55, 2670−2678.
(27) Lai, S.; Tremblay, J.; Deziel, E. Swarming motility: A
multicellular behavior conferring antimicrobial resistance. Environ.
Microbiol. 2009, 11, 126−136.
(28) Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez, B.;
Deprez-Poulain, R. Hydroxamates: relationships between structure and
plasma stability. J. Med. Chem. 2009, 52, 6790−6802.
(29) Lee, J.; Attila, C.; Cirillo, S. L. G.; Cirillo, J. D.; Wood, T. K.
Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa viru-
lence. Microb. Biotechnol. 2009, 2, 75−90.
(30) Wu, H.; Lee, B.; Yang, L.; Wang, H.; Givskov, M.; Molin, S.;
Hoiby, N.; Song, Z. Effects of ginseng on Pseudomonas aeruginosa
motility and biofilm formation. FEMS Immunol. Med. Microbiol. 2011,
62, 49−56.
(31) O'May, C.; Tufenkji, N. The swarming motility of Pseudomonas
aeruginosa is blocked by cranberry proanthocyanidins and other
D
dx.doi.org/10.1021/ml300128f | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
tannin-containing materials. Appl. Environ. Microbiol. 2011, 77, 3061−
3067.
(32) Puerta, D. T.; Griffin, M. O.; Lewis, J. A.; Romero-Perez, D.;
Garcia, R.; Villarreal, F. J.; Cohen, S. M. Heterocyclic zinc-binding
groups for use in next-generation matrix metalloproteinase inhibitors:
Potency, toxicity, and reactivity. J. Biol. Inorg. Chem. 2006, 11, 131−
138.
(33) Jacobsen, F. E.; Buczynski, M. W.; Dennis, E. A.; Cohen, S. M. A
macrophage cell model for selective metalloproteinase inhibitor design.
ChemBioChem 2008, 9, 2087−2095.
E
dx.doi.org/10.1021/ml300128f | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX