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Targeting metalloenzymes: a strategy that works


Richard J White, Peter S Margolis, Joaquim Trias and Zhengyu Yuan

Faced with a wealth of antibacterial drug discovery targets as a In addition to being essential for bacterial growth, it
result of bacterial genomics, we need to carefully select which should be present in a range of relevant pathogens
ones to work with. Choosing bacterial metalloenzymes is one (potential spectrum) and ideally should have no mamma-
possible approach that can increase the probability of success. lian equivalent (basis for selective toxicity) [1]. The
Metalloenzymes can be identified through specific motif recent advent of microbial genomics promised to deliver
searches of bacterial genomes. Current state-of-the-art a large number of previously unexploited targets that
medicinal chemistry allows for the design of inhibitor libraries could form the basis for discovering new classes of anti-
targeting metalloenzymes and the efficient optimization of leads biotics [2]. Indeed, analysis of a typical bacterial genome
identified. This approach has been successfully applied to the indicates that there are 200–300 potential broad-spectrum
discovery of in vivo active antibacterial agents that are inhibitors targets for antibiotic discovery. But faced with this
of bacterial peptide deformylase and UDP-3-O-(R-3- ‘embarrass de richesses’, it is difficult to decide which
hydroxymyristoyl)-N-acetylglucosamine deacetylase. Other one(s) to work with. Historically, antibiotics were dis-
bacterial metalloenzymes are open to the same approach. covered empirically, so the target was not a consideration.
Only recently have chemists, seeking a more rational path
Addresses to drug discovery, attempted a purely synthetic approach
Vicuron Pharmaceuticals, 34790 Ardentech Court, Fremont, using mechanism- and structure-based drug design. This
CA 94555, USA review describes one such approach that focuses on
Correspondence: Z Yuan; e-mail: zyuan@vicuron.com
metalloenzymes — enzymes in which active center metal
ions participate in catalysis. Enzymes in which the metal
Current Opinion in Pharmacology 2003, 3:502–507 ions play a structural role are excluded from this category.
This review comes from a themed issue on
Anti-infectives
Metalloenzymes, especially metalloproteases, are among
Edited by Steven J Projan the best studied of enzyme classes, and there are excel-
lent precedents for the mechanism-based design of their
1471-4892/$ – see front matter
inhibitors [3]. The prime example of this strategy is
ß 2003 Elsevier Ltd. All rights reserved.
provided by the discovery of captopril [4], the first
DOI 10.1016/S1471-4892(03)00115-2 angiotensin-converting enzyme (ACE) inhibitor to be
marketed. Not only did captopril provide a novel treat-
Abbreviations
ment for hypertension but it was also the first drug
ACE angiotensin-converting enzyme developed using mechanistic principles. ACE employs
DXR 1-deoxy-D-xylulose-5-phosphate reductoisomerase a metal ion in catalysis; captopril, a molecule containing
LpxC UDP-(3-O-acyl)-N-acetylglucosamine deacetylase mercaptan and peptidomimetic domains, was designed to
MAP methionine aminopeptidase selectively chelate this metal ion and inhibit ACE activ-
PDF peptide deformylase
VanX zinc-dependent D,D-dipeptidase ity. Supplementing this mechanism-based approach with
structural information on the enzyme further improves
the chances of success. Could this same design principle
Introduction be applied to bacterial targets to provide the necessary
A disturbing increase in the prevalence of antibiotic- potency and requisite specificity? By adding the need to
resistant bacteria, coupled with the recent poor success be a metalloenzyme to the previously mentioned set of
rate of the pharmaceutical industry’s antibiotic discovery criteria, one could define a subset of bacterial targets that
programs, is cause for significant concern. The oxazoli- are amenable to this ‘captopril approach’. Here, we
dinones are the only new class of antibiotic to reach the review the current process of selecting such targets and
North American market in the past 30 years. Although the progress that has been made in designing and/or
some degree of success in overcoming resistance has been identifying inhibitors of these enzymes.
achieved through modification of existing classes, there is
a real need to discover new classes with different mechan- Selecting bacterial metalloenzyme targets
isms of action. Potential metalloenzyme targets can be identified by
searching for metalloenzyme-specific sequence motifs
Typically, an antibiotic inhibits bacteria by interfering within bacterial genomes. Websites devoted to motif anal-
with an essential biological function, such as the activity ysis and annotation already exist, which typically include
of an enzyme. For a bacterial enzyme to represent a sequences of known and unknown function and provide
suitable antibiotic target, it must fulfill certain criteria. links to phylogenetic tools, literature summaries and

Current Opinion in Pharmacology 2003, 3:502–507 www.current-opinion.com


Targeting metalloenzymes: a strategy that works White et al. 503

structural information when available. The Protein Pattern the nascent protein. PDF is essential for bacteria but is
and Domain Databases site (http://www.hgmp.mrc.ac.uk/ not required in mammalian cells, making it an attractive
GenomeWeb/prot-domain.html) is a good starting point. A target for the design of new antibiotics (for a recent
subset of these putative metalloenzymes is conserved review, see [10]). Actinonin, a naturally occurring anti-
among many bacterial genomes and therefore can be biotic, was shown to inhibit bacterial growth through the
considered potential broad-spectrum targets [5]. The inhibition of PDF activity [11], thereby validating defor-
essentiality of the corresponding genes can be efficiently mylase as an antibacterial drug discovery target.
determined by several molecular genetic methods [5–7]. A
similar analysis with multiple ‘signature’ metallo-protein High-resolution three-dimensional structures of PDF are
motifs was carried out in the genome of the archetypal available. The environment around the active-site metal
gram-positive bacterium Bacillus subtilis [8]. In that work, has strong geometric and chemical similarities to that of
approximately 150 known or putative metalloenzymes proteases of the thermolysin family (for review, see [12]).
were identified; 14 genes were both conserved phylogen- Several types of PDF inhibitor have been identified,
etically and required for growth in rich or minimal media. based either on structural and mechanistic design or from
high-throughput screening [10]. Almost all PDF inhi-
Several comments on this approach are pertinent. Firstly, bitors identified share the captopril-like structural feature
a motif-based search will miss some subsets of atypical of a ‘chelator and peptidomimetic’ scaffold. Thus far, only
metalloenzymes. For example, UDP-(3-O-acyl)-N-acetyl- compounds using hydroxamic acid or N-formyl hydro-
glucosamine deacetylase (LpxC) is demonstrably a metal- xylamine as chelators exhibit appreciable antibacterial
loenzyme target for antibacterial drug discovery [9]. activity and in vivo efficacy, including oral activity
However, LpxC, which employs a non-canonical metal- [13–15]. As expected, PDF inhibitors can cure infections
binding domain, can ‘slip through the cracks’ during these caused by bacteria resistant to currently available drugs.
extended analyses. Secondly, claims of essentiality in However, resistance to PDF inhibitors has also been
previous literature are not necessarily borne out upon extensively studied [13,16,17]. The in vitro frequency
re-testing and so should be treated with caution. Finally, of mutation to resistance is low for Streptococcus pneumo-
the majority of possible bacterial metalloenzyme targets niae and Haemophilus influenzae, the two leading respira-
derived from such motif analyses are known proteins, tory infection pathogens. For Staphylococcus aureus, a
already identified by classical biochemistry and genetics. higher resistance frequency was observed, but these
This last observation suggests that most of the essential resistant mutants appear less virulent in vivo. At the
bacterial metalloenzymes may have already been identi- 41st and 42nd Interscience Conference on Antimicrobial
fied. Additional metalloenzymes with a more limited Agents and Chemotherapy (ICAAC), two deformylase
distribution (e.g. Helicobacter-specific) exist, but would inhibitors, VRC4887 (Vicuron Pharmaceuticals) and
correspond to narrower spectrum antibiotics. BB-83698 (British Biotech), were presented as advanced
clinical candidates for the treatment of community-
Identifying inhibitors of bacterial acquired respiratory tract infections [18,19].
metalloenzymes as antibacterial agents
Given the mechanistic resemblance between metalloen- UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine
zymes, the tools and knowledge developed for identifying deacetylase
inhibitors of one metalloenzyme can be easily applied to The lipopolysaccharide layer of the outer membrane of
others, which can eventually reduce the overall time and gram-negative bacteria plays a major role in pathogenesis
cost of identifying inhibitors of each member of this and antibiotic resistance. LpxC, a cytoplasmic zinc hydro-
group. Once candidate metalloenzymes have been iden- lase, catalyzes the first committed step in the biosynthesis
tified, leads can be found through the combination of of lipopolysaccharide lipid A [20]. LpxC is unique to gram-
rational design and random high-throughput screening. negative bacteria, and its gene is essential in Escherichia coli
Because the targets are metalloenzymes, only chemicals and H. influenzae [21,22]. Therefore, LpxC is particularly
with potential chelating function need be considered. In attractive for the discovery of narrow-spectrum gram-nega-
principle, a structurally diverse chelator-based chemical tive-specific antibiotics; this metalloenzyme has been the
library can be used to screen against all metalloenzymes focus of several discovery programs [9,23–26]. All of the
for potential inhibitor leads. This approach has been disclosed LpxC inhibitors contain a chelating moiety; the
employed to identify inhibitors of several bacterial metal- IC50s of the most potent compounds are in the low nano-
loenzymes, as illustrated by the following examples molar range against E. coli and Pseudomonas aeruginosa
(representative inhibitors of these metalloenzymes are enzymes. Several of the compounds are active against E.
listed in Table 1). coli and related organisms, with minimum inhibitory con-
centrations in the 1–4 mg/ml range, but no inhibition of
Peptide deformylase wild-type P. aeruginosa was observed [9,27,28]. VRC5264
Peptide deformylase (PDF), a metal-containing hydro- was reported to be efficacious in vivo against E. coli murine
lase, catalyzes removal of the N-terminal formyl group of septicemias (ED50 ¼28 mg/kg after a single intravenous

www.current-opinion.com Current Opinion in Pharmacology 2003, 3:502–507


504 Anti-infectives

Table 1

List of biological activities of representative inhibitors of bacterial metalloenzymes.


Inhibitor Target IC50 (nM) MIC (mg/ml) MIC (mg/ml)
E. coli S. aureus

HO
N PO3H2
DXR 8.2 0.39 >100
O

Fosmidomycin
Fosmidomycin [34]

HO
O O
H PDF 0.8 >100 4
HO N
N
H
O

Actinonin
Actinonin [11]

O OH
H3CO NH
N
H3CO LpxC 12 1 >100
O

L-161,240
L-161,240 [9]
NH2 O
Ala-Leu-Val-Phe-OM
N MAP 5000 – –
H
O
Bestatin-analog
Bestatin analog [43]

O OH CH3
O O
HO
O
Metallo-b- 2000 – –
HO O lactamase (CfiA)
CH3

SB236049
SB236049 [40]

Sh OH
SH VanX 0.32 – –
2,3 Dimercaptopropan-1-ol
2,3 Dimercaptopropan-1-ol [44]
MIC, minimum inhibitory concentration.

dose) [28]. The lack of anti-pseudomonal activity could be 1-Deoxy-D-xylulose-5-phosphate reductoisomerase


caused by poor permeability, active efflux, lack of target Bacteria synthesize essential isoprenoids through two dis-
access, or low potency against the Pseudomonas enzyme. tinct pathways: the ‘classical’ pathway, involving meva-
Against susceptible organisms, LpxC inhibitors are bacter- lonate, and the so-called ‘non-mevalonate’ pathway
icidal in vitro and the frequency of resistance is low. Two (the glyceraldehyde-3-phosphate/pyruvate pathway) [29].
mechanisms of resistance have been reported in E. coli: Gram-positive cocci use the former pathway, whereas
mutation of LpxC residues close to one of the metal- most gram-negative bacteria use exclusively the latter.
coordinating histidine residues, and mutation of the fabZ Because mammalian cells use only the ‘classical’ pathway,
gene, which encodes R-3-hydroxymyristoyl acyl carrier enzymes of the bacterial ‘non-mevalonate’ pathway
protein dehydrase [27,28]. represent potential targets for new antibiotics [30]. In

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Targeting metalloenzymes: a strategy that works White et al. 505

particular, the second enzyme in this pathway, a reduc- Zinc-dependent D,D-dipeptidase (VanX) is required for
toisomerase, is a Mn2þ-dependent metalloenzyme for resistance to vancomycin in Enterococcus [41]. As with
which the crystal structure has been determined [31,32]. metallo-b-lactamases, this enzyme does not have an essen-
Fosmidomycin, a natural product discovered years ago tial function but is of great interest because of its role in
[33], was recently shown to be a highly potent and specific resistance. Several chelator-based VanX inhibitors have
inhibitor of this enzyme [34]. Notably, both actinonin and been identified, but none accumulate sufficiently inside
fosmidomycin, natural products identified as inhibitors bacteria to be effective. All reported VanX inhibitors con-
of the metalloenzymes PDF and 1-deoxy-D-xylulose-5- tain a metal-chelating bidentate pharmacophore that
phosphate reductoisomerase (DXR), respectively, employ mimics the transition state of the substrate. However,
the strategy used for captopril; that is, a chelating ‘warhead’ the small and constricted active site of VanX leaves little
is coupled to a moiety that mimics the substrate, thereby room for further optimization of these inhibitors [42].
providing selective delivery to the active site. Although
fosmidomycin achieved good safety and efficacy in a Phase Conclusions
II clinical trial for urinary tract infections [35], its devel- The availability of a large number of genome-derived
opment by Fujisawa Pharmaceutical Company was not novel targets has failed to end the antibiotic drought.
continued because of concerns about the high frequency Rather than challenging all the possible targets with a
of resistance when used alone. The resistance problem is panoply of chemical libraries (combinatorial or other-
not, however, a function of the target per se, but a result of wise), one can focus on a family of related targets to
the fact that fosmidomycin, which has a phosphonate increase the probability of success. The subset of essen-
moiety, is imported into bacterial cells by a sugar transport tial bacterial metalloenzymes is a promising group on
system. Loss of this transporter results in high-level resis- which to apply a mechanism-based drug design approach.
tance at no apparent cost to the bacteria; a similar situation This strategy has proved successful so far in rapidly
exists for the related compound fosfomycin. One can generating leads with in vivo activity (e.g. PDF and LpxC
envisage redesigning DXR inhibitors to include chelators inhibitors). Validation of this approach awaits demonstra-
that would penetrate the bacteria by other means. Intrigu- tion of clinical efficacy, which will be known within the
ingly, malarial parasites also use DXR; therefore, fosmi- next few years.
domycin and an analog are being tested for the treatment
of malaria [36]. References and recommended reading
Papers of particular interest, published within the annual period of
Other enzymes review, have been highlighted as:
In addition to the enzymes discussed above, several other  of special interest
bacterial metalloenzymes are considered here.  of outstanding interest

1. Trias J, Gordon EM: Innovative approaches to novel


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functionality was critical for inhibition. for antibacterial drug discovery.

www.current-opinion.com Current Opinion in Pharmacology 2003, 3:502–507


506 Anti-infectives

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