|In Silico Pathway Analysis Predicts Metabolites that are Potential
|Malabika Sarker1, Sidharth Chopra2, Kristien Mortelmans2, Krishna Kodukula3, Carolyn Talcott1 and Amit K. Galande3*
|1Computer Science Laboratory, SRI International, Menlo Park, CA 94025
|2Center for Infectious Disease and Biodefense Research, SRI International, Menlo Park, CA 94025
|3Center for Advanced Drug Research, SRI International, Harrisonburg, Virginia 22802, USA
||Dr. Amit K. Galande
Center for Advanced Drug Research
SRI International, Harrisonburg, Virginia 22802, USA
Tel: (540) 438 6621
(540) 568 5758
|Received April 04, 2011; Accepted April 20, 2011; Published April 25, 2011
|Citation: Sarker M, Chopra S, Mortelmans K, Kodukula K, Talcott C, et al. (2011)
In Silico Pathway Analysis Predicts Metabolites that are Potential Antimicrobial
Targets. J Comput Sci Syst Biol 4: 021-026. doi:10.4172/jcsb.1000071
|Copyright: © 2011 Sarker M, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License,which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
|Antibiotic discovery aimed at conventional targets such as proteins and nucleic acids faces challenges from
mutations and antibiotic resistance. Small molecule metabolites, however, can be considered resistant to change,
as they do not undergo rapid mutations. Developing analogs or scavengers of essential microbial metabolites as
antibiotics is a promising strategy that can delay drug resistance. The objective of this work was to identify microbial
metabolites that are most suitable targets for antimicrobial discovery. We performed extensive literature mining
and systems level pathway analysis to identify bacterial metabolites that fulfill the criteria for drug targets. The
BioCyc interactive metabolic pathway maps and Pathway Tools software were used to corroborate our finding.
We identified ten metabolites as potential candidates for developing novel antibiotics. These metabolites are Lipid
II, meso-diaminopimelate, pantothenate, shikimate, biotin, L-aspartyl-4-phosphate, dTDP-α-L-rhamnose, UDP-Dgalacto-
1,4-furanose, des-N-acetyl mycothiol, and Siroheme. The article describes the selection criteria, analysis
of metabolic pathways, and the potential role of each of the ten metabolites in therapeutic intervention as broadspectrum
antibiotics with emphasis on M. tuberculosis. Our study revealed previously unexplored targets along with
metabolites that are well established in antibiotic discovery. Identification of established metabolites strengthen our
analyses while the newly discovered metabolites could lead to novel antimicrobials.
|Bacteria; Metabolites; BioCyc; Metabolic pathways;
Essentiality; Drug target
|Many bacterial infections that were once controlled effectively
with antibiotics are becoming increasingly resistant to multiple drugs,
leading to treatment failure and death . Mutations are among the
most common causes of the development of antibiotic resistance.
Under the stress of exposure to a given antibiotic, genes encoding errorprone
polymerases are up-regulated, leading to the introduction of
mutations that confer antibiotic resistance. In conventional antibiotic
therapy, the biopolymers that have been used for drug targeting are
prone to mutations that lead to antibiotic resistance. Mutations in the
quinolone resistance-determining region of gyrA and the mutation of
d-Ala-d-Ala to d-Ala-d-Lac in the cell wall peptidoglycan precursor
that confers resistance to vancomycin are classical examples . Unlike
biopolymers, such as proteins and nucleic acids, which readily respond
to evolutionary pressure, small molecule metabolites can be considered
immutable. Targeting selective metabolites that are essential for the
survival of a bacterium can thus be a much more robust strategy for
|Metabolite analogs have long been used as potent and selective
inhibitors of microbial growth. These inhibitory analogs are structurally
similar to the microbial metabolites and interfere with the functions
of the corresponding native metabolites. Recent studies also indicate
that “Bacterial-Metabolite-Likeness” can be used as an effective
cheminformatic filter in the design and analysis of pharmaceutical
libraries for drug discovery [1,3,4].
|Antimicrobials used in the treatment of infectious diseases inhibit
essential metabolic pathways that exist in the microbial pathogens
but not in the hosts. Examples of drugs that act in this way are the
sulfonamides, trimethoprim and sulfamethoxazole, which have
for three decades had a central role in the treatment of numerous
commonly encountered infections . Sulfamethoxazole is a structural
analog of para-aminobenzoic acid that inhibits synthesis of the intermediary dihydrofolic acid from its precursors. Trimethoprim is
a structural analog of the pteridine portion of dihydrofolic acid that
competitively inhibits dihydrofolate reductase, and thus the production
of tetrahydrofolic acid from dihydrofolic acid. This sequential blockade
of two enzymes in one pathway results in effective antimicrobial action.
Host cells do not synthesize their own folic acid, but obtain it as a
vitamin. Since hosts do not make folic acid, they are not affected by
these drugs, despite their toxicity for bacteria . Isoniazid, the firstline
drug used for tuberculosis treatment, is an analog of pyridoxine
(Vitamin B6). Isoniazid inhibits mycolic acid synthesis and pyridoxinecatalyzed
reactions in mycobacteria . Para-aminosalicylic acid (PAS),
a second-line tuberculosis drug, is an antifolate, similar in activity to
the sulfonamides . D-cycloserine, a structural analog of D-alanine,
acts as a broad-spectrum antimicrobial by inhibiting alanine racemase
and D-alanine ligase, which are involved in peptidoglycan synthesis
. Roseoflavin, an analog of flavin mononucleotide (FMN) interferes
with the FMN function and obtains antimicrobial activity by directly
binding to FMN riboswitch aptamers .
|The success of these metabolite analogs as antibiotics suggests that
efforts to discover similar agents could lead to useful new drugs. In
this study, we have performed in-depth literature mining and systems
level in silico analysis of metabolite pathways of a broad-spectrum of
pathogenic bacteria (M.tuberculosis, E. coli, P. aeruginosa, S. aureus, S. enterica, S. typhirium, K. pneumoniae, K. aerogenes, and B. subtilis)
and the human host to identify novel essential metabolites that can
be targeted for developing broad-spectrum antibiotics. The research
focused on metabolites for which direct experimental evidence
supports the conclusion that they are essential for the growth and
survival of the relevant bacteria. To identify potential drug targets, we
have further screened these metabolites to eliminate any found in the
human host. Comparative computational whole genome metabolic
pathway analysis was performed using BioCyc Pathway Tools.
|Materials and Methods
|Comprehensive and intelligent manual literature mining was
performed to select the appropriate bacterial metabolites based on the
following selection criteria:
|a. Whether direct experimental evidence shows that the metabolite
is essential for growth of bacteria.
|b. Whether the metabolite has one or more important biological
functions in the bacteria.
|c. Whether the metabolite is absent in the human host.
|d. Whether the enzymes of the metabolite biosynthesis have
suitable synthetic or natural inhibitors that are antimicrobial.
|e. Whether the metabolite is physio-chemically suitable for
designing the inhibitors.
|f. Whether the metabolite analogs have been tested for
|g. Whether the metabolite can be a broad-spectrum target.
|Comparative analysis using BioCyc pathway tools cellular
|To expand on the results of the literature mining, we made
use of the computational software capabilities provided by BioCyc
. BioCyc Pathway Tools software produces a pathway-based
visualization of cellular biochemical networks, called the cellular
overview diagram, which supports interrogation and exploration of
system-biology analyses of whole organism. The cellular overview
includes metabolic, transport, and signaling pathways, and other
membrane and periplasmic proteins. BioCyc provides overview
diagrams for more than 200 organisms from bacteria to human beings
. Pathway Tools automatically generate a cellular overview diagram
for an organism from a Pathway/Genome Database (PGDB) describing
the genome and biochemical networks of the organism. A PGDB can in
turn be automatically generated from the annotated genome sequence
of that organism . The cellular overview diagram can be explored
for comparative analyses of the complete metabolic networks of two
or more organisms. In the display of an overview for one organism,
the software can highlight all reactions that are either shared or not
shared with other combinations of organisms for which PGDBs are
available. For the present work, the entire metabolic network of human
from HumanCyc  was compared with the networks of pathogens
of interest to search for metabolites that are absent from human
beings. Moreover, the reactions around the selected metabolites were
compared for their presence or absence among the bacteria of choice
to determine whether the metabolites are shared and hence can have
broadspectrum action. These comparisons are not performed at the
sequence level, since the question of whether the organisms share
common enzymatic activities is orthogonal to whether the enzymes that catalyze those activities share sequence similarity. Rather, two
organisms are considered to share a reaction if the PGDBs for the
organisms both specify that the same enzyme catalyzes that reaction.
|Pathway analysis using BioCyc pathway genome database
|The study of a metabolite in the context of the relevant biological
pathway is important since it provides knowledge about any alternative
compensatory pathway that might exist. Nevertheless, the pathway
information depicts the genes of the pathways, especially the ones
that encode the enzymes that catalyze both the formation and the
consumption of the particular metabolite. This information helps to
determine whether that metabolite could be considered a suitable
target. BioCyc bacterial metabolic pathways were extensively used for
studying the relevant pathways and reactions, including the relevant
enzymes. BioCyc provides 673 PGDBs, each containing the predicted
metabolic network of an organism, including metabolic pathways,
enzymes (and the genes encoding them), metabolites (with structural
details), and reaction details .
|Results and Discussions
|Based on the pathway analyses, the following ten metabolites were
identified that matched all of the above-mentioned selection criteria
and can be proposed as potential candidates for developing novel
antibiotics. Previous identification of the first five metabolites as targets
for antibiotic discovery validates our in silico approach and suggests
that the five other metabolites could be promising novel candidates as
well. The following subsections provide more detailed information on
the known or potential role of all ten of these metabolites in therapeutic
|A. Lipid II (N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminoheptane-Dalanyl-
|Lipid II is a membrane-anchored peptidoglycan precursor that is
essential for bacterial cellwall biosynthesis. It is the target for at least
four classes of antibiotics, including glycopeptides, lipopeptides, and
lantibiotics . Lipid II is a broad-spectrum target, and antibiotics
that interact with lipid II cause bacterial cell death in many ways.
Examples include equimolar stoichiometric complex formation with
lipid II followed by inhibition of cell wall biosynthesis (plectasin),
and permeabilization of membranes by binding to lipid II, followed
by assembly and pore formation (nisin). Lipid II is a chemically
complex molecule, with a hydrophilic head group that consists of
a GlcNAc-MurNAc-pentapeptide linked via a pyrophosphate to a
long lipidic bactoprenol tail . Building synthetic analogs of lipid
II for antimicrobial testing would require considerable effort .
Nonetheless, such analogs present an untapped opportunity in the field
of antibiotic discovery.
|B. meso-diaminopimelate (meso-DAP):
|Meso-DAP is a unique
metabolite that functions as a precursor of lysine and as a structural
component of the pentapeptide linker in the peptidoglycan layer of
most bacterial cell walls, except gram-positive cocci. The metabolite
is present in most algae, fungi, and higher plants, but absent in
mammals. DAP cross-links provide stability to the cell wall and confer
resistance to intracellular osmotic pressure. The four variants of the
biosynthetic pathways of meso-DAP differ in the routes leading from
tetrahydrodipicolinate to meso-diaminopimelate. The presence of
multiple biosynthetic pathways for DAP, at least in some bacteria, is
probably an indication of the importance of DAP to bacterial survival.
Various DAP analogs are potent antibacterials. For example, 3-chloro DAP, an inhibitor of DapF, the DAP epimerase that catalyzes the
formation of meso-DAP, is active against E. coli. Other inhibitors
include 3-methyl-DAP, which may inhibit DAP transport .
Phosphono-DAP analogs, which inhibit DAP biosynthesis, are active
against a wide range of bacteria. γ-Methylene-DAP inhibits the growth
of E. coli and P. aeruginosa .
|Pantothenic acid (Vitamin B5) is the universal
precursor for the synthesis of the 4’-phosphopantetheine moiety of
coenzyme A (CoA) and acyl carrier protein (ACP). CoA and ACP
play important roles as acyl-group carriers in fatty acid metabolism,
the tricarboxylic acid cycle, biosynthesis of polyketides, and several
other reactions associated with intermediary metabolism. The de novo
biosynthetic pathway to pantothenate consists of four enzymes encoded
by panB, panE, panD, and panC; all four are absent in mammals. Bacteria,
plants, and fungi synthesize pantothenate de novo from amino acid
intermediates, but Human beings need to acquire it through diet .
Various pantothenate analogs show antibiotic activity against many
bacteria, including E. coli and S. aureus . Pantothenate cannot be a
broad-spectrum target because many microorganisms that synthesize
pantothenate are also capable of absorbing preformed pantothenate
from the extracellular environment. For example, pantothenate uptake
is mediated by pantothenate permease PanF in E. coli K-12  and
pantothenate transporter PanT in S. pneumoniae . Pantothenate,
however, can be a promising target for Mycobacterium tuberculosis.
Vaccination with a ΔpanCD strain of Mycobacterium tuberculosis
elicited an immune response that protected immuno-compromised
mice against virulent M. tuberculosis more effectively than a BCG
vaccination did, without causing widespread infection by itself. A
sulfamoyl adenylate inhibitor of PanC is also being tested for activity in
a cell-based assay against Mycobacterium tuberculosis .
|Biotin or Vitamin H is a co-factor for a small number
of enzymes that facilitate the transfer of CO2 during carboxylation,
decarboxylation, and transcarboxylation reactions involved in
fatty acid and carbohydrate metabolism. The biotin-requiring
enzymes identified to date (including acetyl-CoA carboxylase, 3-
methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and
pyruvate carboxylase) play essential roles in cell metabolism .
Biotin is present in microorganisms and higher plants, and absent from
mammals, which must acquire it from their diets. Like pantothenate,
biotin cannot be considered a broad-spectrum target since a biotin
transporter has been found in E. coli, S. aureus, and S. pneumoniae
. It can be a potential target for M. tuberculosis, however, as biotin
functions as a precursor for synthesis of mycolate, the major cell wall
component essential for survival and pathogenesis of M. tuberculosis
 and for degradation of cholesterol, which is an alternative carbon
source during their persistence within the human host . Two
antibiotics, actithiazic acid and amiclenomycin, irreversibly inactivate
BioA, which is involved in biotin biosynthesis and needed for the longterm
survival of mycobacteria. Mycobacteria might be able to reverse
the effect of such antibiotics by taking up external biotin, although a
transporter that could perform this function has not been identified
in the annotated genes of M. Tuberculosis . Biotin antagonists
such as 4-(Imidazolidone-2) caproic acid, homobiotin, norbiotin,
and hexahydro-2-oxo-4-hydroxybutyl-1-furo- [26,12] imidazole
have shown inhibitory effects on M. tuberculosis (Pope, 1952 #74}.
α-Dehydrobiotin, a naturally occurring biotin analog, exhibits
antimicrobial properties against E. coli, B. subtilis, and several strains
of mycobacteria. It is a product of biotin catabolism that coordinately
represses the 7,8-diaminopelargonic acid aminotransferase and the
dethiobiotin synthetase enzymes .
|Shikimate leads in three enzymatic reaction steps to
the production of chorismate, a key precursor of biosynthesis of several
essential metabolites, including aromatic amino acids, ubiquinones,
folates, naphthoquinones, menaquinones, and mycobactins. Shikimate
is present in algae, higher plants, bacteria, and fungi, but absent in
mammals . It could be a broad-spectrum target. Genes for seven
enzymes of the shikimate pathway-aroD, aroB, aroK, aroF, aroG, aroE,
and aroA-have been shown to be essential for mycobacterial viability.
Even in the presence of exogenous supplementation, none of these aro
mutants could be obtained in M. tuberculosis . A shikimate analog,
(6S)-6-fluoroshikimic acid, acts as a broad-spectrum antibacterial
agent. It is active against E. coli, presumably because inhibition of
aromatic biosynthesis results from the irreversible inhibition of
4-amino-4- deoxychorismate synthase caused by 2-fluorochorismate
|L-aspartyl-4-phosphate is present in
plants and bacteria, but absent from mammals. It is produced from
ATP-dependent phosphorylation of L-aspartate through a reaction
catalyzed by aspartokinase, an enzyme that feeds a branched network of
many biochemical pathways, including the biosynthesis of the aspartate
family of amino acids: methionine, threonine, lysine, and isoleucine.
All these are essential amino acids for humans and are absorbed
through diet . Consequently, L-aspartyl-4-phosphate is expected
to be a broad-spectrum target. Transposon site hybridization analysis
identified the aspartokinase-encoding (ask) gene as one of the genes
required for the growth of M. tuberculosis . In E. coli, unsaturated
and fluorinated analogues of aspartyl-4-phosphate act as potential
inhibitors of the enzyme aspartate semialdehyde dehydrogenase, which
catalyzes the formation of L-aspartate semialdehyde from aspartyl-4-
|An L-rhamnosyl residue (a 6-deoxyhexose
sugar) plays an important structural role in the cell wall of many human
pathogens, including M. tuberculosis and S. typhimurium . A
nucleotide linked conjugate of this sugar consisting of deoxythymidine
diphosphate dTDP-L-rhamnose is a key intermediate in cell wall
synthesis because it donates L-rhamnose. Neither rhamnose nor the
genes for its synthesis have been identified in humans. The rhamnose
pathway is ubiquitous and highly conserved in both gram-positive and
gram-negative bacteria. L-rhamnose is a common component of the
O-antigen of lipopolysaccharides (LPS) of gram-negative pathogens
such as S. enterica, S. flexneri, and E. coli. L-rhamnose has been found
to occupy important anchoring positions in M. tuberculosis, where
it covalently links the arabinogalactan to the peptidoglycan layer.
Inhibitors (e.g., 2,3,5 trisubstituted-4-thiazolidinone compounds
against RmlC) of L-rhamnose-synthesizing enzymes have been shown
to be active against whole M. tuberculosis cells, and the pathway was
shown to be essential . L-rhamnose can be used as the sole carbon
and energy source by many groups of microorganisms. It cannot be
classified as a broad-spectrum target, however, because the utilization
of L-rhamnose requires an L-rhamnose-H+ symporter (RhaT) to cross
the cytoplasmic membrane in E. coli and S. typhimurium . Since
rhamnose has no role in mammalian metabolism, L-rhamnose mimics
have been developed as selective antibacterials to inhibit incorporation
of dTDP-rhamnose into the cell wall .
|H. UDP-D-galacto-1, 4-furanose (Galf):
|Galf has been reported
in a number of microorganisms, such as bacteria, protozoa, and fungi.
It is not present in mammals, and Galf-containing epitopes have been
shown to be highly antigenic. Galf residues are formed in nature by
a ring contraction of uridine diphosphate galactopyranose (UDP Galp) to UDP-galactofuranose (UDP-Galf) catalyzed by the enzyme
UDP-galactopyranose mutase (UGM). UGM is present in several
microorganisms, including E. coli, Mycobacteria, and Klebsiella sp.
Galf is transferred from UDP-Galf to the respective glycoconjugate
molecules by specific galactofuranosyl transferases. Galf has been
shown to be present in numerous structures considered to be essential
for virulence in many pathogenic organisms. These include the LPS
Oantigen of an increasing number of gram-negative bacteria, the
T1-antigen polysaccharide of Salmonella sp., and extracellular or
capsular polysaccharides of a variety of both gram-positive and gramnegative
bacteria. Galf is a critical and abundant component of the
arabinogalactan of mycobacteria. Mycobacterial arabinogalactan, a
polysaccharide consisting largely of Galf, covalently links the highly
impermeable mycolic acid outer layer of the mycobacterial cell wall
with the inner layer of peptidoglycan. The metabolism of arabinan,
which is directly linked to the Galf component of arabinogalactan,
is the target of the proven antituberculosis drug ethambutol .
Therefore, Galf metabolism is a particularly promising target in the
search for new antimycobacterial drugs. From a focused library of
synthetic aminothiazoles, several compounds that block the UGM
from K. pneumoniae or M. Tuberculosis were identified. A pyrazole
compound with a similar structure has been shown to be an inhibitor
of UGM from M. tuberculosis and K. pneumonia; it was also effective
against M. bovis BCG and M. tuberculosis, but it was ineffective against
other bacterial strains tested. This compound showed potency against
mycobacteria in infected macrophages, but it also exhibited moderate
cellular toxicity and was ineffective against nonreplicating persistent
|The major lowmolecular-
mass thiol found in the actinomyceteae group of bacteria
is mycothiol (MSH; AcCys-GlcNIns). This thiol, which is unique to
these organisms, has the structure (1-D-myoinosityl- 2(N-acetyl-Lcysteinyl)
amino-2-deoxy-α-D-glucopyranoside. MSH is the functional
equivalent of glutathione and is present at millimolar levels in M.
tuberculosis. It functions as a reserve of cysteine and is used for the
detoxification of alkylating agents, reactive oxygen, nitrogen species,
and antibiotics. Mycothiol also acts as a thiol buffer, maintaining the
highly reducing environment within the cell and protecting against
disulfide stress. MSH biosynthesis is absent in human beings. Current
studies indicate that dormant M. tuberculosis cells are metabolically
active and therefore must maintain a reducing intracellular redox
environment. Since MSH and its disulfide reductase form the thiol
redox buffer in mycobacteria, MSH biosynthesis drug targets may
be particularly relevant to the treatment of dormant tuberculosis
infection . Des- N-acetyl mycothiol or Cys-GlcN-Ins is the
immediate precursor of MSH. The enzymes of the MSH biosynthesis
are MshA/MshA2, MshB, MshC, and MshD. MshC has been found to
be an essential enzyme for producing Cys-GlcN-Ins, which in turn can
undergo transacetylation to make N-formyl-Cys-GlcN-Ins (a weak
surrogate of MSH) even in the presence of low MSH (in the case of the
mshD mutant) . Therefore des-N-acetyl mycothiol may be a better
target than MSH. The mshA deletion mutants are defective in MSH
biosynthesis and ethionamide resistant, as well as slightly defective
for growth in immunocompetent mice . The mshB mutant had a
heightened sensitivity to the toxic oxidant cumene hydroperoxide and
to rifampin. Viable mutants with the native mshC gene inactivated
could be obtained only when a second copy of mshC was present,
thus indicating that MSH is essential for growth. The mshD mutant
produces only 1% of normal MSH levels; it produces N-formyl-Cys-GlcNIns (as a weak surrogate of MSH), but not in sufficient quantities
to support normal growth of M. tuberculosis under stress conditions
such as those found within the macrophage .
|Dequalinium chloride, an ATP-competitive inhibitor of MshC,
has been shown to inhibit the growth of M. tuberculosis under aerobic
and anaerobic conditions . In vivo, the 1-L-Ins-1-P required for
MSH biosynthesis is usually obtained from glucose-6-phosphate by
1-L-inositol-1-phosphate synthase (Ino1). Antisense inhibition of
Ino1 in M. tuberculosis results in a marked depletion of intracellular
MSH levels and increased sensitivity to vancomycin, rifampicin, and
isoniazid. A thioglycosidic analogue of mycothiol has recently been
shown to have good specific activity against M. tuberculosis .
|Sulfur and nitrogen metabolism are ancient, essential
biosynthetic pathways. Human beings cannot process sulfur and
nitrogen directly, but rather depend on bacteria and plants that possess
the necessary metabolic pathways to reduce inorganic sulfur and
nitrogen to the correct redox state for human consumption. Siroheme is
an iron-containing isobacteriochlorin, a modified tetrapyrrole similar
in structure to both heme and chlorophyll, which was discovered in 1973
. It is an unusual but useful prosthetic group of several enzymes,
including sulfite and nitrite reductases, which catalyze the six-electron
reductions of sulfite to sulfide and nitrite to ammonia, respectively.
Assimilatory sulfite reductases are found in bacteria, fungi, and plants,
but not in animals, while dissimilatory sulfite reductases are found in
diverse sulfate-reducing eubacteria and some species of thermophilic
archaebacteria . Siroheme can therefore be considered a broadspectrum
target. Siroheme is covalently coupled to an iron-sulfur
cluster ([FeS]) to form an electronically integrated metallo-co-factor
for delivering electrons to a substrate. It is formed by methylation,
oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen
III (Uro-III). The enzymes catalyzing this pathway have many
variations. In some bacteria the transformation of uroporphyrinogen-
III into siroheme is catalyzed by three separate enzymes (uroporphyrin
III methyltransferase, dihydrosirohydrochlorin dehydrogenase, and
sirohydrochlorin ferrochelatase) . In other organisms, such as
E. coli and S. enterica, a single trifunctional enzyme (uroporphyrin
III C-methyltransferase [multifunctional], CysG) catalyzes all three
reactions . In either case, cysG mutants cannot reduce sulfite
to sulfide and require a source of sulfide or cysteine for growth. In
addition, CysG-mediated methylation of Uro-III is required for de
novo synthesis of cobalamin (coenzyme B12) in S. enterica . cysF,
encoding an alternative siroheme synthase homologous to CysG, has
been identified in K. aerogenes. In contrast, Klebsiella cysG mutants fail
to synthesize coenzyme B12. The cysF gene is absent from the E. coli and S. enterica genomes .
|In this study, we have performed in-depth literature mining to
find the essential metabolites for a wide range of pathogenic bacteria.
The research focused on metabolites for which direct experimental
evidence supports the conclusion that these are essential for the growth
and survival of the relevant bacteria. To identify potential drug targets,
we have further screened these metabolites to eliminate any found
in the human host. Comparative whole genome metabolic pathway
analysis was performed computationally using BioCyc Pathway Tools.
The results of our analysis were validated when we found literaturebased
evidence of antibacterial activity in analogs of lipid II, meso-
DAP, pantothenate, biotin, and shikimate. This validation suggests
that the remaining five relatively underexplored metabolites from
our list are attractive targets for antibiotic discovery. These findings indicate that computational pharmacophore approaches can be useful
for in silico antimicrobial design efforts to identify essential metabolite
mimics that are similar to known drugs. Our analysis suggests that
novel synthetic analogs of the metabolites reported here could provide
effective antibiotics. Moreover, novel intervention strategies such as in
vitro selection can be used to identify metabolite “scavenging” peptide
aptamers as a new class of antibiotics.
|The establishment of Center for Advanced Drug Research (CADRE) was
made possible by funding support to SRI International from the Commonwealth of
Virginia. This work was supported by grant 53123 from the Bill & Melinda Gates
Foundation. Encouragement and guidance from Dr. Walter Moos, Vice President of
the Biosciences Division of SRI International, are gratefully acknowledged.
- Nikaido H (2009) Multidrug resistance in bacteria. Annu Rev Bioche 78:119-
- Lambert PA (2005) Bacterial resistance to antibiotics: modified target sites. Adv
Drug Deliv Rev 57: 1471-1485.
- ShiveW (1952) Biological activities of metabolite analogues. Annu Rev
Microbiol 6: 437-466.
- Cherkasov A (2006) Can ‘Bacterial-Metabolite-Likeness’ model improve odds
of ‘in silico’ antibiotic discovery? J Chem Inf Model 46:1214-1222.
- Masters PA, O’Bryan TA, Zurlo J, Miller DQ, Joshi N (2003) Trimethoprim
sulfamethoxazole revisited. Arch Intern Med 163: 402-410.
- Vilcheze C, Jacobs WR Jr (2007) The mechanism of isoniazid killing: clarity
through the scope of genetics. Annu Rev Microbiol 61: 35-50.
- Rengarajan J, Sassetti CM, Naroditskaya V, Sloutsky A, Bloom BR, et al. (2004)
The folate pathway is a target for resistance to the drug para-aminosalicylic
acid (PAS) in mycobacteria. Mol Microbiol 53: 275-282.
- Halouska S, Chacon O, Fenton RJ, Zinniel DK, Barletta RG, et al. (2007) Use
of NMR metabolomics to analyze the targets of D-cycloserine in mycobacteria:
role of D-alanine racemase. J Proteome Res 6: 4608-4614.
- Lee ER, Blount KF, Breaker RR (2009) Roseoflavin is a natural antibacterial
compound that binds to FMN riboswitches and regulates gene expression. RNA Biol 6: 187-194.
- Karp PD, Paley S, Romero P (2002) The Pathway Tools software. Bioinformatics
- Paley SM, Karp PD (2006) The Pathway Tools cellular overview diagram and
Omics Viewer. Nucleic Acids Res 34: 3771-3778.
- Karp PD, Ouzounis CA, Moore-Kochlacs C, Goldovsky L, Kaipa P, et al. (2005)
Expansion of the BioCyc collection of pathway/genome databases to 160
genomes. Nucleic Acids Res 33: 6083-6089.
- Romero P, Wagg J, Green ML, Kaiser D, Krummenacker M, et al. (2005)
Computational prediction of human metabolic pathways from the complete
human genome. Genome Biol 6: R2.
- Caspi R, Altman T, Dale JM, Dreher K, Fulcher CA, et al. (2010) The MetaCyc
database of metabolic pathways and enzymes and the BioCyc collection of
pathway/genome databases. Nucleic Acids Res 38: D473-479.
- Schneider T, Sahl HG (2010) Lipid II and other bactoprenol-bound cell wall
precursors as drug targets. Curr Opin Investig Drugs 11: 157-164.
- Breukink E, de Kruijff B (2006) Lipid II as a target for antibiotics. Nat Rev Drug
Discov 5: 321-332.
- Van Heijenoort J (2007) Lipid intermediates in the biosynthesis of bacterial
peptidoglycan. Microbiol Mol Biol Rev 71: 620-635.
- Baumann RJ, Bohme EH, Wiseman JS,Vaal M, Nichols JS (1988)
Inhibition of Escherichia coli growth and diaminopimelic acid epimerase by
3-chlorodiaminopimelic acid. Antimicrob Agents Chemother 32: 1119-1123.
- Cox RJ (1996) The DAP pathway to lysine as a target for antimicrobial agents. Nat Prod Rep 13: 29-43.
- Sambandamurthy VK, Wang X, Chen B, Russell RG, Derrick S, et al. (2002) A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated
and protects mice against tuberculosis. Nat Med 8: 1171-1174.
- McIlwain H (1942) Bacterial inhibition by metabolite analogues: Analogues of
pantothenic acid. Biochem J 36: 417-427.
- Jackowski S, Alix JH (1990) Cloning, sequence, and expression of the
pantothenate permease (panF) gene of Escherichia coli. J Bacteriol 172: 3842-
- Rodionov DA, Hebbeln P, Eudes A, ter Beek J, Rodionova IA, et al. (2009)
A novel class of modular transporters for vitamins in prokaryotes. J Bacteriol
- Ciulli A, Scott DE, Ando M, Reyes F, Saldanha SA, et al. (2008) Inhibition
of Mycobacterium tuberculosis pantothenate synthetase by analogues of the
reaction intermediate. Chembiochem 9: 2606-2611.
- Mann S, Ploux O (2006) 7,8-Diaminoperlargonic acid aminotransferase from
Mycobacterium tuberculosis, a potential therapeutic target. Characterization
and inhibition studies. FEBS J 273: 4778-4789.
- Hebbeln P, Rodionov DA, Alfandega A, Eitinger T (2007) Biotin uptake in
prokaryotes by solute transporters with an optional ATPbinding cassettecontaining
module. Proc Natl Acad Sci U S A 104: 2909-2914.
- Kurth DG, Gago GM, de la Iglesia A, Bazet Lyonnet B, Lin TW, et al. (2009) ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and
mycolic acid biosynthesis in mycobacteria. Microbiology 155: 2664-2675.
- Savvi S, Warner DF, Kana BD, McKinney JD, Mizrahi V, et al. (2008) Functional
characterization of a vitamin B12-dependent methylmalonyl pathway in
Mycobacterium tuberculosis: implications for propionate metabolism during
growth on fatty acids. J Bacteriol 190: 3886-3895.
- Piffeteau A, Dufour MN, Zamboni M, Gaudry M, Marquet A (1980) Mechanism
of the antibiotic action of alpha-dehydrobiotin. Biochemistry 19: 3069-3073.
- Dosselaere F, Vanderleyden J (2001) A metabolic node in action: chorismateutilizing
enzymes in microorganisms. Crit Rev Microbiol 27: 75-131.
- Bulloch EM, Jones MA, Parker EJ, Osborne AP, Stephens E, et al. (2004)
Identification of 4-amino-4-deoxychorismate synthase as the molecular target
for the antimicrobial action of (6s)-6-fluoroshikimate. J Am Chem Soc 126:
- Viola RE (2001) The central enzymes of the aspartate family of amino acid
biosynthesis. Acc Chem Res 34: 339-349.
- Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial
growth defined by high density mutagenesis. Mol Microbiol 48: 77-84.
- Cox RJ, Gibson JS, Hadfield AT (2005) Design, synthesis and analysis of
inhibitors of bacterial aspartate semialdehyde dehydrogenase. Chembiochem
- Dong C, Beis K, Giraud MF, Blankenfeldt W, Allard S, et al. (2003) A structural
perspective on the enzymes that convert dTDP-d-glucose into dTDPlrhamnose. Biochem Soc Trans 31: 532-536.
- Babaoglu K, Page MA, Jones VC, McNeil MR, Dong C, et al. (2003) Novel
inhibitors of an emerging target in Mycobacterium tuberculosis; substituted
thiazolidinones as inhibitors of dTDP-rhamnose synthesis. Bioorg Med Chem
Lett 13: 3227-3230.
- Tate CG, Muiry JA, Henderson PJ (1992) Mapping, cloning, expression, and
sequencing of the rhaT gene, which encodes a novel Lrhamnose-H+ transport
protein in Salmonella typhimurium and Escherichia coli. J Biol Chem 267:
- Estevez JC, Saunders J, Besra GS, Brennan PJ, Nash RJ, et al. (1996) Mimics
of L-Rhamnose: Synthesis of CGlycosides of L-Rhamnofuranose and an
alpha-azidoester as divergent intermediates for combinatorial generation of
rhamnofuranose libraries. Tetrahedron: Asymmetry 7: 383-386.
- Pedersen LL, Turco SJ (2003) Galactofuranose metabolism: a potential target
for antimicrobial chemotherapy. Cell Mol Life Sci 60: 259-266.
- Borrelli S, Zandberg WF, Mohan S, Ko M, Martinez-Gutierrez F, et al. (2010)
Antimycobacterial activity of UDP-galactopyranose mutase inhibitors. Int J
Antimicrob Agents 36: 364-368.
- Dykhuizen EC, May JF,Tongpenyai A, Kiessling LL (2008) Inhibitors of UDPgalactopyranose
mutase thwart mycobacterial growth. J Am Chem Soc 130:
- Newton GL, Buchmeier N, Fahey RC (2008) Biosynthesis and functions of
mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol Biol Rev
- Sareen D, Newton GL, Fahey RC, Buchmeier NA (2003) Mycothiol is essential
for growth of Mycobacterium tuberculosis Erdman. J Bacteriol 185: 6736-6740.
- Vilcheze C, Av-Gay Y, Attarian R, Liu Z, Hazbon MH, et al. (2008) Mycothiol
biosynthesis is essential for ethionamide susceptibility in Mycobacterium
tuberculosis. Mol Microbiol 69: 1316-1329.
- Buchmeier NA, Newton GL, FaheyRC (2006) A mycothiol synthase mutant of
Mycobacterium tuberculosis has an altered thioldisulfide content and limited
tolerance to stress. J Bacteriol 188: 6245-6252.
- Gutierrez-Lugo MT, Baker H, Shiloach J, Boshoff H, Bewley CA (2009) Dequalinium, a new inhibitor of Mycobacterium tuberculosis mycothiol ligase
identified by high-throughput screening. J Biomol Screen 14: 643-652.
- Witczak ZJ, Culhane JM (2005) Thiosugars: new perspectives regarding
availability and potential biochemical and medicinal applications. Appl Microbiol
Biotechnol 69: 237-244.
- Murphy MJ, Siegel LM (1973) Siroheme and sirohydrochlorin. The basis for a
new type of porphyrin-related prosthetic group common to both assimilatory
and dissimilatory sulfite reductases. J Biol Chem 248: 6911-6919.
- Crane BR, Getzoff ED (1996) The relationship between structure and function
for the sulfite reductases. Curr Opin Struct Biol 6: 744-756.
- Johansson P, Hederstedt L (1999) Organization of genes for tetrapyrrole
biosynthesis in gram--positive bacteria. Microbiology 145: 529-538.
- Warren MJ, Bolt EL, Roessner CA, Scott AI, Spencer JB et al. (1994) Gene
dissection demonstrates that the Escherichia coli cysG gene encodes a
multifunctional protein. Biochem J 302: 837-844.
- Woodcock SC, Raux E, Levillayer F, Thermes C, Rambach A, et al. (1998) Effect
of mutations in the transmethylase and dehydrogenase/chelatase domains of
sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis. Biochem
J 330: 121-129.
- Kolko MM, Kapetanovich LA, LawrenceJG (2001) Alternative pathways for
siroheme synthesis in Klebsiella aerogenes. J Bacteriol 183: 328-335.