TB DRUG TARGETS

Jimma University
Website: www.ju.edu.et JUCAVM
College of Agriculture and Veterinary Medicine
School of Veterinary Medicine
Senior seminar presentation
By
Ebsa Bushura
Review on mycobacterium metabolic pathway as drug
target
April, 2016
Jimma, Ethiopia1

Jimma University
Introduction
 Mycobacterium is rod shaped acid fast ,non spore forming
bacteria with special cell wall structure .
 The genus Mycobacterium encompasses 71 validly named
species and 32 species are known to be pathogenic to humans
or animals
 The cell wall makes a substantial contribution to the hardiness
of this genus.
 Tuberculosis is a mycobacterium infection that affects a wide
range of mammals including humans.
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Jimma University
cont.…
 The increasing emergencies of drug resistance tuberculosis and
latent infection pose further challenges for effective control of
the disease
 Ethiopia is one of the 27 high MDR-TB countries; it is ranked
15th with more than 5000 estimated MDR-TB patients each
year, in the world
 objective of this paper is to review on mycobacterium
metabolic pathways as drug targets
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Jimma University
Metabolic Pathways Used as Drug Targets
 Mycobacterium metabolic pathways which do not appear in the
host but present in the pathogen are identified as pathways
unique to mycobacterium as compared to the host
 Enzymes in these unique pathways and other metabolic
pathways are important to identify novel drug targets
 These biochemical pathways which are absent in the host and
therefore unique to the pathogen; Peptidoglycan biosynthesis,
Mycobactins biosynthesis, d-alanine metabolism, thiamine
metabolism and polyketide sugar unit biosynthesis, all absent in
the host.
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Jimma University
Mycobactin biosynthesis
 To overcome iron deficiency imposed by the host defensive
system, bacteria have evolved iron acquisition systems where
small molecules called siderophores, which bind extracellular
iron, are secreted.
 The Mtb siderophore pathway is well studied and consists of
secreted siderophore termed mycobactins
 Mycobactin G (Mycobactin lysine-N6-hydroxylase), which
catalyzes the hydroxylation of lysine moiety in mycobactin
synthesis is the potential target in this pathway.
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Jimma University
Con…
 An Mtb heme-uptake system has been defined that consists of
the secreted protein known as hypothetical protein (Rv0203)
and possible membrane transport protein (MmpL3 and
MmpL11) this protein are the possible drug target.
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Peptidoglycan biosynthesis
 The cell wall of mycobacterium is made up of cross-linked
Peptidoglycan covalently linked to Arabinogalactan chain via
polyketide linkage unit. Arabinogalactan in turn esterified to
mycolic acid
 PG which plays a critical role in protecting the bacteria against
osmotic lysis
 The unique nature of the MAPc leads to the conclusion that
enzyme that involves in synthesis these structures yield many
number potential drug targets
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Jimma University
Con…
 D-alanine is a necessary precursor in the bacterial
Peptidoglycan biosynthetic pathway
 The d-alanine–d-alanine ligase (ddlA) and alanine racemase
(alr) from this pathway have no similarity to any of the host
proteins.
 Alanine racemace is Catalyzes the racemization of L-alanine into
D-alanine
 Both alanine racemase and D-Ala-D-Ala ligase are targets of D-
cycloserine, a second-line anti -TB drug
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Jimma University
Polyketide sugar unit biosynthesis
 Arabinogalactan is connected via a linker disaccharide called
polyketide (a-L-Rha--a-D-Glc-NAc-1 -phosphate) to the sixth
position of a muramic acid residue in the Peptidoglycan
 L-rhamnose transferase (WbbL) is an enzyme that utilizes dTDP-
Rha as a substrate for the formation of final product L-rhamnose
which plays a crucial role in the linkage of cell wall (add Rha to
the prenyl diphosphoryl GlcNAc. )
 The biosynthesis of dTDP-rhamnose is catalysed by four enzymes
coded by the genes; RmlA (Rv0334), RmlB (Rv03464), RmlC
(Rv3465) and RmlD (Rv3266) and ultimately synthesizes dTDP
rhamnose from glucose-1-phosphate
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Targets from other pathways
 Even amongst the pathways shared by the host and the
pathogen there are several proteinswhich do not bear similarity
to host proteins.
 some of them are known to be associated with virulence or
important for persistence or vital for mycobacteria metabolism,
 The glyoxylate by pass allows the bacterium to synthesize
carbohydrates from fatty acids. Succinate and glyoxylate
produced by this cycle are supplied to the TCA cycle and
gluconeogenesis.
 the enzymes of the glyoxylate cycle are activated during
adaptation to the low oxygen environment of the granuloma.
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Jimma University
Identification of unique pathways and potential
drug
 As bacterial genome sequences become available, there is
increasing interest in developing new antibacterial agents using
genomics-based approaches
 After functional analysis unique enzymes involved in cellular
components like cell wall, cytoplasm, extra cellular region, and
plasma membrane, their biological processes and their functions
have been retrieved The recent developments in
microarray technologies provide important tools to
identify new drug targets
 also used to identify genes that are switched on in
the Wayne “dormancy” model under hypoxic and
nitric oxide stress conditions that controlled by
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Jimma University
Con…
 Recent developments in mycobacterial molecular
genetics tools facilitate the identification and
validation of new drug targets essential for the
survival and persistence of tubercle bacilli not only
in vitro but also in vivo.
 The sub cellular localization analysis of all
supposed essential and unique enzymes of M.
tuberculosis were evaluated by Universal protein
resource.
 the functional analysis using Uniprot showed
involvement of all the unique enzymes in the
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Possible drug targets
 The gene products that control vital aspects of mycobacterial
physiology like, metabolism, persistence, virulence, two
component system and cell wall synthesis would be attractive
targets for new drugs.
 it is important to develop new drugs that inhibit novel targets
that are different from those of currently used drugs.
 To avoid significant toxicity, the targets of inhibition should be
present in bacteria but not in the human host
 Similarly, targeting existing TB drug targets for drug
development may be limited value because of potential cross-
resistance13

Jimma University
Con…
 There are many enzymes from the glyoxylate by pass, which is
important for mycobacterial persistence. Among these
enzymes the most important are Isocitrate Lyase and Malate
Dehydrogenase which are activated during adaptation to the
low oxygen environment of the granuloma
 The glyoxylate by pass allows the bacterium to synthesize
carbohydrates from fatty acids.
 Isocitrate lyase splits the C6-unit into succinate and glyoxylate
which is condensed by malate synthase with acetyl-CoA
generating free CoA-SH and malate. Malate is used by malate
dehydrogenase to continue the cycle and succinate is released
as net product
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Jimma University
Con…
 novel methyl transferase (pcaA)involved in the modification of
mycolic acids in mycobacterial cell wall was identified
 RelA(ppGpp synthase) is critical for the successful
establishment of persistent infection in mice by altering the
expression of antigenic and enzymatic factors that may
contribute to successful latent infection.
 DosR used to control a 48-gene regulon involved in MTB
survival under hypoxic conditions and could be good targets for
development of drugs that target persistent bacilli
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Jimma University
Targeting essential genes
 Essential genes are genes whose inactivation leads to non-
viability or death of the bacteria
 Transposon mutagenesis and signature-tagged mutagenesis
have been used to identify genes essential for M. tuberculosis
growth in vitro and survival in vivo
 Transposon mutagenesis is a biological process that allows
genes to be transferred to a host organism's chromosome,
interrupting or modifying the function of an extant gene on the
chromosome and causing mutation.
 Signature tagged mutagenesis is a genetic approach that was
developed to identify novel bacterial virulence factors.16

Jimma University
Targeting Mycobacterial persistence
 Mb persistence refers to the ability of tubercle bacillus to
survive in the face of chemotherapy and/or immunity in the
macrophage
 Nature persistent bacteria is unclear but might consist of
stationary phase bacteria, post-chemotherapy residual
survivors or dormant bacteria that do not form colonies upon
plating
 The presence of such persistent bacteria is considered to be the
major reason for lengthy therapy
 A lot of research activity is currently aimed at understanding
the biology of persistence of the tubercle bacillus and
developing new drugs that target the persistent bacteria
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Targeting energy production
pathway
 Although, energy production pathways in
Mycobacterium are not well characterized, their
importance as drug targets is demonstrated by
the recent finding that PZA acts by disrupting
membrane potential and depleting energy in M.
tuberculosis.
 PZA is a frontline TB drug that is more active
against non-growing persistent bacilli than
growing bacilli and shortens TB therapy
 The target for diarylquinoline was proposed to be
the mycobacterial ATP synthase, which is a new
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Jimma University
Con…
 The target Rv1854c (gene ndh) in oxidative phosphorylation
pathway is the target for INH and several mutations in this gene
account for INH resistant cases
 Targeting virulence factors
 Undoubtedly, one requisite to classify a gene as a virulence
factor is that its absence attenuates the virulence of the
microorganism in an in vivo model inhibition of virulence
factors may not be lethal because they may not essential to the
pathogen
 However, inhibitors of these virulence gene products may be
used in combination with existing drugs to improve the regime
of chemotherapy
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Jimma University
Targeting two-component systems
 mycobacterium genes that can mastermind the intracellular
survival and multiplication within macrophages as also the
shutdowns of mycobacteria during persistence are signal
transduction systems, in particular TCS.
 The mycobacterial genome encodes several two-component
systems, which consist of histidine kinases and their associated
response regulators which control the expression of target
genes in response to stimuli
 magnesium transporter (MtrA) and histidine kinases (SenX3)
that are essential for mycobacterial virulence and persistence in
mice, could also be good targets for the development of new
drugs for persistent TB bacteria
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targeting cell wall synthesis
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 The cell wall is the most common target of antituberculosis
drugs, and many compounds target enzymes that synthesize
distinct layers of the cell wall
 The first committed step in the synthesis of decaprenyl
phosphoryl-D-arabinose, the lipid donor of mycobacterial D-
arabinofuranosyl residues during AG biosynthesis
 This step is catalyzed by a ribosyltransferase that has recently
been characterized and shown to be essential for growth Other
enzymes essential for arabinogalactan biosynthesis including
UDP-galactopyranose mutase (glf gene), galactofuranosyl
transferase and dTDP-6-deoxy-Llyxo- 4-hexulose reductase,

Jimma University
ANTI-TUBERCULOSIS DRUGS IN CURRENT
CLINICAL PRACTICE
 Current TB therapy, also known as DOTS :
 INH and ethonamide -Enoyl acyl carrier protein reductase
(InhA)
 Rifampin -RNA polymerase
 Pyrazinamide -Membrane energy metabolism pncA
 Ethambutol - Arabinsyltransfe embCAB
 Fluoroquinolone -DNA gyrase
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 Streptomycin, Kanamycin and Capreomycin – inhibit
protein synthesis by modification of ribosomal subunit
16s rRNA
 Ethonamide -Acyl carrier protein reductase
 PAS - Thymidylatesynthase(TlyA) ,TB drug active
against non growing mycobacterium bacteria
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Limitations of current TB drugs
 The length of therapy makes patient compliance difficult
 most of the TB drugs available today are ineffective against
persistent bacilli, except for RIF and PZA
 RIF is active against both actively growing and slow metabolizing
non-growing bacilli and PZA is active against semi-dormant non-
growing bacilli
 However, there are still persistent bacterial populations that are
not killed by any of the available TB drugs
 there is a need to design new drugs that are more active against
slowly growing or non-growing persistent bacilli
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Jimma University
CONCLUTION AND RECCOMENDATION
 There are many Mycobacterium metabolic pathways that can
be used as drug target.
 These metabolic pathways should be unique and essential to
the pathogen to develop effective drugs.
 Although many essential genes for mycobacterium are
identified by modern genetic tools, there is gap to identify exact
function for most of essential genes.
 better understanding on the physiology of mycobacterium
during the latent period will help in the identification of new
drug targets
 Identification of these targets will to help produce new drugs
against tuberculosis
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Jimma University
Con…
Based on the above conclusion the following recommendation
forwarded:
 The lists of potential drug targets encoded in the genome of
Mycobacterium should be explored
 More research should be done to identify exact function of all
essential gene for latent infection and virulence
 Research should involve testing new or reformulated drug,
combination of different drugs to shorten therapy,
supplementation and enhancements of existing drugs
 Existing drugs should be modified because of continuous
development of drug resistance and to make less toxic
 More research should be conducted on molecular targets of
Mycobacterium
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TB DRUG TARGETS

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