MT-Einstein UNIVERSITY OF RWANDA Pharmacy STUDENT YEAR4 2018-19

DRUG RESISTANCE MECHANISMS

MYCOBACTERIUM TUBERCULOSIS

Abstract: Tuberculosis (TB) is a serious public health

problem worldwide. Its situation is worsened by the presence
of multidrug resistant (MDR) strains of Mycobacterium
tuberculosis, the causative agent of the disease. In recent
years, even more serious forms of drug resistance have been
reported. A better knowledge of the mechanisms of drug
resistance of M. tuberculosis and the relevant molecular
mechanisms involved will improve the available techniques
for rapid drug resistance detection and will help to explore
new targets for drug activity and development. This review
article discusses the mechanisms of action of anti-
tuberculosis drugs and the molecular basis of drug resistance
in M. tuberculosis.

Keywords: drug resistance; molecular mechanisms;

Mycobacterium tuberculosis

1. INTRODUCTION

Tuberculosis (TB) remains as an important infectious disease and

public health concern worldwide. According to the latest World
Health Organization (WHO) report, there were an estimated 8.6
million incident cases of TB in 2012 and 1.3 million deaths were
attributed to the disease. More than half a million cases occurred in
children and 320,000 deaths were reported among HIV-infected
persons. However, even more disturbing is the emergence of drug
resistance. In 2012, there were an estimated 450,000 cases of
multidrug resistant (MDR)-TB and 170,000 deaths were due to it.
MDR-TB is caused by strains of Mycobacterium tuberculosis that are
resistant to at least rifampicin and isoniazid, two key drugs in the
treatment of the disease. Since 2006, it has been recognized the
presence of even more resistant strains of M. tuberculosis labelled as
extensively drug resistant (XDR)-TB . These strains in addition to
being MDR are also resistant to any fluoroquinolone and to at least
one of the injectable second-line drugs: kanamycin, capreomycin or
amikacin. More recently, a more worrying situation has emerged with
the description of M. tuberculosis strains that have been found
resistant to all antibiotics that were available for testing, a situation
labelled as totally drug resistant (TDR)-TB . Early detection of all
forms of drug resistance in TB is a key factor to reduce and contain
the spread of these resistant strains. A better knowledge of the
mechanisms of action of anti-TB drugs and the development of drug
resistance will allow identifying new drug targets and better ways to
detect drug resistance. The following sections will review the mode
of action and resistance mechanisms of the main anti-TB drugs as
well as new drugs recently described with anti-TB activity.

2. FIRST-LINE ANTI-TB DRUGS

2.1. Rifampicin
Rifampicin is a rifamycin derivative introduced in 1972 as an anti-
tuberculosis agent. It is one of the most effective anti-TB antibiotics
and together with isoniazid constitutes the basis of the multidrug
treatment regimen for TB. Rifampicin is active against growing
and non-growing (slow metabolizing) bacilli. The mode of action of
rifampicin in M. tuberculosis is by binding to the β-subunit of the
RNA polymerase, inhibiting the elongation of messenger RNA .The
majority of rifampicin-resistant clinical isolates of M. tuberculosis
harbor mutations in the rpoB gene that codes for the β-subunit of
the RNA polymerase. As a result of this, conformational changes
occur that decrease the affinity for the drug and results in the
development of resistance. In about 96% of M. tuberculosis isolates
resistant to rifampicin, there are mutations in the so-called ―hot-spot
region‖ of 81-bp spanning codons 507–533 of the rpoB gene. This
region is also known as the rifampicin resistance-determining region.
Mutations in codons 516, 526 and 531 are the most commonly
associated mutations with rifampicin resistance in the majority of
studies. Although less frequent, some reports have also noted the
occurrence of mutations outside of the hot-spot region of rpoB. Cross-
resistance with other rifamycin can occur. Mutations in some codons
(e.g., 518 or 529) have been associated with low-level resistance to
rifampicin but still susceptible to other rifamycins, such as rifabutin
or rifalazil. This is important for TB patients that need to receive
antiretroviral therapy since rifabutin is a less effective inducer of the
cytochrome P450 CYP3A oxidative enzyme. On the other hand,
mono resistance to rifampicin is quite rare and almost all rifampicin-
resistant strains are also resistant to other drugs, especially to
isoniazid. This is the reason why rifampicin resistance is considered
as a surrogate marker for MDR-TB
Recent genome sequencing studies have uncovered the acquisition of
compensatory mutations in rpoA and rpoC, encoding the α and β'
subunits of RNA polymerase, in rifampicin-resistant strains with
mutations in rpoB. These compensatory mutations would be
responsible for restoring the fitness of these strains in vivo and have
also been associated with a higher transmissibility in some settings.

2.2. Isoniazid
Isoniazid was introduced in 1952 as an anti-TB agent and it remains,
together with rifampicin, as the basis for the treatment of the disease.
Unlike rifampicin, isoniazid is only active against metabolically-
active replicating bacilli. Also known as isonicotinic acid hydrazide,
isoniazid is a pro-drug that requires activation by the
catalase/peroxidase enzyme KatG, encoded by the katG gene, to
exert its effect. Isoniazid acts by inhibiting the synthesis of mycolic
acids through the NADH-dependent enoyl-acyl carrier protein
(ACP)-reductase, encoded by inhA although simple in its structure,
resistance to this drug has been associated with mutations in several
genes, such as katG, inhA, ahpC, kasA and NDH. The two main
molecular mechanisms of isoniazid resistance are associated with
gene mutations in katG and inhA or its promoter region. Indeed,
numerous studies have found mutations in these two genes as the
most commonly associated with isoniazid resistance.

Among these, the most prevalent gene mutation has been identified
as S315T in katG resulting in an isoniazid product deficient in
forming the isoniazid-NAD adduct needed to exert its antimicrobial
activity. This mutation has been consistently associated with high-
level resistance (MIC > 1 µg/mL) to isoniazid and occurs more
frequently in MDR strains. The second most common mutation
occurs in the promoter region of inhA causing an overexpression of
InhA or less frequently, a mutation in its active site, which decreases
its affinity for the isoniazid-NAD adduct. The most prevalent
mutation found is at position −15C/T and is more commonly
associated with low level resistance to isoniazid (MIC < 1 µg/mL).
Mutations in inhA not only cause resistance to isoniazid but also to
the structurally related drug ethionamide, which shares the same
target .A recent study found that a mutation in the inhA regulatory
region together with a mutation in the inhA coding region produced
high-level isoniazid resistance and also cross-resistance to
ethionamide. One recent interesting finding showed that the 4R
isomer of the isoniazid-NADP adduct causes inhibition of the
dihydrofolate reductase (DfrA) in M. tuberculosis, suggesting that
mutations in dfrA could possibly play a role in resistance to
isoniazid. Moreover, an analysis of the proteome of isoniazid targets
in M. tuberculosis identified 16 other proteins, in addition to InhA
and DfrA, that were bound by these adducts with high affinity, which
could signal other not yet clearly defined actions of isoniazid on the
bacteria. Two recent studies, however, have failed to identify any
mutation in dfrA associated with resistance to isoniazid. In M.
tuberculosis, ahpC encodes an alkyl hydroperoxidase reductase that
is implicated in resistance to reactive oxygen intermediates and it was
initially proposed that mutations in the promoter of ahpC could be
used as proxy markers for isoniazid resistance It is now better
understood that mutations in the promoter of ahpC are compensatory
mutations for the loss of catalase/peroxidase activity rather than the
cause for isoniazid resistance. Moreover, overexpression of AhpC
does not confer resistance to isoniazid. Several studies have found
single nucleotide polymorphisms in other genes in isoniazid resistant
clinical isolates of M. tuberculosis, including kasA and the oxyR-
ahpC and furA-katG intergenic regions However, their direct role as
a cause of isoniazid resistance has not been fully demonstrated. On
the other hand, co-resistance to isoniazid and ethionamide has been
clearly demonstrated to be caused by mutations in ndh in M.
smegmatis and M. bovis BCG, by altering the NADH/NAD ratios
inside the cell, leading to a competitive inhibition of the INH-NAD
adduct. A recent study has also found that a silent mutation in mabA
conferred isoniazid resistance through upregulation of inhA in M.
tuberculosis.

2.3. Ethambutol
Ethambutol was first introduced in the treatment of TB in 1966 and
is part of the current first-line regimen to treat the disease.
Ethambutol is bacteriostatic against multiplying bacilli interfering
with the biosynthesis of arabinogalactan in the cell wall. In M.
tuberculosis, the genes embCAB, organized as an operon, code for
arabinosyl transferase, which is involved in the synthesis of
arabinogalactan, producing the accumulation of the intermediate D-
arabinofuranosyl-P-decaprenol. The recognized mechanism of
resistance to ethambutol has been linked to mutations in the gene
embB with mutations at position embB306 as the most prevalent in
most of the studies performed. Some studies, however, have also
found mutations in embB306 in ethambutol susceptible isolates.
Moreover, a study with a large number of M. tuberculosis isolates
found that mutations in embB306 were not necessarily associated
with resistance to ethambutol but with a predisposition to develop
resistance to increasing number of drugs and to be transmitted. In
fact, allelic exchange studies have shown that individual mutations
causing certain amino acid substitutions produced ethambutol
resistance, while other amino acid substitutions had little or no effect
on ethambutol resistance. The same authors have more recently
reported that mutations in the decaprenylphosphoryl-B-D-arabinose
(DPA) biosynthetic and utilization pathway genes, Rv3806c and
Rv3792, together with mutations in embB and embC accumulate,
giving rise to a range of MICs of ethambutol depending on mutation
type and number. These findings could have influence on the correct
detection of ethambutol resistance by current molecular methods.
Mutations in embB306 then, cause variable degrees of ethambutol
resistance and are required but are not enough to cause high-level
resistance to ethambutol. There remain about 30% ethambutol
resistant strains that do not present any mutation in embB stressing
the need to identify other possible mechanisms of drug resistance to
this drug.

2.4. Pyrazinamide
Pyrazinamide was introduced into TB treatment in the early 1950s
and constitutes now part of the standard first-line regimen to treat the
disease. Pyrazinamide is an analog of nicotinamide and its
introduction allowed reducing the length of treatment to six months.
It has the characteristic of inhibiting semi-dormant bacilli residing in
acidic environments such as found in the TB lesions.
Pyrazinamide is also a pro-drug that needs to be converted to its active
form, pyrazinoic acid, by the enzyme
pyrazinamidase/nicotinamidase coded by the pncA gene .The
proposed mechanism of action of pyrazinamide involves conversion
of pyrazinamide to pyrazinoic acid, which disrupts the bacterial
membrane energetics inhibiting membrane transport. Pyrazinamide
would enter the bacterial cell by passive diffusion and after
conversion to pyrazinoic acid it is excreted by a weak efflux pump.
Under acid conditions, the protonated pyrazinoic acid would be
reabsorbed into the cell and accumulated inside, due to an inefficient
efflux pump, resulting in cellular damage. One study has also found
that pyrazinoic acid and its n-propyl ester can inhibit the fatty acid
synthase type I in replicating M. tuberculosis bacilli.
A recent study, however, has challenged the previous model by
proposing that pyrazinoic acid inhibits trans-translation, a process of
ribosome-sparing in M. tuberculosis. The study was performed in
pyrazinamide-resistant strains lacking mutations in pncA but that had
mutations in rpsA identifying the ribosomal protein 1 (RpsA) as the
proposed target. Overexpression of RpsA conferred increased
resistance to pyrazinamide and pyrazinoic acid was confirmed to be
bound to RpsA. While a very intriguing hypothesis as a target for
pyrazinamide, the failure to perform allelic transfers in this study
makes it difficult to conclude that in fact mutations in rpsA are the
target of pyrazinamide.
Mutations in the gene pncA remain as the most common finding in
pyrazinamide resistant strains. These mutations, however, are
scattered throughout the gene but most occur in a 561-bp region in
the open reading frame or in an 82-bp region of its putative promoter.
Some few studies have reported the occurrence of pyrazinamide
resistant strains without any mutation in pncA stating that the
resistance could be due to mutations in another not yet identified
regulatory gene. Based on the current evidence, the contribution of
mutations in rpsA to pyrazinamide resistance remains limited.
2.5. Streptomycin
Originally isolated from the soil microorganism Streptomyces
griseus, streptomycin was the first antibiotic to be successfully used
against TB. Unfortunately, as soon as it was prescribed, resistance to
it emerged, a result of being administered as monotherapy.
Streptomycin is an aminocyclitol glycoside active against actively
growing bacilli and its mode of action is by inhibiting the initiation
of the translation in the protein synthesis. More specifically,
streptomycin acts at the level of the 30S subunit of the ribosome at
the ribosomal protein S12 and the 16S rRNA coded by the genes rpsL
and rrs, respectively.
Consequently, mutations in rpsL and rrs are the major mechanisms
of resistance to streptomycin but account for 60%–70% of the
resistance found. Among the mutations reported in rpsL, a
substitution in codon 43 from lysine to arginine has been the most
commonly reported. This mutation produces high-level resistance to
streptomycin. In rrs the most common mutations occur around
nucleotides 530 and 915. There remain an important percentage of
strains resistant to streptomycin that lack mutations in either of these
two genes, suggesting additional mechanisms of resistance.

In the last years, it has also been reported that mutations in gidB, a
gene encoding a conserved 7-methylguanosine methyltransferase
specific for the 16S rRNA, confers low-level resistance to
streptomycin.
3. SECOND-LINE ANTI-TB DRUGS

3.1. Fluoroquinolones
Fluoroquinolones are currently in use as second-line drugs in the
treatment of MDR-TB. Both ciprofloxacin and ofloxacin are
synthetic derivatives of the parent compound nalidixic acid,
discovered as a by-product of the antimalarial chloroquine. Newer-
generation quinolones such as moxifloxacin and gatifloxacin,
levofloxacin, norfloxacin are being evaluated in clinical trials and
proposed as first-line antibiotics with the purpose of shortening the
length of treatment in TB.
The mode of action of fluoroquinolones is by inhibiting the
topoisomerase II (DNA gyrase) and topoisomerase IV, two critical
enzymes for bacterial viability. These proteins are encoded by the
genes gyrA, gyrB, and parC and parE, respectively In M.
tuberculosis, only type II topoisomerase (DNA gyrase) is present
and, thus, is the only target of fluoroquinolone activity .Type II
topoisomerase is a tetramer formed by two α and β subunits, coded
by gyrA and gyrB, respectively, which catalyzes the supercoiling of
DNA. The main mechanism of development of fluoroquinolone
resistance in M. tuberculosis is by chromosomal mutations in the
quinolone resistance-determining region of gyrA or gyrB. The most
frequent mutations found are at position 90 and 94 of gyrA but
mutations at position 74, 88 and 91 have also been reported. A
recent systematic review of fluoroquinolone resistance-associated
gyrase mutations in M. tuberculosis has been published. One
interesting finding in M. tuberculosis is the presence of a natural
polymorphism at position 95 in gyrA that is not related to
fluoroquinolone resistance since it is also found in
fluoroquinolone-susceptible strains. Another interesting finding has
been the report that the simultaneous occurrence of mutations T80A
and A90G in gyrA led to hyper susceptibility to several quinolones.
This finding could point out that the problem of fluoroquinolone
resistance in M. tuberculosis might be more complex than was
thought initially.

Cross-resistance is assumed to occur between fluoroquinolones

although isolated reports have acknowledged the presence of strains
resistant to gatifloxacin and moxifloxacin that were still susceptible
to ofloxacin. Also, the involvement of efflux mechanisms has been
suggested as a possible cause for fluoroquinolone resistance in M.
tuberculosis.

3.2. Kanamycin, Capreomycin, Amikacin, Viomycin

These four antibiotics have the same mechanism of action by
inhibiting the protein synthesis but, while kanamycin and amikacin
are aminoglycosides, capreomycin and Viomycin are cyclic peptide
antibiotics. All four are second-line drugs used in the management of
MDR-TB. Kanamycin and amikacin inhibit protein synthesis by
alteration at the level of 16S rRNA. The most common mutations
found in kanamycin-resistant strains are at position 1400 and 1401
of the rrs gene, conferring high-level resistance to kanamycin and
amikacin. However, mutations at position 1483 have also been
reported. Full cross-resistance between kanamycin and amikacin is
not complete, as previously thought. Some studies have shown
variable levels and patterns of resistance suggesting that other
mechanisms of resistance might be possible. In concordance with
this, a low-level resistance to kanamycin has been associated with
mutations in the promoter region of the eis gene, encoding an
aminoglycoside acetyltransferase. Mutations at position −10 and
−35 of the eis promoter led to an overexpression of the protein and
low-level resistance to kanamycin but not to amikacin. These
mutations were found in up to 80% of clinical isolates showing low-
level resistance to kanamycin.
Capreomycin and Viomycin, on the other hand, have a similar
structure and bind at the same site in the ribosome, at the interface
of the small and large subunits. They show full cross-resistance as
reported in previous studies. Mutations in the tlyA gene have also
been associated with resistance to capreomycin and Viomycin. TlyA
is an rRNA methyltransferase specific for 2'-O-methylation of
ribose in rRNA. Mutations in tlyA determine the absence of
methylation activity. Although some studies did not find this
association, a recent meta-analysis, evaluating the association of
genetic mutations and resistance to second-line drugs, has confirmed
the presence of tlyA mutations in addition to mutations in rrs and
eis.

3.3. Ethionamide
Ethionamide is a derivative of isonicotinic acid structurally similar to
isoniazid. It is also a pro-drug requiring activation by a
monooxygenase encoded by the ethA gene. It interferes with the
mycolic acid synthesis by forming an adduct with NAD that inhibits
the enoyl-ACP reductase enzyme. EthA is regulated by the
transcriptional repressor EthR. Resistance to ethionamide occurs by
mutations in etaA/ethA, ethR and also mutations in inhA, which cause
resistance to both isoniazid and ethionamide. Moreover, studies
with spontaneous isoniazid- and ethionamide-resistant mutants of
M. tuberculosis found that they map to mshA, encoding an enzyme
essential for mycothiol biosynthesis.

3.4. Para-Amino Salicylic Acid

Although it was one of the first anti-tuberculosis drugs used in the
treatment of the disease, together with isoniazid and streptomycin,
para-amino salicylic acid or PAS is now considered as a second-line
drug part of the treatment regimen for MDR-TB. Until recently, its
mechanism of action was not completely defined. It has been
proposed that being an analog of para-amino benzoic acid, it must
compete with it for dihydropteroate synthase, interfering in the
process of folate synthesis. A study using transposon mutagenesis
identified mutations in the thyA gene associated with resistance to
PAS that were also present in clinical isolates resistant to PAS. A
recent study has also identified various missense mutations in folC
encoding dihydrofolate synthase that conferred resistance to PAS in
laboratory isolates of M. tuberculosis. In a panel of 85 clinical MDR-
TB isolates, mutations in folC were identified in five isolates
resistant to PAS. Nevertheless, just less than 40% of PAS-resistant
strains had mutations in thyA indicating that still other mechanisms
of resistance to the drug might exist.

3.5. Cycloserine
Cycloserine is an oral bacteriostatic second-line anti-tuberculosis
drug used in MDR-TB treatment regimens. It is an analog of D-
alanine that by blocking the activity of D-alanine: D-alanine ligase
inhibits the synthesis of peptidoglycan. It can also inhibit D-alanine
racemase (AlrA) needed for the conversion of L-alanine to D-
alanine. Although the actual target of cycloserine in M. tuberculosis
is not completely elucidated, in previous studies in M. smegmatis it
was shown that overexpression of alrA led to resistance to
cycloserine in recombinant mutants. More recently, it has also been
shown that a point mutation in cycA, which encodes a D-alanine
transporter, was partially responsible for resistance to cycloserine
in M. bovis BCG.

3.6. Thioacetazone
Thioacetazone is an old drug that was used in the treatment of TB due
to its favorable in vitro activity against M. tuberculosis and it’s very
low cost. It has toxicity problems, however, especially in patients co-
infected with HIV. It belongs to the group 5 drugs of the WHO and
acts by inhibiting mycolic acid synthesis.

3.7. Macrolides
Macrolides are more frequently recommended for the treatment of
other mycobacterial infections due to their limited activity against
M. tuberculosis. Among them, clarithromycin is considered as part
of the group 5 drugs of the WHO. Intrinsic resistance to macrolides
has been attributed to low cell wall permeability and the expression
of emr37, a gene that codifies for a methylase at a specific site in
the 23S rRNA, blocking the binding of the antibiotic. In studies
performed with M. tuberculosis and Mycobacterium microti, it was
found that this intrinsic resistance was inducible with sub-inhibitory
concentrations of clarithromycin, leading to four- to eight-fold
increase in MIC values. Moreover, in studies performed with clinical
isolates of M. tuberculosis, sub-inhibitory concentrations of
ethambutol reversed resistance to clarithromycin, signaling a
permeability barrier as the cause of the intrinsic resistance to the
macrolide.

3.8. Clofazimine
Clofazimine is a riminophenazine compound reported long ago as
having anti-TB activity. Due to the availability of other effective anti-
TB drugs at the time and some undesirable side-effects, such as
pigmentation of the skin, its use was more limited to the treatment of
leprosy. It is now considered in the group 5 drugs of the WHO for
the management of MDR-TB. Until recently, the exact mode of
action of this antibiotic was not completely understood. Recent
studies, however, have signaled the outer membrane as the possible
target of Clofazimine. Another study found that in M. tuberculosis
Clofazimine is reduced by NADH dehydrogenase and subsequently
after spontaneous re-oxidation liberates bactericidal levels of
reactive oxygen species (ROS) .
Resistance to Clofazimine has not yet been fully characterized;
however, a recent study has found that in spontaneous mutants of
the reference strain H37Rv,

.
3.9. Linezolid
Also part of the category 5 drugs of second-line anti-TB drugs,
linezolid is an oxazolidinones originally approved for clinical use in
the treatment of skin infections and nosocomial pneumonia caused
by Gram-positive bacteria. The mode of action of linezolid is by
inhibition of an early step in the synthesis of proteins, binding to the
50S ribosomal subunit. Resistance to linezolid in M. tuberculosis is
still unusual, but a study analyzing 210 MDR strains found 1.9% of
strains being resistant to linezolid. Further analysis of in vitro
selected linezolid-resistant mutants found that strains with mutations
in the 23S rRNA had MICs of 16–32 µg/mL, while strains with MICs
of 4–8 µg/mL or susceptible strains showed no mutations. A more
recent study using next-generation sequencing has also found the
mutation T460C in rplC, encoding the 50S ribosomal L3 protein, in
in vitro-selected mutants and clinical isolates of M. tuberculosis
resistant to linezolid. Previous studies have also found evidence of
the possible involvement of efflux pumps in the resistance of M.
tuberculosis to linezolid.

SUMMARY TABLE OF DRUG 1ST AND SECOND LINE

ANTI TB DRUG. GENES THAT IS MUTATED FOR
RESISTANCE CREATIONS
Table 1 gives an overview of the first- and second-line anti-
tuberculosis drugs currently in use and target of action.

Table 1. First- and second-line TB drugs, genes involved in their

activation and mechanisms involved.
Drug Gene Mechanism Involved
Isoniazid katG, inhA Catalase/peroxidase; enoyl reductase
Rifampicin rpoB RNA polymerase
Pyrazinamide pncA, rpsA Pyrazinamidase; ribosomal protein 1
Ethambutol embB Arabinosyl transferase
Streptomycin rpsL, rrs, gidB S12 ribosomal protein, 16A rRNA, 7-
methylguanosine methyltransferase
Quinolones gyrA, gyrB DNA gyrase
Capreomycin rrs, tlyA 16S rRNA, rRNA methyltransferase
Kanamycin/Amikacin rrs 16S rRNA
Ethionamide ethA Enoyl-ACP reductase
Para-amino salicylic thyA, folC Thymidylate synthase A
acid (PAS)

4. NEW ANTI-TB DRUGS

Notwithstanding the alleged lack of interest of the pharmaceutical

industry for the development of new antibiotics, there are several anti-
tuberculosis drugs in the pipeline and some of them are already being
evaluated in clinical trials and in new combinations with the purpose
of reducing the length of TB treatment.

4.1. Bedaquiline
Formerly known as TMC207 or R207910, bedaquiline is a new
antibiotic belonging to the class of diarylquinolines with specific
activity against M. tuberculosis, which has also shown in vitro
activity against other non-tuberculous mycobacteria. Bedaquiline was
discovered after a high-throughput evaluation of thousands of
compounds using Mycobacterium smegmatis in a whole-cell assay.
The drug showed in vitro and in vivo activity against M. tuberculosis
and then entered into clinical evaluation for drug susceptible and
MDR-TB. Based on the results of two phase II clinical trials,
bedaquiline has recently received conditional approval for the
treatment of MDR-TB under the trade name Sirturo. A black box
warning is, however, accompanying this authorization due to the
reported unexplained deaths and QT interval prolongation. Recent
reviews and evaluation of this new drug have been published. A phase
III clinical trial was scheduled to begin in 2013 but has not yet started.
Bedaquiline is also being evaluated in new combination regimens
with the purpose of reducing the length of treatment.
The mode of action of bedaquiline is by inhibiting the ATP synthase
of M. tuberculosis, which was a completely new target of action for
an antimycobacterial drug. This mode of action was discovered by
analyzing M. tuberculosis and M. smegmatis mutants resistant to
bedaquiline. By sequencing the genome of these mutants and
comparing to that of the susceptible strains, the only mutation found
was in the atpE gene, which encodes the c part of the F0 subunit of
the ATP synthase. This is a complex structure that generates the ATP
needed by the mycobacterial cell for which bedaquiline has a favored
specificity compared to mitochondrial ATP synthase. The structure
of bedaquiline is shown in Figure 1.

Figure 1. Structure of bedaquiline.

The most prevalent mutation in the atpE gene found in bedaquiline
resistant mutants is A63P but also I66M has been found. The latter
introduces a modification that interferes the proper binding of
bedaquiline to its target. Nevertheless, in a study to further assess
the mechanisms of resistance to bedaquiline in M. tuberculosis, it
was found that only 15 out of 53 resistant mutants had mutations in
atpE. The other 38 strains lacked mutations in atpE or even in the F0
or F1 operons, which suggests that other mechanisms of resistance
are still possible.

4.2. Delamanid
Delamanid, previously known as OPC-67683, is a derivative of nitro-
dihydro-imidazooxazole with activity against M. tuberculosis that
acts by inhibiting the synthesis of mycolic acid and is undergoing
clinical evaluation in a phase III trial. The structure of delamanid is
shown in Figure 2. Delamanid was previously shown to have a very
good in vitro and in vivo activity against drug-susceptible and drug-
resistant M. tuberculosis, as well as good early bactericidal activity
comparable to that of rifampicin. Delamanid has more recently shown
its safety and efficacy in a clinical evaluation for MDR-TB.

Figure 2. Structure of delamanid.

The specific mode of action of delamanid is by inhibition of the
mycolic acid synthesis but it differs from isoniazid in that, it only
inhibits methoxy- and keto-mycolic acid while isoniazid also
inhibits α-mycolic acid .
Delamanid also requires reductive activation by M. tuberculosis to
exert its activity. In experimentally generated delamanid-resistant
mycobacteria, a mutation was found in the Rv3547 gene, suggesting
its role in the activation of the drug.

4.3. PA-824
PA-824 is a bicyclic derivative of nitro-imidazole that showed
specific activity against M. tuberculosis. The structure of PA-824 is
shown in Figure 3. This small-molecule compound showed a very
good in vitro and in vivo activity in animal models and it also showed
to be safe and well tolerated. PA-824 is currently undergoing further
clinical evaluations.

Figure 3. Structure of PA-824.

PA-824 needs to be activated by a nitro-reductase to exert its activity
and it inhibits the synthesis of protein and cell wall lipids. The
mechanism of resistance to PA-824 has been shown to be most
commonly associated with loss of a specific glucose-6-phosphate
dehydrogenase (FGD1) or the dezaflavin cofactor F420.

More recently, a nitro-imidazo-oxazine-specific protein causing

minor structural changes in the drug has also been identified.

4.4. SQ-109
Compound SQ-109 is a synthetic analogue of ethambutol that has
shown in vitro and in vivo activity against drug-susceptible and drug-
resistant M. tuberculosis. The structure of SQ-109 is shown in Figure
4. It has also been shown to possess synergistic in vitro activity when
combined with first-line drugs, and more interestingly, when
combined with bedaquiline and the oxazolidinone PNU10048. SQ-
109 is currently being evaluated in a phase II clinical trial.
The mode of action of SQ-109 is by interfering with the assembly of
mycolic acids into the bacterial cell wall core, resulting in
accumulation of trehalose monomycolate, a precursor of the trehalose
dimycolate. Transcriptional studies have shown that, similar to other
cell wall inhibitors such as isoniazid and ethambutol, SQ-109 induces
the transcription of the iniBAC operon required for efflux pump
functioning. Moreover, by producing spontaneously generated
resistant mutants to SQ-109 analogs and performing whole-genome
sequencing, mutations in the mmpL3 gene were identified, suggesting
MmpL3 as the target of SQ-109 and signaling MmpL3 as transporter
of trehalose monomycolate.
Figure 4. Structure of SQ-109.
4.5. Benzothiazinones
A new class of drug with antimycobacterial activity, 1,3-
benzothiazin-4-one or benzothiazinone (BTZ), was recently
described. The lead compound, 2-[2-S-methyl-1,4-dioxa-8-
azaspiro[4.5]dec8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-
benzothiazin-4-one (BTZ043) was found to have in vitro, ex vivo and
in vivo activity against M. tuberculosis. It was also found to be active
against drug-susceptible and MDR clinical isolates of M.
tuberculosis. Structure of BTZ043 is shown in Figure 5.
Figure 5. Structure of BTZ043.

By transcriptome analysis, the mode of action of BTZ043 was

initially spotted at the cell wall biogenesis level. By further genetic
analysis, using in vitro generated mutants, the target of the drug was
identified at the level of the gene rv3790, which together with rv3791
encode proteins that catalyze the epimerization of
decaprenylphosphoryl ribose (DPR) to decaprenylphosphoryl
arabinose (DPA), a precursor for arabinan synthesis needed for the
bacterial cell wall. DprE1 and DprE2 were proposed as names for
these two key enzymes. More recent studies have characterized more
precisely the mechanism of action of BTZ043 by showing that the
drug is activated in the bacteria through reduction of an essential nitro
group to a nitro derivative, which can react with a cysteine residue in
DprE1. In studies with M. smegmatis, an alternative mechanism of
resistance has been suggested. The overexpression of a nitro-
reductase NfnB led to the inactivation of the drug by reducing an
essential nitro group to an amino group. Although M. tuberculosis
apparently lacks nitro-reductases able to reduce the drug, this
finding could be important for development of new BTZ analogues
with improved activity.
Just recently a series of piperazine-containing BTZs has been
reported. The lead compound PBTZ169 has improved activity, safety
and efficacy in animal models and has shown in vitro synergy with
bedaquiline signaling it as an attractive new candidate for further
clinical development.

5. CONCLUDING REMARKS

Drug resistance in TB remains a man-made phenomenon. It emerges

as a result of spontaneous gene mutations in M. tuberculosis that
render the bacteria resistant to the most commonly used anti-TB
drugs. Among the reasons for this, the non-compliance with the
treatment regimens is signaled as the first cause. The standard
treatment of TB calls for a six-month regimen of four drugs that in
the case of MDR-TB is extended to 18–24 months involving second-
line drugs. This makes compliance with the treatment regimens very
challenging and the rates of non-adherence could be high, resulting in
poor outcomes and further dissemination of MDR strains.
Notwithstanding the fact that mutations in a number of genes are
clearly associated with drug resistance in M. tuberculosis, there are
still many cases where resistant strains do not harbor any known
mutation. For example, a recent study using whole-genome
sequencing identified new genes and intergenic regions that were
associated with drug resistance and its evolution, showing that TB
drug resistance is a phenomenon more complex than previously
assumed. More clarification is needed on the role of specific gene
mutations and the development of MDR- or XDR-TB, or the relation
between drug resistance and fitness of the bacteria. A better
knowledge is also required on the role of efflux pump mechanisms
and the development of clinical drug resistance, or the role of porins,
if any, on the intrinsic resistance to certain antibiotics. It is, thus, quite
important to further our knowledge of additional mechanisms of drug
resistance to the available anti-TB drugs. This could have a major
impact on the dynamics of TB transmission and for the discovery and
development of new anti-TB drugs.