CHAPTER-1 INTRODUCTION

ABSTRACT:

Tuberculosis is highly contagious disease which has cursed humanity for decades & its
resistance is a global threat, caused mainly by Mycobacterium tuberculosis. It has been
estimated that about one-fourth of the global population are suffering from tuberculosis,
which accounts for 9.6 million of new cases and 1.5 million of deaths annually1. The rise
of drug-resistant, multidrug-resistant, extensively drug-resistant and the recent cases of
total drug resistant TB has led to increase the challenges to eradicate TB globally. Searching
for new scaffolds against various DR-TB, MDR-TB, and XDR-TB strains are essential1.
As mycobacterium tuberculosis has gained resistance, there need of new molecular design
like hybrid drugs which are potent as well as used to overcome the resistance by various
mechanisms, so keeping this idea in mind the combination of two molecules/drugs can lead
to development of drugs which can be used for the treatment of tuberculosis1. Hybrid drug
is the combination of 2 or more drugs which are used to target the multiple target at a time.
The molecular hybridized potent molecules is a useful tool for drug discovery to target
multiple diseases2. It opens up door for better opportunities of new hybrid drugs for the
treatment of a number of diseases including cancer, malaria, tuberculosis and AIDS and
also can provide multifunctional therapies in a single dose and therefore, by doing so, more
specific and powerful hybrid drugs can be synthesised. The above developed approach is a
great way for overcoming resistance in research field2.

INTRODUCTION:

The death due to TB is among the top ten leading cause’s globally, which is caused by
bacterial infection. Tuberculosis (TB) is an endemic disease caused by a single bacterial
infection, which is considered to be one of the lethal communicable diseases worldwide, which
is caused by Mycobacterium tuberculosis (MTB) whose reservoir is human dominant. The
epidemic of tuberculosis (TB) is catastrophic, which has a great influence on human health
globally, grasping global scrutiny which has hiked the number of cases worldwide,
endangering both developed and developing nations. It is Predicted that one fourth of the global
population are infected with TB. Two billion commuters worldwide, are considered to be
carrying non-eradicated intra-granulomatous TB bacilli as latent tuberculosis infection (LTBI).
The MTB is an aerobic Acid fast pathogen, which causes infection in the human lungs. The
Mycobacterium tuberculosis complex comprises of seven species: M. tuberculosis, M. bovis,
M. africanum, M. microti, M. canetti, M. caprae and M. pinipedi. Among this, M. tuberculosis

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is the most dominant bacterial species, which is responsible for the causes of Tuberculosis (TB)
in human & a less dominant of TB infection is governed by M. bovis and M. africanum.
According to WHO, in 2018, 10.4 million people were affected with TB, from which 1.7
million perish (with inclusion of 0.4 million people with HIV). The epidemiology of TB among
the nations with 10,000 incident cases, conclude that, over 95% of TB deaths exists in
developing nations. Seven global nations credits for 64% of the entire TB cases, from which
India is at the top in leading the count, pursued by Indonesia, China, Philippines, Pakistan,
Nigeria, and South Africa . TB is a prominent killer of HIV-positive patients, in 2018, 40% of
HIV deaths were due to TB. HIV infected patients are 20 to 40 times more endangered to
develop active TB than the normal individuals.

TB incidence in 2018, for the nations with at least 100,000 incident cases:

DRUG RESISTANCE TB (DRTB)

As from the past few decades, resistance among the drugs for the particular diseases has given
a critical warning alarm to human health. There arises a serious public health crisis in
Tuberculosis also which leads to health security threats. Drug-resistant TB (DR-TB) among
the patients is a serious & critical phase, which researchers are facing in recent times. As per
WHO report, in 2018 there were 600,000 modern cases with resistance to TB. Drug resistance

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TB rises due to inaccurate use of antibiotics in chemotherapy drug susceptible TB patients.

Types of DRTB includes:
1. Mono-resistance: Resistance to one, first-line anti-TB drug only.
2. Poly-resistance: Resistance to more than one, first-line anti-TB drug, other than both
isoniazid and rifampicin.
3. Multidrug resistance (MDR): Resistance to at least both isoniazid and rifampicin.
4. Extensive drug resistance (XDR): Resistance to any fluoroquinolone, and at least one
of three second-line injectable drugs (capreomycin, kanamycin and amikacin), in
addition to multidrug resistance.
5. Totally drug-resistant tuberculosis (TDR-TB): It is a comprehensive term
for tuberculosis strains that are resistant to a board range of drugs than strains,
collectively classified as extensively drug-resistant tuberculosis. TDR-TB has been
diagnosed in three nations; India, Iran, and Italy.

WHO interprets three, high burden countries (HBC) lists for the course of 2018–2022,
one for TB, one for MDR-TB and one for TB/HIV, each accounts for 30 nations. These all
represents as the top 20 nations in premises of the total number of predicted incident cases,
plus including further 10 more nations with the most harsh burden, which has maximum
incidence cases per capita, which was not included in the previous top 20 nations list, and that
accommodate a least possible outcome in terms of their total numbers of incident cases (10,000
per annum for TB, and 1000 per annum for TB/HIV and MDR-TB). Each series accounts for
about 90% of the national burden, from which most of the involved nations accounted, belongs
from the top 20 nations list.

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MDR-TB incidence for nations with 1000 incident cases:

These are the nations with immense rate of drug resistance rate with 1000 incident cases, which
comprises of, most of the Asian countries like India, Pakistan, and other countries like China
and Russia.

MECHANISM OF DRUG RESISTANCE:

The intrinsic ability of Mycobacterium Tuberculosis to sustain hosts immunity by gaining
resistance to most of the anti-tubercular agents available in the market has provoked the
severity of the disease. Due to this the drugs available for the chemotherapeutic treatment for
TB are limited to its use. Prolonged administration of the same drugs leads to poor patient
compliance, which results in increase in the resistance of Anti-TB drugs. The lack of use &
irrational use of antibiotics has insured M. tuberculosis by gaining resistance due to rapid
evolution of TB bacteria, patrolling from mono drug resistant to multidrug resistant (MDR),
extensively drug resistant (XDR), and eventually totally drug resistant (TDR), through
mutation. For the development of new classes of anti TB drugs, researcher first of all have to
understand the mechanism of drug resistance in TB.

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INTRINSIC RESISTANCE:
Intrinsic drug resistance is generally associated to the bizarre structure of the cell wall
composed of mycolic acid which is responsible for the low permeability for most of the
antibiotics used for TB. The recent studies, shows that the efflux mechanisms in an important
factor in accruing natural resistance to mycobacteria against antibiotics such as tetracycline,
fluoroquinolones and aminoglycosides. β-lactam antibiotics inhibit the activities of penicillin-
binding proteins by bind with them, which are involved in cell wall biosynthesis, but due to
possession of β-lactamase enzymes by mycobacteria, they degrades these drugs. Hence due to
this degradation, the mycobacteria has gained resistance to β-lactam antibiotics. Recent
research have shown that in M. tuberculosis, Rv1698 & MspA have the same function which
contributes to intrinsic resistance of hydrophilic compounds. With the help of genetic studies&,
bioinformatics analysis, both Rv1698 and Rv1973 are identified which are the mycobacterial
outer membrane proteins (OMPs) which is also responsible for the intrinsic resistance of the
antibiotics.
A part from this, the Physiological changes occurring within the individual is also responsible
for the resistance & antibiotic tolerance. Moreover, in M. tuberculosis, the MDR determinant
whiB7, whose removal give rise to a multidrug-susceptible phenotype, which plays an
important role in MDR TB. The cure of this intrinsic resistance is essential as due to this many
antibiotics cannot exert their effect as resistance gained by M. tuberculosis.
From past few decades Mycobacterium tuberculosis has evolved to greater extent & has gained
resistance to most of the drugs through various molecular mechanisms which helps the bacteria
to neutralize the effect of Anti TB drugs. Due to all this factors the treatment of TB is getting
very difficult & there arises a great challenge for the researchers for the development of new
drugs in treatment of TB. Furthermore, there are many more factors which also contribute to
the resistance of the M. tuberculosis.
1. Cell wall permeability: As, the cell wall of Mycobacterium Tuberculosis is made up
of mycolic acid, which act as a barrier & decrease the cell wall permeability of many
drugs, for example, the penetration of β-lactum antibiotics through the bacterial cell
wall is 100-fold slower than the E. coli cell wall. As M. tuberculosis is acid-fast Gram-
positive bacteria, the bacterial cell wall is highly thick and multilayered, which is
responsible for the drug penetration. As the intimate layer of peptidoglycan is concealed
by an additional layer of arabinogalactan, as both of this layer are highly hydrophilic,
thus affecting the penetration of hydrophobic moieties. These two layers are covalently

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linked to mycolic acids, long-chain fatty acids that form a hydrophobic barrier
restricting the entry of hydrophilic molecules
2. Specialized resistance mechanisms: While the mycobacterial cell wall hinders
antibiotic penetration specialized resistance mechanisms promotes the detoxification of
drug molecules & decrease the efficacy of the drugs. There are many more specialized
resistance mechanism which mycobacteria adapt in order to hinder the activity of drugs.
 Target alteration: In order to rescue the effect of antibiotic Mycobacterium
Tuberculosis alter the specified target. Which decrease the affinity of binding
site. By this mechanism mycobacteria reduces the binding affinity of macrolides
and lincosamides to their ribosomal target site. These drugs bind reversibly to a
specific site of the ribosomal RNA within the 50S subunit of bacterial
ribosomes, thus inhibiting translocation of peptidyl-tRN.This activity
suppresses protein synthesis, thereby inhibiting growth. Mycobacterial species
including M. tuberculosis and M. bovis are naturally resistant to macrolides and
lincosamides. Interestingly, the Pasteur vaccine strain BCG (Bacillus of
Calmette and Guérin) derived from M. bovis is uniquely susceptible to these
antibiotics. Comparative genomics revealed a chromosomal deletion causing
the loss of the erm37 (or rv1988) gene in the BCG genome). erm37, encoding a
ribosomal RNA methyltransferase, is located within a larger chromosomal locus
known as the RD2 (Region of Difference 2) which was deleted in BCG during
its culture passage. The intrinsic resistance to macrolides and lincosamides
could be restored to BCG by in Trans expression of the M. tuberculosis erm37,
which alters ribosomal structures by methylating the 23S ribosomal RNA. This
reduces the affinity of macrolides to ribosomes, thus lowering the inhibitory
activity of the drug on protein synthesis. Other ermgenes conferring macrolide
and lincosamide resistance were found in M. smegmatis and M. fortuitum,
whose expressions are inducible by exposure to macrolides and lincosamides.
The inducible expression of Erm37 was later found to be regulated by the MDR
transcription regulator WhiB7, which is discussed elsewhere in this chapter.
These studies suggest that the function of Erm37 and related ribosomal RNA
methyltransferase is specialized for macrolide and lincosamide resistance in
mycobacteria. Interestingly, a more recent study showed that Erm37, besides its
role in protecting the mycobacterial ribosome from macrolides and
lincosamides, is also involved in an epigenetic mechanism hijacking host
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macrophages gene expression control. In this study, Erm37 was shown to be

secreted by M. tuberculosis and localized to the host nucleus where it interacts
with chromatin and methylates histone H3 at H3R42, repressing the expression
of genes involved in the first-line defense against pathogenic mycobacteria The
antibiotic-induced expression of mycobacterial factors such as Erm37 or Eis
(see “Drug inactivation” below), which are involved in both virulence and drug
resistance, represents a dangerous evolution of infectious diseases against
antibiotic chemotherapies. Similar mechanism is used by M. tuberculosis to
neutralize activity of cyclic peptide antibiotics such as capreomycin and
viomycin, which are commonly used to treat MDR TB. Studies in M. smegmatis

and M. tuberculosis mapped resistance mutations to tlyA that encodes a 2′-O-

methyltransferase whose activity correlates with capreomycin and viomycin

resistance TlyA methylates both 16S and 23S ribosomal RNA at nucleotide
C1409 and C1920, respectively (Johansen et al. 2006), thereby rendering
ribosomes susceptible to the binding of capreomycin and viomycin
 Target mimicry: Molecular mimicry is a fascinating mechanism employed by
M. tuberculosis to neutralize the action of fluoroquinolones, synthetic
antibiotics that have recently become important for treating drug resistant TB
cases (Duncan and Barry 2004). Fluoroquinolones are bactericidal drugs that
kill bacterial cells by inhibiting DNA replication, transcription and repair. These
antibiotics bind DNA gyrase or topoisomerase in their complexes with DNA,
thereby stabilizing breaks while inhibiting resealing of the DNA strands. These
events eventually result in DNA degradation and cell death (Andriole 2005).
Whereas acquired fluoroquinolone resistance is commonly mapped to
mutations in DNA gyrase-encoded genes gyrA and gyrB, in M. tuberculosis),
its intrinsic resistance is attributed to a pentapeptide repeat protein called MfpA.
A study in M. smegmatis and M. bovis first found that expression of MfpA
correlates with fluoroquinolone resistance (Montero et al. 2001). MfpA is most
homologous to pentapeptide repeat proteins, within the sequences of which
every fifth amino acid is either leucine or phenylalanine. When the structure of
the M. tuberculosis MfpA was resolved, it was found that it closely resembles
the 3D structure of a DNA double helix (Ferber 2005; Hegde et al. 2005), with
tandems of pentapeptide repeats coiling around in a right-handed helix of the

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same width as DNA (Hegde et al. 2005; Morais Cabral et al. 1997). It was
proposed that MfpA sequesters fluoroquinolones in the mycobacterial
cytoplasm by mimicking the DNA structure, thus freeing DNA from the drug’s
action (Ferber 2005). Although the physiological function of MfpA and its
significance in fluoroquinolone resistance remain unknown, this finding reveals
a fascinating mechanism that bacterial pathogens may use to avoid succumb to
antibiotics.
 Drug modification: Mycobacterial species can also directly inactivate drugs
through chemical modifications such as acetylation. Aminoglycosides are
broad-spectrum antibiotics that constitute an important position in the history of
TB treatment. While streptomycin was the first effective antibiotic for TB,
kanamycin and amikacin are second-line drugs that are commonly used to treat
MDR TB cases. Resistance to these drugs in MDR strains is the hallmark to
define XDR TB. The precise mode of action of aminoglycosides remains to be
understood although they are thought to act mainly as inhibitors of protein
synthesis. The intrinsic resistance of M. tuberculosis to aminoglycosides is
provided by an acetyltransferase (Zaunbrecher et al. 2009) termed Eis
(enhanced intracellular survival), which was initially found to play a role in
mycobacterial survival in host macrophages (Wei et al. 2000). This protein was
more recently found to play an important role in manipulating the host innate
immunity against mycobacterial infection. Eis is secreted by M. tuberculosis to
the infected macrophage’s cytosol where it acetylates MKP7, a host
phosphatase that regulates the phosphorylation status of the protein kinases JNK
and p38 of the MAPK pathway. These modulations on the host signaling lead
to the suppression of host immune responses, namely autophagy, inflammation
and apoptosis, to mycobacterial infection (Kim et al. 2012). In a screen for
aminoglycoside resistance determinants, a cosmid library was constructed from
the genomic DNA of a kanamycin-resistant M. tuberculosis strain (Zaunbrecher
et al. 2009), then transformed to a susceptible strain and selected for kanamycin
resistance. Mapping of the cosmid conferring kanamycin resistance identified
mutations within the promoter region of eis, whose expression was previously
found to be regulated by the MDR transcription regulator WhiB7 (Burian et al.
2012, 2013; Morris et al. 2005). The mutations, which were later found in 80 %
of low-level kanamycin-resistant isolates (Campbell et al. 2011; Engstrom et
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al. 2011; Zaunbrecher et al. 2009), as well as in MDR strains (Huang et al.
2011), increased eis transcription by 180-fold (Zaunbrecher et al. 2009). In vitro
biochemical studies showed that Eis acetylates multiple amine groups of
aminoglycosides using acetyl coenzyme A as an acetyl donor (Chen et al. 2011),
thereby inactivating the antibiotics. A recent study further suggested that Eis
acetylates not only aminoglycosides but also capreomycin, a cyclic peptide
antibiotic now commonly used to treat MDR TB (Houghton et al. 2013). The
fact that Eis dually functions in protecting mycobacteria against both the host
immunity and antibiotics implies a sinister coevolution of virulence and
antibiotic resistance in M. tuberculosis.
 Drug degradation: Another method that bacterial pathogens including M.
tuberculosis use to subvert antibiotics’ action is to degrade those using
hydrolases. This mechanism is well studied in the case of β-lactams, drugs that
have virtually no effect on M. tuberculosis and related mycobacteria. These
antibiotics bind and inhibit penicillin-binding proteins (PBPs), which are
required for the assembly of the peptidoglycan network, thereby disrupting cell
wall synthesis and causing cell death. The genome of M. tuberculosis encodes
at least four major PBPs, all of which bind β-lactams at clinically achievable
concentrations (Chambers et al. 1995), indicating that low target affinity is not
the cause of β-lactam resistance in mycobacteria. Target accessibility due to the
impermeable cell wall, however, plays a clear role in mycobacterial β-lactams
resistance. In this regard, the long generation time of M. tuberculosis serves as
both pros and cons to the bacillus’ resistance. Carbapenems, for example, are
rather unstable and therefore loose activity much faster compared with the
mycobacterial growth rate. Nevertheless, continued intakes from daily regimens
could still allow sufficient accumulation to lethal concentrations that inhibit the
slow cell division machinery (Watt et al. 1992). For β-lactams, the paramount
resistance determinant was shown in mycobacteria to be β-lactamases,
hydrolytic enzymes that hydrolyze the β-lactam ring of the drugs (Chambers et
al. 1995). This knowledge is mostly obtained from studies in M. fallax, a
Mycobacterium species uniquely β-lactam susceptible (Kasik 1979; Quinting et
al. 1997). Permeability assays demonstrated that β-lactams penetrate through
the M. fallax cell wall at rates which are similar to those of other mycobacteria
and should allow accumulation of β-lactams to lethal concentrations (Quinting
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et al. 1997). When M. fallax was engineered to in trans express a β-lactamase

from M. fortuitum, its resistance immediately increased to levels comparable to
other species (Quinting et al. 1997), indicating that a lack of effective β-
lactamases underlies the bacillus β-lactam susceptibility. Although
mycobacterial β-lactamases are generally less effective than those of other
pathogenic bacteria, the slow penetration of β-lactams across their impermeable
cell wall renders this low β-lactamase activity good enough to protect
mycobacteria from β-lactams (Jarlier et al. 1991). BlaC, the most important β-
lactamase in M. tuberculosis, belongs to the Ambler class -A β-lactamases, of
which enzymatic activities and structures have been thoroughly studied
(Voladri et al. 1998; Wang et al. 2006). Possibly due to its large and flexible
substrate-binding site (Wang et al. 2006), BlaC exhibits broad substrate
specificity, including carbapenems that generally are β-lactamases-resistant
(Hugonnet and Blanchard 2007; Tremblay et al. 2010). β-Lactamase inhibitors
such as clavulanic acid are also less effective against BlaC than to other class A
enzymes. Expression of BlaC in M. tuberculosis is controlled by BlaI, a β-
lactam-induced, wingedhelix transcription repressor. In the absence of β-
lactams, BlaI forms homodimers that bind the promoter of blaC, thereby
inhibiting its transcription (Sala et al. 2009). When M. tuberculosis is exposed
to β-lactams, BlaI dissociates from its DNA binding site, thus derepressing blaC
transcription and leading to β-lactamase production (Sala et al. 2009). In
addition to BlaC, M. tuberculosis encodes at least three more β-lactamases,
BlaS, Rv0406c and Rv3677c, which provide lower β-lactamase activities
(Flores et al. 2005; Nampoothiri et al. 2008).
 Drug efflux: A method commonly used by bacterial pathogens to avoid
succumbing to antibiotics is to expel them from the cytoplasm through efflux
pumps. These transmembrane proteins usually play roles in antibiotic-unrelated
physiology or metabolism, such as transporting nutrients, wastes, toxins or
signaling molecules across the cell wall. Their functions in antibiotic resistance
may be secondary due to non-specific transport activities. For examples, 20 out
of the total of 36 genes encoding membrane transport proteins in the E. coli
genome confer some levels of resistance to one or more antibiotics (Nishino and
Yamaguchi 2001). It is unlikely that all these transporters have evolved to
become specialized drug transporters. Instead, what have evolved are likely
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regulatory proteins that control these transporters expression, thereby

specializing their function toward drug resistance. For example, the major E.
coli MDR determinant AcrB is a transporter with a broad substrate specificity,
but its expression is controlled by three antibiotic-responsive regulatory
systems: Mar, Sox and Rob (Alekshun and Levy 1997). At least 18 transporters
in mycobacteria have been found to confer low-level antibiotic resistance
(Viveirosa et al. 2012). Some of these transporters are expressed under the
control of antibiotic-responsive transcription regulators. For example,
expression of IniBAC and EfpA are negatively regulated by Lsr2, a nucleoid-
associated transcription regulator that binds AT-rich sequences (Colangeli et al.
2007). Whereas IniBAC confers resistance to isoniazid and ethambutol, EfpA
provides non-specific transport (Colangeli et al. 2005, 2007). Importantly, the
transcriptional control of iniBAC and efpA by Lsr2 is inducible by isoniazid or
ethambutol (Colangeli et al. 2007), thus specializing these transporters’ function
to antibiotic resistance. Besides drug resistance, recent studies suggested that
Lsr2 is involved in mycobacterial adaptation to changes in oxygen level, and
persistence in hosts (Bartek et al. 2014), thus connecting antibiotic resistance
and the pathogenesis of Tuberculosis. Another example is Tap, a transporter
responsible for mycobacterial efflux of aminoglycosides, spectinomycin,
tetracycline and PAS (Ainsa et al. 1998; Morris et al. 2005; Ramon-Garcia et
al. 2012). Transcription of the Tap-encoding gene (rv2158c) is controlled by the
antibiotic-responsive MDR regulator WhiB7 (Ainsa et al. 1998; Morris et al.
2005; Ramon-Garcia et al. 2012). A recent study showed that expression of
several efflux pumps including Tap is induced in mycobacterial cells residing
within host granulomas, indicating that these molecular pumps may contribute
to the drug tolerance of M. tuberculosis during latent TB (Adams et al. 2011).
For more information on mycobacterial transporters and their role in antibiotic
resistance, readers are referred to many recent excellent reviews (da sSilva et al.
2011; Viveirosa et al. 2012).

ACCQUIRED RESISTANCE:
Acquired antibiotic resistance may occur in bacteria through either mutations or horizontal
gene transfer mediated by phages, plasmids or transposon elements. In M. tuberculosis,
horizontal transfer of drug resistance genes has not been reported; but resistance mostly arises

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from chromosomal mutations under the selective pressure of antibiotic use. M. tuberculosis
genes to which mutations confer drug resistance are listed in Table. The progression of drug
resistance in M. tuberculosis epitomized the Darwin’s theory of evolution. Antibiotic resistance
becomes predominant traits in M. tuberculosis populations because they bring survival
advantages to the arising mutants under selective pressure. The continuous drug exposure
during lengthy regimens, together with patients’ non-adherence, has pushed evolution to select
for resistant mutants that otherwise would never predominate the population because of their
reduced fitness. This process, occurring during combination therapies, has created a steady
evolution of M. tuberculosis strains, gradually becoming resistant to all existing drugs. In
addition to the survival and selection, sub lethal exposures to bactericidal antibiotics might
have mediated radical-induced mutagenesis (Kohanski et al. 2010a), thus promoting the
emergence of multidrug resistance phenotypes in pathogenic bacteria including M. tuberculosis.
TB drugs such as isoniazid and ethionamide are prodrugs, which require activation by redox
enzymes in the mycobacterial cytoplasm to become cytotoxic. The prodrug activation process
produces reactive oxygen and radicals that exert the mycobactericidal activity (Ito et al. 1992;
Wang et al. 1998). However, if these reactive oxygen and radicals fail to kill the mycobacterial
cell, they will turn bad by promoting cellular mutagenesis and the emergence of drug resistance
mutations.

INFECTIOUS AGENT:
Mycobacterium tuberculosis belongs to the pathogenic bacterial species of actinomycetales,
family Mycobacteriaceae, which is the causative agent of tuberculosis. First discovered in 1882
by Robert Koch, M. tuberculosis has an unusual, waxy coating on its cell surface primarily due
to the presence of mycolic acid. This coating makes the cells impervious to Gram staining, and
as a result, M. tuberculosis can appear either Gram-negative or Gram-positive. Acid-fast stains
such as Ziehl-Neelsen, or fluorescent stains such as auramine are used instead to identify M.
tuberculosis with a microscope. The physiology of M. tuberculosis is highly aerobic and
requires high levels of oxygen. Primarily a pathogen of the mammalian respiratory system, it
infects the lungs.

Mycobacteria are aerobic, slender, non-motile, non-encapsulated, non-spore forming

and rod-shaped bacilli (2-4 micrometres (μ) in length and 0.2-0.5 μ in width). These intra-

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cellular pathogens infect alveolar macrophages (i.e. are macrophage inhabitants) and are found
within membrane-bound particles or phagosomes. Mycobacteria’s are resilient bacilli capable
of adapting to different environmental conditions, they are resistant to phagocytosis and remain
dormant for decades within phagocytic or non-phagocytic tissue cells with the ability to re-
activate. Their lipid rich (lipoid) cell wall makes it impermeable and resistant to bacterial
standard stains e.g. Gram stain. Acid-fast stains depend on the ability of mycobacteria to retain
dye when treated with mineral acid or an acid-alcohol solutions such as the Ziehl-Neelsen (ZN
stain) for acid fast bacilli (AFB) that is used for M. tuberculosis. (Glickman et al., 2001;
Hernandez-Pando et al., 20006)

PATHOPYSIOLOGY OF TB:
Once inhaled, the infectious droplets settle throughout the airways. The majority of the bacilli
are trapped in the upper parts of the airways where the mucus-secreting goblet cells exist. The
mucus produced catches foreign substances, and the cilia on the surface of the cells constantly
beat the mucus and its entrapped particles upward for removal. This system provides the body
with an initial physical defense that prevents infection in most persons exposed to tuberculosis.

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Macrophages are the primary cells infected by M. tuberculosis. Early in infection, tuberculosis
Bacilli replicate essentially unchecked, while later in infection, the cell response stimulates
Macrophages to contain the proliferation of the bacteria.
1. M. tuberculosis enters macrophages by endocytosis mediated by several macrophage
Receptors: mannose receptors bind lipoarabinomannan, a glycolipid in the bacterial cell
wall, and complement receptors (already discussed) bind opsonized mycobacteria.
2. Once inside the macrophage, M. tuberculosis organisms replicate within the phagosome
by blocking fusion of the phagosome and lysosome. M. tuberculosis blocks
phagolysosome formation by inhibiting Ca 2+ signals and the recruitment and assembly
of the proteins that mediate phagosome-lysosome fusion. Thus, during the earliest stage
of primary tuberculosis (<3 weeks) in the no sensitized individual, bacteria proliferate
in the pulmonary alveolar macrophages and airspaces, resulting in bacteremia and
seeding of multiple sites. Despite the bacteremia, most people at this stage are
asymptomatic or have a mild flulike illness.
3. The genetic makeup of the host may influence the course of the disease. In some people
with polymorphisms in the NRAMP1 gene, the disease may progress due to the absence
of an effective immune response. NRAMP1 is a transmembrane protein found in
endosomes and lysosomes that pumps divalent cations (e.g. Fe 2+) out of the lysosome.
NRAMP1 may inhibit microbial growth by limiting availability of ions needed by the
bacteria.
4. About 3 weeks after infection, a T-helper 1 (TH1) response is mounted that activates
macrophages to become bactericidal. The response is initiated by mycobacterial
antigens that enter draining lymph nodes and are displayed to T cells. Differentiation
of TH1 cells depends on IL-12, which is produced by antigen-presenting cells that have
encountered the mycobacteria. M. tuberculosis makes several molecules that are
ligands for TLR2, and stimulation of TLR2 by these ligands promotes production of
IL-12 by dendritic cells.
5. Mature TH1 cells, both in lymph nodes and in the lung, produce IFN-γ. INF-γ is the
critical mediator that enables macrophages to contain the M. tuberculosis infection.
IFN-γ stimulates formation of the phagolysosome in infected macrophages, exposing
the bacteria to an inhospitable acidic environment. IFN-γ also stimulates expression of
inducible nitric oxide synthase, which produces nitric oxide, capable of destroying
several mycobacterial constituents, from cell wall to DNA.

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6. In addition to stimulating macrophages to kill mycobacteria, the TH1 response

orchestrates the formation of granulomas and caseous necrosis. Macrophages activated
by IFN-γ differentiate into the “epithelioid histiocytes” that characterize the
granulomatous response, and may fuse to form giant cells. In many people this response
halts the infection before significant tissue destruction or illness. In other people the
infection progresses due to advanced age or immunosuppression, and the ongoing
immune response results in tissue destruction due to caseation and cavitation. Activated
macrophages also secrete TNF, which promotes recruitment of more monocytes. The
importance of TNF is underscored by the fact that patients with rheumatoid arthritis
who are treated with a TNF antagonist have an increased risk of tuberculosis
reactivation.
7. In addition to the TH1 response, NK-T cells that recognize mycobacterial lipid antigens
bound to CD1 on antigen-presenting cells, or T cells that express a γδ T-cell receptor,
also make IFN-γ. However, it is clear that TH1 cells have a central role in this process,
since defects in any of the steps in generating a TH1 response result in absence of
resistance and disease progression.
In summary, immunity to M. tuberculosis is primarily mediated by TH1 cells, which stimulate
macrophages to kill the bacteria. This immune response, while largely effective, comes at the
cost of hypersensitivity and accompanying tissue destruction. Reactivation of the infection or
re-exposure to the bacilli in a previously sensitized host results in rapid mobilization of a
defensive reaction but also increased tissue necrosis. Just as hypersensitivity and resistance are
correlated, so, too, the loss of hypersensitivity (indicated by tuberculin negativity in a
previously tuberculin-positive individual) may be an ominous sign that resistance to the
organism has faded. (Robbins and Cotran pathologic basis of disease. – 8th ed. / Vinay
Kumar… [et al.]; with illustrations by James A. Perkins8.)

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CLINICAL SIGNIFICANCE OF TUBERCULOSIS:

Although your body may harbour the bacteria that cause tuberculosis, your immune system
usually can prevent you from becoming sick. For this reason, doctors make a distinction
between:

 Latent TB: In this condition, you have a TB infection, but the bacteria remain in your
body in an inactive state and cause no symptoms. Latent TB, also called inactive TB
or TB infection, isn't contagious. It can turn into active TB, so treatment is important
for the person with latent TB and to help control the spread of TB. An estimated 2
billion people have latent TB.
 Active TB: This condition makes you sick and can spread to others. It can occur in
the first few weeks after infection with the TB bacteria, or it might occur years later.

The sign & symptoms of TB includes:

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 Coughing that lasts three or more weeks

 Coughing up blood
 Chest pain, or pain with breathing or coughing
 Unintentional weight loss
 Fatigue
 Fever
 Night sweats
 Chills
 Loss of appetite

Tuberculosis can also affect other parts of your body, including your kidneys, spine or brain.
When TB occurs outside your lungs, signs and symptoms vary according to the organs
involved. For example, tuberculosis of the spine may give you back pain, and tuberculosis in
your kidneys might cause blood in your urine.

RISK FACTORS FOR TB:

HIV coinfection is the most important and potent risk factor for TB infection and disease. The
growing population (especially in countries like China and India) is likely to inflate the number
of TB cases in future. Smoking rates are high among men in these endemic countries, and,
together with rising rates of diabetes, the risk of progression to TB disease will also increase.
Interventions such as smoking cessation and early screening for TB can be advocated, but the
impact of these interventions in reducing TB risk remains negligible at population level.
Malnutrition and indoor air pollution are recognized risk factors which are confounded with
the socioeconomic status, which leads to TB.

The risk for TB disease includes one or multiple factors, which are as follows:

Narasimhan, P.; Wood, J.; MacIntyre, C.; Mathai, D. Risk Factors for Tuberculosis
(accessed May 1, 2018)10.

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DIAGNOSIS OF TB:

To diagnose TB, the prophylactic therapy remain the most important tools to reduce the risk of
progression to TB disease among high risk individuals. The newly available IGRAs of
diagnostic tests detect latent TB infection are highly specific but has reduced sensitivity.
Diagnosis and treatment (through DOTS) of smear-positive cases remains the key to TB control
by reducing transmission from infectious cases. In addition to passive case-finding practices,

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early diagnosis of smear-positive cases can be improved through untargeted case-finding

strategies in endemic countries.

The diagnosis of pulmonary disease is based in part on the history and on physical and
radiographic findings of consolidation or cavitation in the apices of the lungs. Acid-fast smears
and cultures of the sputum of patients suspected of having tuberculosis should be performed.
Conventional cultures require up to 10 weeks, but culture in liquid media can provide an answer
within 2 weeks. PCR amplification of M. tuberculosis DNA allows for even more rapid
diagnosis. PCR assays can detect as few as 10 organisms in clinical specimens, compared with
more than 10,000 organisms required for smear positivity. However, culture remains the gold
standard because it also allows testing of drug susceptibility. Multidrug resistance is now seen
more commonly than it was in past years; hence, all newly diagnosed cases in the United States
are assumed to be resistant and are treated with multiple drugs. The prognosis is generally good
if infections are localized to the lungs, except when they are caused by drug-resistant strains or
occur in aged, debilitated, or immunosuppressed individuals, who are at high risk for
developing military tuberculosis. Narasimhan, P.; Wood, J.; MacIntyre, C.; Mathai, D. Risk
Factors for Tuberculosis (accessed May 1, 2018)11.

There are two type of diagnostic test for TB:

1. TB skin test:
 The TB skin test is also called the Mantouxs tuberculin skin test (TST). The TB skin
test is the preferred TB test for children under the age of five. The TB skin test is
performed by injecting a small amount of fluid (called tuberculin) into the skin on the
lower part of the arm. A person given the tuberculin skin test must return within 48 to
72 hours to have a trained health care worker look for a reaction on the arm. The result
depends on the size of the raised, hard area or swelling.
 Positive skin test: This means the person’s body was infected with TB bacteria.
Additional tests are needed to determine if the person has latent TB infection or
TB disease. It does not tell whether or not the person has progressed to TB
disease. Other tests, such as a chest x-ray and a sample of sputum, are needed
to see whether the person has TB disease.
 Negative skin test: This means the person’s body did not react to the test, and
that latent TB infection or TB disease is not likely.

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2. TB blood test:
 TB blood tests are also called interferon-gamma release assays or IGRAs. Two TB
blood tests are approved by the U.S. Food and Drug Administration (FDA) and are
available in the United States:
1. The QuantiFERON–TB Gold In-Tube test (QFT-GIT)
2. The T-SPOT.TB test (T-Spot).
 This TB blood test are preferred TB test for peoples who had received TB vaccines
(BCG).
 Positive TB blood test: This means that the person has been infected with TB
bacteria. Additional tests are needed to determine if the person has latent TB
infection or TB disease.
 Negative TB blood test: This means that the person’s blood did not react to
the test and that latent TB infection or TB disease is not likely.

TREATMENT OF TUBERCULOSIS:

WHO guidelines (2011), the choice of drugs was based on efficacy and toxicity in a step-down
manner, from group1 to group 5 (Table 1). Group 1 included first-line drugs and groups2–5
included second-line drugs. Group 5 included the drugs with (at the time) potentially limited
efficacy or limited clinical trials.

According to the new WHO drug classification (2016), patients with rifampicin-resistant or
MDR-TB require a regimen with at least five effective TB medicines during the intensive phase:
pyrazinamide and four core second-line TB drugs (see Table), one each from group A and
group B, and at least two from group C. If the minimum number of effective TB medicines
cannot be composed, an agent from group D2 and other agents from D3 should be added to
bring the total to five. If pyrazinamide is compromised or cannot be used, the regimen can be
reinforced with a drug from group C or D (preferably D2, and if not, from D3). Agents from
group D1 are added if they are considered to add benefit (e.g., high-dose isoniazid in patients
without high-level isoniazid resistance).1Thetotal number of TB medicines included in the
regimen needs to balance the expected benefit with the risk of harm and non-adherence

1. First line Anti TB drugs:

 Isoniazid

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 Rifampicin
 Pyrazinamide
 Ethambutol
 Streptomycin

2. Second line Anti TB drugs:

The second line drugs are only used to treat disease that is resistant to first line therapy
(i.e., for extensively drug-resistant tuberculosis (XDR-TB) or multidrug-resistant
tuberculosis (MDR-TB)). A drug may be classed as second-line instead of first-line for
one of three possible reasons: it may be less effective than the first-line drugs (e.g., p-
amino salicylic acid); or, it may have toxic side-effects (e.g., cycloserine); or it may be
effective, but unavailable in many developing countries (e.g., fluoroquinolones)
 Aminoglycosides: e.g., amikacin (AMK), kanamycin (KM);
 Polypeptides: e.g., capreomycin, viomycin, enviomycin
 Fluoroquinolones e.g., ciprofloxacin (CIP), levofloxacin, moxifloxacin (MXF)
 Thioamides: e.g. ethionamide, prothionamide
 Cycloserine
 Terizidone

3. Third line Anti TB drugs:

Third-line drugs include drugs that may be useful, but have doubtful or unproven
efficacy:

 Rifabutin

 Macrolides: e.g, clarithromycin (CLR);

 Linezolid (LZD);

 Thioacetazone (T);

 Thioridazine

 Arginine

 Vitamin D

 Bedaquiline

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4. Newer Anti TB drugs: This drugs are used useful in MDR, TDR TB.
 Pretomanid
 Delamanid
 Sutezolid
 SQ-109
 Q-203

INTRODUCTION OF HYBRID:

Hybrid drug is the combination of 2 or more drugs which are used to target the multiple target
at a time. The hybridization of biologically active molecules is a powerful tool for drug
discovery used to target a variety of diseases. It offers the prospect of better drugs for the
treatment of a number of illnesses including cancer, malaria, tuberculosis and AIDS. Hybrid
drugs can provide combination therapies in a single multi-functional agent and, by doing so,
be more specific and powerful than conventional classic treatments. This research field is in
great expansion and attracts many researchers worldwide. (Hu.Y.Q. et al,
Eur.J.Med.chem,2017,10.1016/j.ejmech.2017.04.00214)

CLASSIFICATION OF HYBRID:

1. Directly linked hybrid drugs:

• Directly linked hybrid drugs are connected via a functional group of each molecule
resulting in mostly an enzymatically hydrolysable ester, carbamateoramide.

• For e.g.:

Isoniazid & para amino salicylic Pyrazinamide &

acid hybrids as anti-tubercular ciprofloxacin hybrids.
drug

2. Spacer linked hybrid drugs:

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• These molecules can be classified as cleavable and non-cleavable.

• The majority of reported examples of cleavable conjugates contain an ester linker that
is cleaved by plasma esterase to release two individual drugs that then act
independently.

• Non-cleavable linkers are connected with non-hydrolysable chemical bonds to create

chemically as well as enzymatically stable linkers.

Ethambutol & Para amino salicylic acid hybrids

3. Merged or overlapped hybrid drugs:

• These types of hybrid agents are obtained by overlapping structural motifs or

pharmacophores of two drugs. These hybrid agents differ significantly in their
structures compared to drugs from which they were designed.
• The hybrid agents may retain the functional properties of either or both of overlapped
drugs.

CHEMISTRY OF HYBRID DRUG:

Hybrid drugs can be designed using ‘post hoc’ and ‘ad hoc’ approaches. In the ‘post hoc’
approach, the hybrid drugs are derived from the already developed drugs. Examples of this
class of hybrid agents are estramustine NO–aspirin.

In ‘ad hoc’ design involves the use of lead molecules suffering from some drawback such as
in vivo instability or lack of drug-like properties. In general hybrid drug design strategy could
be useful for lead optimization and should be implemented at the early stages of drug
development process. (Gediya.L.K.et al, Expert opin.Drug Discov,11,2009,1099-111115)

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Biquinolone-isoniazid hybrid

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