I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3
.sc ienced i rec t .comHO ST E D  BY Avai lab le a t wwwScienceDirect
journal homepage: www.elsevier .com/ locate / IJMYCOReviewCurrent perspectives in drug discovery against
tuberculosis from natural products* Corresponding author at: Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, University
Ghana.
Peer review under responsibility of Asian African Society for Mycobacteriology.
http://dx.doi.org/10.1016/j.ijmyco.2015.05.004
2212-5531/ 2015 Asian African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. All rights reserved.Joseph Mwanzia Nguta a,b,*, Regina Appiah-Opong a, Alexander K. Nyarko a,
Dorothy Yeboah-Manu c, Phyllis G.A. Addo d
a Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana
b Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Nairobi, Kenya
c Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana
d Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research, University of Ghana, GhanaA R T I C L E I N F O A B S T R A C TArticle history:
Received 6 March 2015
Received in revised form
3 May 2015
Accepted 6 May 2015
Available online 3 June 2015
Keywords:
Drug discovery
Natural products
Mycobacterium tuberculosis
Dormancy
Bioassay-guided fractionation
Natural products chemistryCurrently, one third of the world’s population is latently infected with Mycobacterium tuber-
culosis (MTB), while 8.9–9.9 million new and relapse cases of tuberculosis (TB) are reported
yearly. The renewed research interests in natural products in the hope of discovering new
and novel antitubercular leads have been driven partly by the increased incidence of
multidrug-resistant strains of MTB and the adverse effects associated with the first- and
second-line antitubercular drugs. Natural products have been, and will continue to be a
rich source of new drugs against many diseases. The depth and breadth of therapeutic
agents that have their origins in the secondary metabolites produced by living organisms
cannot be compared with any other source of therapeutic agents. Discovery of new chem-
ical molecules against active and latent TB from natural products requires an interdisci-
plinary approach, which is a major challenge facing scientists in this field. In order to
overcome this challenge, cutting edge techniques in mycobacteriology and innovative nat-
ural product chemistry tools need to be developed and used in tandem. The present review
provides a cross-linkage to the most recent literature in both fields and their potential to
impact the early phase of drug discovery against TB if seamlessly combined.
 2015 Asian African Society for Mycobacteriology. Production and hosting by Elsevier Ltd.
All rights reserved.ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Trends in discovery of TB drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166The four pioneer first-line drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
New TB drugs in the pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168of Ghana,
166 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3Drug discovery against TB from natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
The need for tuberculosis drug development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Bioassay-guided (bioactivity) fractionation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
High-throughput, inexpensive, time-saving assay using M. smegmatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
The target organism, M. tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Challenges facing antimycobacterial drug discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Combination of whole cell and target-based screening approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Anti-TB in vitro bioassays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172Agar diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Macro- and micro-agar dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Radiorespirometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Micro-broth dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Nitrate reductase assay/Greiss method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Reporter gene assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Dormant tubercle bacilli bioassay/low oxygen bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
High-performance liquid chromatography mycolic acid analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Numerical evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Macrophage bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Anti-TB ex vivo bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Patient Peripheral Blood Mononuclear Cell (PBMC) bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Anti-TB in vivo bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Toxicity evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Selectivity and criteria for antimycobacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Natural products chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176Minor considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Structure elucidation of natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Dereplication and NMR fingerprinting of natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Countercurrent separation of natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177New perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178Introduction
Tuberculosis (TB), an old, highly infectious disease, declared a
global health emergency by the World Health Organization
(WHO) in 1993, is still the second leading killer in the world,
with an approximate 2 billion people being latently infected.
These latently infected individuals with Mycobacterium tuber-
culosis (MTB) represent one third of the world’s population.
It still remains one of the world’s deadliest infectious dis-
eases. WHO estimates that there were approximately 9.0 mil-
lion new cases and 1.5 million cases of mortality in 2013–
360,000 of whom were positive for HIV [1]. TB treatment is
generally comprised of 2 months with isoniazid, rifampicin,
ethambutol and pyrazinamide (the intensive phase), followed
by four additional months of isoniazid and rifampicin therapy
(the continuation phase) [1]. Unfortunately, lack of adherence
to prescribed treatment procedures and inefficient healthcare
structures have contributed to the development of multidrug-
resistant TB (MDR-TB, defined as resistance to at least isoni-
azid and rifampicin, two front-line drugs used for the treat-
ment of TB) that requires at least 20 months of treatment
with second-line drugs comprised of capreomycin, kanamy-
cin, amikacin and fluoroquinolones; these are more toxic
and less efficient, with cure rates in the range of 60–75% [2].Riccardi et al. [3] notes that in 2012, 450,000 people developed
MDR-TB in the world. It is estimated that about 9.6% of these
cases were extensively drug resistant (XDR-TB), showing
additional resistance to at least one fluoroquinolone and
one injectable second-line drug [1,4]. In patients affected by
XDR-TB, the chances of successful treatment are quite low
[3], underpinning the need for urgent discovery of novel com-
pounds with activity against MTB strains resistant to second-
line drugs. Recently, a few reports have claimed the emer-
gence of a ‘totally drug-resistant TB’ strain with a limited
chance of successful therapy [3,5–8]. Moreover, there is an
urgent need to come to an agreement on the definition of
these strains of MTB, mainly in terms of their severity [9].
Hence, the search for new antitubercular drugs is a priority
so as to overcome the problem of drug resistance and to
finally eradicate TB.
Trends in discovery of TB drugs
The four pioneer first-line drugs
Pyridine-4-carboxy hydrazide, isoniazid (INH; isonicotinyl
hydrazide, Fig. 1a) was discovered at the same time in 1952
by three different pharmaceutical companies: BAYER
I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3 167
Fig. 1 – Chemical structure of isoniazid (a) and rifampicin (b).(Leverkusen, Germany); Hoffmann LaRoche (Nutley, NJ, USA);
and ER Squibb & Sons (Princeton, NJ, USA). INH cured many
patients and was defined as a ‘magic drug’ [3]. Since 1952,
INH continues to be an essential drug in the fight against TB
[10].
Isoniazid is a prodrug that requires activation by MTB
catalase-peroxidase katG enzyme to form an INH-NAD com-
plex which inhibits the nicotinamide adenine dinucleotide
(NADH)-dependent enoyl-ACP reductase (encoded by inhA
gene) of the fatty acid synthase type II system, a key player
in the mycolic acid biosynthetic pathway of MTB [3]. The inhi-
bition of enoyl-ACP reductase (encoded by inhA gene) causes
an accumulation of long-chain fatty acids and cell death [11].Fig. 2 – Chemical structure of pyrazinamide (a) and
ethambutol (b).Mutations in the katG and inhA genes have been shown to
contribute approximately 70% and 80% respectively to isoni-
azid resistance in MTB clinical isolates [3,10]. Since isoniazid
is a prodrug, its activity is greatly influenced by mutations in
the katG enzyme, and, as such, a reasonable way to bypass
this mechanism of resistance is designing drugs that do not
require the katG enzyme activation, but mainly target the
inhA enzyme. Triclosan inhibits the inhA enzyme [3], but its
usefulness as an antitubercular drug has not been successful
because of its sub-optimal bioavailability [12]. A series of tri-
closan derivatives have been synthesized using a structure-
based drug design approach [13]. It is interesting to note that
these derivatives have been shown to be effective against
MTB isoniazid resistant clinical and laboratory strains. The
best triclosan derivative inhibitor had a minimum inhibitory
concentration (MIC) value of 4.7 lg/ml, which represents a
tenfold improvement compared with the activity of the par-
ent compound, triclosan, but less potent than isoniazid with
an MIC value of 50 ng/ml [3,13]. Investigators are searching
for new anti-TB drugs targeting the inhA gene which do not
require activation by the katG enzyme with a susceptibility
pattern similar to that of isoniazid. This will be a hard task
because isoniazid is a very potent antitubercular drug [3].
New inhibitors of the inhA enzyme have been synthesized
of late, but their effectiveness is not as good as isoniazid [14].
Pyridomycin, a natural compound produced by
Dactylosporangium fulvum with specific ‘cidal’ activity against
mycobacteria, has been recently demonstrated to target the
inhA enzyme [15]. Moreover, biochemical and structural
approaches have showed that pyridomycin inhibits the inhA
enzyme directly via the competitive inhibition of the NADH
binding site, without activation by the katG enzyme [3].
168 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3Interestingly, the majority of the MTB isoniazid-resistant clin-
ical isolates are sensitive to pyridomycin, underpinning the
potency of this drug [15].
Pyrazinamide (PZA), an analog of nicotinamide (Fig. 2a), is
a prodrug that requires conversion by MTB pyrazinamidase
(coded by the pncA gene) to pyrazinoic acid [16]. RpsA has
recently been shown to be a pyrazinamide cellular target
[17], and its over-expression (wild-type RpsA) has been impli-
cated in PZA resistance in MTB. Binding of the activated
pyrazinoic acid to RpsA interferes with its binding to the mes-
senger RNA. In addition, some PZA-resistant MTB strains
without mutations in pncA have shown mutations in RpsA
[3]. This enzyme (RpsA) is essential for translation, and its C
terminus is also involved in trans–translation [18] in MTB.
Trans–translation in MTB is dispensable during active growth
conditions, but it is also required by some bacterial strains for
survival under stress conditions and also for disease progres-
sion. The inhibition of the trans–translation step in MTB may
interfere with its survival under the dormancy state, and this
could explain how diverse stress conditions, such as starva-
tion, acidic pH and hypoxia, can potentiate the activity of
pyrazinamide [3,17,19–21].
Ethambutol (EMB) (Fig. 2b), interferes with mycobacterial
cell wall synthesis in MTB by inhibiting polymerization of ara-
binogalactan, an important cell wall component in MTB [22].
Moreover, it also interrupts the utilization of the arabinose
donor by inhibiting either arabinosyltransferase enzymatic
activity or the formation of an arabinose acceptor in
mycobacteria [22]. The embCAB operon has been shown to
be responsible for ethambutol resistance in MTB [23]. It is
worth noting that ethambutol acts at the same pathway that
is blocked by benzothiazinones, but not at the same step of
metabolism.
Rifampicins comprise a group of antibacterial drugs and
include the following derivatives: rifampicin, rifapentine, rifa-
butin and rifalazil [3]. They bind to the bacterial beta RNA
polymerase subunit, thus interfering with transcription [3].
Resistance to rifampicins in MTB is conferred by mutations
in the 81-bp region of the rpoB gene (encodes beta RNA poly-
merase) [24]. Both rifampicin (Fig. 1b) and isoniazid are
essential and commonly used first-line drugs for TB therapy
in combination with other molecules [3].
Following the introduction of rifampicin into clinical use,
the treatment of active TB was reduced from 9–12 months
to 6 months, while the duration for treatment of latent TB
was reduced from 9 months to 3 months [10]. It is important
to note that rifampicins are among the few drugs that can kill
the dormant (non-replicating) strains of MTB. Rifampicin (RIF)
was developed by blind whole-cell screening in an extensive
program of chemical modification of the rifamycins, the nat-
ural metabolites of Amycolatopsis mediterranei under the super-
vision of Professor Piero Sensi [25]. Since rpoB is an essential
gene in MTB, and RNA polymerase is a proven target for
antibacterial and anti-TB therapy, it would be reasonable to
search for new RNA polymerase inhibitors binding at sites dif-
ferent from that utilized by rifampicin [26]. In 1989 Professor
Piero Sensi wrote: ‘‘In the last two decades, no new major
anti-TB drug has been developed. Although dramatic
improvements in chemotherapy for TB have been achieved
through careful studies of drug regimens, there is still a needfor new agents that are highly active. The antimycobacterial
drugs used at present in therapy for TB were obtained by
either blind screening or chemical modification of active com-
pounds. Other approaches based on the knowledge of the bio-
chemistry of the mycobacterial cell should be tried. Certain
constituents of the cell, such as mycolic acids, arabinogalac-
tan, peptidoglycan and mycobactin, may represent specific
targets for new anti-TB drugs [27].’’ As an outstanding scien-
tist, Professor P. Sensi understood what research scientists
in the field of TB drug discovery would realize many years
later. Afterwards, a lot of compounds have been discovered
that inhibit specific steps involved in either arabinogalactan
or mycolic acid biosynthesis [28]. Novel efficacious and safe
anti-TB drugs are currently needed so as: (1) to shorten the
duration of TB therapy; (2) to be able to treat MDR, XDR and
totally drug resistant (TDR) TB strains; (3) to be able to treat
latent TB; (4) to be able to act in a synergistic manner with
other co-administered anti-TB drugs; and, finally, (5) to be
able to be safely co-administered with anti-HIV agents.
New TB drugs in the pipeline
After a long period of inactivity, the last few years have seen
an increase in the number of new anti-TB drugs in the pipe-
line. As it can be seen in Fig. 3, there are currently adequate
numbers of drug candidates in the lead optimization stage,
preclinical development, phase II and phase III clinical trials.
However, there is a worrying gap within phase I that needs to
be filled to have a constant delivery of molecules in case of
failure of advanced drug candidates. Many molecules under
clinical evaluation, such as fluoroquinolones, were developed
to treat other infectious diseases and have now been repur-
posed for TB treatment and this call for increased efforts
towards discovery of new compounds against different phys-
iological states of tubercle bacilli.
Bedaquiline (TMC-207), a diarylquinoline, was approved by
the FDA (Food and Drug Administration) in December 2012 as
part of the combination therapy for the treatment of adult
patients affected by MDR-TB, and it is now in phase III of clin-
ical development (Fig. 3). Bedaquiline can be considered to be
the first major drug approved by the FDA for TB therapy in the
last four decades (40 years). It came out following a pheno-
typic screening of compounds against MTB, while the corre-
sponding target was identified through the whole-genome
sequencing of MTB and Mycobacterium smegmatis spontaneous
mutants that were resistant to chemical molecules.
Interestingly, the resistant mutants showed missense muta-
tions in the atpE gene (encoding the c subunit of ATP syn-
thase) [29]. Bedaquiline acts by inhibiting ATP synthase and
has activity against active and dormant MTB strains.
Currently, it is well known that TB patients with pulmonary
TB can have both active and dormant tubercle bacilli, the lat-
ter being difficult to eliminate with the currently used anti-TB
drugs, hence favoring the development of resistant strains
and latent infection [30]. It is well known that human mito-
chondrial ATP synthase is 20,000-fold less sensitive to
diarylquinoline than the mycobacterial one, thus validating
the enzyme as an important drug target against MTB [31].
Bedaquiline has been associated with an increased risk of
inexplicable mortality and QT prolongation, but it still
I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3 169
Lead opmizaon Preclinical Phase 1 Phase 2 Phase 3
development
Diarylquinolines CPZEN-45 (Caprazene TBA-354 Sutezolid (OZ) Bedaquiline (TMC-
nucleoside (nitroimidazol 207) (DQ)
InhA inhibitors e)
Rifapenne Delamanid OPC-
LLeuRSSiLnhibitors SQ609 (Dipiperidine) (RIF) 67683) (IO)
Pyrazinamide Analogs AZD 5847 (OZ)
SQ641 Rifapenne (RIF)
TL 1 inhibitors (Capuramycin)
Bedaquiline Pretomanid-
DprE inhibitors Q203 (DQ) Moxifloxacin-
Ureas (Imidazopyridine)Ureas Pretonamid- Pyrazinamide (PA-
Ruthenium (II) PBTZ 169 Pyrazinamide
824)(IO)
phosphine/diimine/pi (Benzithiazinone) SQ-109 
colinate complexes (ethylenediami
Specnamides ne)
SQ-109 
Indazoles
(ethylenediam
Macrolides ines)
Cyclopepdes
SPR-113
Fig. 3 – Current global drug pipeline (modified by www.newtbdrugs.org). OX, oxazolidinone; RIF, rifamycin; DQ, diarylquinoline;
IO, imidazooxazole. Source: @2015 Working group on New TB Drugs. Stop TB Partnership. (http://www.newtbdrugs.org/
pipeline.php).represents a great addition for the treatment of MDR- and
XDR-TB strains, especially in TB-endemic regions of the world
[32].
Delamanid (OPC67683) and pretomanid–moxifloxacin–
pyrazinamide combination (PA-824) are two new imidazooxa-
zoles in phase III clinical development (Fig. 3). Both molecules
are pro-drugs whose activation depends on a F420-
deazaflavin-dependent nitroreductase (Ddn) which is present
in MTB. The active form of PA-824 is the corresponding des-
nitroimidazole molecule, which releases reactive nitrogen
species, such as nitric oxide [33], causing respiratory poison-
ing which appears to be crucial for its anaerobic activity
[34]. PA-824 has activity against both active and latent TB
infection, which could shorten the duration of TB therapy
[35]. Delamanid inhibits mycolic acid biosynthesis and has
been associated with increased sputum-culture conversion
in MDR-TB patients [36]. In addition, Delamanid has been
shown to be effective with acceptable toxicity when com-
bined with other anti-TB drugs in an MDR-TB regimen [37].
Rifapentine, a semi-synthetic cyclopentyl rifamycin
derivative, acts by binding the b-subunit of the RNA poly-
merase in MTB, a mechanism of action that is also utilized
by rifampicin [38]. It is more effective than rifampicin against
MTB, both in vitro and in vivo with an MIC value in the range of
0.02–0.06 lg/ml [39]. Both rifamycin and rifapentine exhibit
cross resistance. The United States Food and Drug
Administration (US FDA) in 1998 approved rifapentine at a
dosage rate of 10 mg/kg (oral administration) once or twice
weekly for the therapy of active and latent TB. There is goodclinical evidence that supports the use of rifapentine plus iso-
niazid for 3 months (once-weekly regimen) against latent TB
[40], but quite different for the treatment of active TB, where
it is approved by the FDA at a dose of 600 mg orally, twice
weekly during the intensive phase of TB treatment
(2 months), and then once weekly during the continuation
phase (4 months) [41]. Recently, animal studies have sug-
gested that more frequent administration of rifapentine
might cure both active and latent TB in 3 months or less, how-
ever, the observed findings could not be reproduced in clinical
trials involving human subjects. Moreover, in animal studies,
the administration of the drug via inhalation appeared to
improve tubercle clearance in the lungs, but clinical data
has not yet been generated [39].
SQ109, a 1,2-ethylenediamine {N0-(2-adamantyl)-N-[(2E)-3,
7-dimethylocta-2,6-dienyl]ethane-1,2-diamine} is in phase II
clinical trials (Fig. 3). SQ109 is active against sensitive MTB,
MDR-TB and XDR-TB strains [42,43]. This compound was
found while screening a 63,238 chemical library, designed
around the active 1,2-ethylenediamine pharmacophore of
ethambutol, an essential first-line antitubercular drug, with
the hope of identifying an ethambutol-like chemical mole-
cule, probably more effective and safer than ethambutol.
Interestingly, both ethambutol (EMB) and SQ109 have differ-
ent chemical structures and different mechanisms of action,
with SQ109 targeting MmpL3 (an essential membrane trans-
porter belonging to the resistance, nodulation and division
[RND] family), whose main function (MmpL3) in MTB is to
transport the trehalose monomycolate into the envelope thus
170 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3interfering with mycolic acid synthesis in the mycobacterial
cell [44]. This membrane transporter (MmpL3) also assists
with iron acquisition for mycobacteria survival, and together
with Rv0203 plays an important role in mycobacterial heme
uptake. Currently, MmpL3 is considered as one of the hottest
targets in drug discovery against MTB, as several other com-
pounds under preclinical investigation have also been
reported to inhibit the transporter, such as the ureas in lead
optimization stage (Fig. 3).
Other compounds have recently moved from phase I to
phase II clinical trials. These include PNU-100480 (Sutezolid)
(Fig. 3), a close analog of linezolid and AZD5847 (Fig. 3), a
member of the oxazolidinone class.Drug discovery against TB from natural products
The urgent need for the development of new drugs to help
reduce the global burden of TB is well documented in the cur-
rent biomedical literature [45–47]. Novel antimycobacterial
scaffolds from natural products have recently been reviewed
[51]. Uplekar et al. [48] points out that in order to attain the
WHO’s ambitious targets of 95% reduction in TB deaths and
90% reduction in TB incidence by 2035, the need for better
and safer drug regimens to shorten treatment is key.
Because natural products are a proven template for the devel-
opment of new scaffolds of drugs, they have received consid-
erable attention as potential anti-TB agents [46]. There are
excellent reviews on antitubercular compounds derived from
natural products [49–53]. In a recent review, Mdluli et al. [54]
highlights some recent notable examples of natural product
compounds that may prove to be useful leads for TB drug dis-
covery. A number of medicinal plants with promising activity
against TB have recently been reported [55].
Antimycobacterial bioactive chemical molecules have been
found from many natural product skeletons, mainly from
plant biodiversity, but also from other organisms, such as
fungi and marine organisms. The plethora of structures
reported to have anti-TB activity is summarized in a recent
review focusing on naturally occurring compounds with
reported growth inhibitory activity in vitro towards sensitive
and resistant MTB [56]. Another noteworthy source of infor-
mation is a recent comprehensive compilation of plant spe-
cies for which promising anti-TB activity has been reported
[57]. Considering that none of the several screened non-
microbial natural products with activity against MTB has pro-
gressed towards the clinical trial stage in anti-TB drug devel-
opment, it seems reasonable to evaluate the reasons for the
failure. This could possibly be caused by: (i) low yields of puri-
fied compounds; (ii) structural complexity exhibited by natu-
ral products, such as the occurrence of multiple
stereoisomers, e.g., triterpenes which contain ten or more
chiral centers; (iii) most studies are purely academic and are
not focused on drug development; (iv) low activity exhibited
by the isolated compounds with MIC P 1 lg/ml; (v) the pres-
ence of pan inhibitors (non-specific compounds or pan-
inhibitors); (vi) difficulties in isolating novel cidal compounds
acting on new targets that can potentially reduce the duration
of therapy; and (vii) difficulties in identification of anti-TB
compounds with exceptional safety profiles without thedrug-drug-interaction problem presently confronting concur-
rent TB and HIV therapy. In addition, the current literature
has no indication on the safety profile of isolated compounds
as shown by the selectivity index (SI) (anti-TB activity vs.
mammalian cytotoxic activity) [58], hence there is a need to
evaluate the toxicological profile of purified and semi-
purified natural products [59,60]. It will be a hard task to meet
the aforementioned difficulties without increased funding for
anti-TB drug discovery and construction of a more robust
drug development pipeline through well-coordinated interna-
tional efforts.
The classic pathway towards anti-TB drug discovery from
natural products and indeed other infectious diseases must
be able to overcome a number of challenges. The first is to
reliably detect efficacious and safe hits and be able to identify
already known compounds at the early stages of the drug dis-
covery program. The second major challenge is the de novo
structure elucidation of new molecular entities. The latter
challenge has been revolutionized by current advances in
spectroscopic techniques, specifically the high resolution
neutron magnetic resonance (NMR) technologies. Many
approaches have been developed to solve the major hurdle,
but it still remains a major challenge in anti-TB drug discov-
ery from natural products [58]. In order to impact the early
phases of anti-TB drug discovery from natural products, inno-
vative technologies need to be leveraged for rapid navigation
of natural product hits through the detection, validation, iso-
lation, hit-to-lead and lead optimization phases [46]. In the
present review, the aforementioned bottlenecks are
approached from a different perspective, so as to reflect on
the truly interdisciplinary nature of the scientific challenges
encountered at the initial phase of anti-TB drug discovery
from natural products. Accordingly, the present review puts
more emphasis on the recent advances in the field of
mycobacteriology and natural product chemistry, specifically
to provide an overview of the methods that are currently
available, point out how both fields can impact the early
phase of anti-TB drug discovery if seamlessly combined,
obstacles faced even in an environment where mycobacteri-
ologists and natural product chemists are working together
and finally demonstrate some perspectives for drug discovery
against TB from natural products.The need for tuberculosis drug development
The needs, challenges and recent advances towards develop-
ment of novel chemical molecules against TB have been
reviewed recently [2]. Approximately 2 billion people of the
world’s population are latently infected with MTB and are at
risk of reactivation to active disease [61]. Even though an
inexpensive and effective quadruple drug therapy regimen
comprising isoniazid, rifampicin, ethambutol and pyrazi-
namide was introduced 40 years ago, TB continues to spread
in every corner of the globe [62]. TB remains a global emer-
gency according to the seventeenth World Health
Organization (WHO) report on the worldwide incidence of
the disease [63]. Globally, there are approximately 8 million
new cases and 2 million deaths yearly associated with TB;
hence, the disease is responsible for more human mortality
I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3 171than any other single microbial infection. A major break-
through in TB therapy came after the introduction of strepto-
mycin, followed by p-aminosalicylic acid (1949), isoniazid
(1952), pyrazinamide (1954), cycloserine (1955), ethambutol
(1962) and rifampin (1963) over 40 years ago. The current
treatment regimen has several drawbacks, including pro-
longed treatment time to completely eradicate the bacteria
(sterilization). This increases the opportunity for develop-
ment of MTB-resistant strains documented in almost every
country where the disease is prevalent. These obstacles, in
addition to an increasing prevalence of MDR, XDR and cur-
rently TDR strains, call for an urgent need to search for and
develop novel agents against TB. Pulmonary TB remains a
major health hazard in Asia, Africa and the Western Pacific
region, despite its sharp decline in the Western world since
the beginning of the 20th century [47]. A number of chal-
lenges, including the lack of economic incentive due to the
predominance of the disease in the developing world, have
continued to face drug discovery towards TB. However, there
has been a renewed interest by scientists, funding bodies and
high-profile advocacy by the WHO’s STOP TB department and
other organizations towards discovery of new agents against
TB, as well as the creation of a roadmap for their development
[46]. These efforts have recently cul minated in the approval
of two new drugs: delamanid (previously known as
OPC67683) and bedaquiline (also known as TMC207 or
R207910) for the treatment of MDR strains of MTB [45,64].
Bioassay-guided (bioactivity) fractionation
Recent reviews have described bioassay-guided fractionation
in TB drug discovery programs [46,65]. Bioassay-directed frac-
tionation is the state-of-the-art process that is currently being
utilized to isolate and identify bioactive principles from natu-
ral product crude extracts. This process consists of alternat-
ing steps of evaluating the activity of natural products using
bioassays and chemical fractionation; hence, multiple transi-
tions of samples and mutual design of protocols at the
mycobacteriology-natural product chemistry interface is
required. The sensitivity of natural product fractionation pro-
cedures has increased dramatically over the recent years due
to enormous technological advancements in chromatograph
and spectroscopy, opening new alleys not only for unstudied
materials, but also for previously investigated genera, provid-
ing access to unexpected chemical types and novel com-
pounds [66]. Thus the development and application of new
natural product chemistry methods is key in a bioassay-
guided anti-TB drug discovery program. In order to provide
valid guidance, the mycobacteriological assay is the second
key point to be addressed and has to be chosen wisely with
regard to the ultimate endpoint, i.e., the activity of the
anti-TB agent against virulent MTB in vivo. Using this
method, three potent antimycobacterial compounds have
been isolated from Dracaena angustifolia [65].
High-throughput, inexpensive, time-saving assay
using M. smegmatis
Mycobacterial strains can be broadly classified depending
on their in vitro growth as follows: (i) fast-growing,non-pathogenic strains; and (ii) slow-growing, pathogenic
strains. Slow-growing pathogenic mycobacterium will be a
difficult organism to screen a large number of candidates
within a short period of time. Therefore, preference has been
given to M. smegmatis mainly because of the following
reasons: (i) it is non-pathogenic and can be handled easily;
(ii) the growth rate of M. smegmatis is approximately eight
times faster than MTB; (iii) M. smegmatis is widely used to
understand the biology of MTB, such as in cell culture, gene
expression and persistence in the face of nutrient starvation;
and (iv) MTB has been found to display a drug susceptibility
profile similar to MDR MTB [67]. Therefore, cell viability assay
with M. smegmatis could serve as a ‘surrogate’ for MDR MTB.
This bioassay usually serves to prioritize the candidates
which can be tested further in more specific in vitro assays
on pathogenic MTB, MDR and XDR strains. M. smegmatis has
reportedly been used in primary screening for the selection
of compounds with activity against MTB [68]. Recently, it
has been reported that the susceptibility pattern of
M. smegmatis to the two front-line essential drugs against
TB – isoniazid and rifampicin – is identical to that of MDR
clinical isolates of MTB [46]. The sensitivity of M. smegmatis
based on screening should be extremely specific so that hits
generated in this bioassay can be a potential target for both
sensitive as well as MDR strains of MTB [68].The target organism, M. tuberculosis
MTB, the actual etiologic agent for TB, is the ideal target
organism in an anti-TB drug discovery effort. MTB H37Rv
(ATCC 27294), a well-characterized virulent strain available
from the American Type Culture Collection (ATCC, Rockville,
MD), has a drug susceptibility profile which is quite similar
to majority of those clinical MTB isolates which have not
developed drug resistance as a result of prior treatment with
one or more clinical TB drugs (susceptible clinical isolates).
Testing of chemical molecules against drug-resistant and
MDR strains of MTB (strains resulting from specific stepwise
mutations to individual drugs) is not critical in primary
screening, since these strains are not ‘‘superbugs’’, which
are resistant to multiple anti-TB drugs by virtue of a single
mechanism, such as the effusion pumps found in other bac-
teria and, hence, would be expected to be susceptible to any
novel compound, acting in a different site from that utilized
by an existing anti-TB drug. Since MTB H37Rv is a virulent
strain, it should only be handled in a biosafety level 3 labora-
tory (BSL-3) that requires a pass-through autoclave, a negative
air pressure relative to an anteroom and hallway, and a class 2
biosafety cabinet. Laboratory personnel working within the
BSL-3 laboratories must be well-trained, must wear protective
gear, and most importantly a respirator, which will minimize
the risk of infection from aerosolized MTB. Most investigators
in anti-TB drug discovery either collaborate with an institu-
tion with a BSL-3 facility, or work with an avirulent surrogate
organism, such as M. smegmatis (ATCC 19420), since few insti-
tutions have a BSL-3 laboratory. The majority of natural pro-
duct researchers have chosen to work with these rapidly
growing, avirulent, saprophytic mycobacteria, erroneously
172 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3referred to as MTB 607 in several publications. However, M.
smegmatis only possess a limited degree of similarity to MTB
with regard to drug susceptibility. Alternatively, one can also
use either MTB H37Ra (ATCC 25177), or the commonly used
vaccine strain, Mycobacterium bovis BCG (ATCC 35745), both
of which are slow-growing and non-pathogenic and, most
importantly, are more closely related to MTB H37Rv than the
rapidly growing mycobacteria with respect to drug suscepti-
bility profile and genetic composition. To work with these
strains, one only requires a class 2 biosafety cabinet and
sound microbiological techniques [46].
Challenges facing antimycobacterial drug
discovery
The existence of MTB in different physiological states during
infection, its pathogenesis and complex biology pose specific
challenges for drug discovery against TB. The first major chal-
lenge is the perceived heterogeneity of the population of
tubercle bacilli in the human host with respect to the meta-
bolic state as reflected in the multiplication rate, which
greatly impact on the choice of in vitro and ex vivo models
used to screen new anti-TB compounds [69]. A particular out-
come of the slow growth rate is ambiguity about the ‘vulner-
ability’ of the metabolic pathways, raising critical questions.
For example: (i) which metabolic pathway is crucial for sur-
vival to a persisting organism? and (ii) during persistence,
are metabolites required at much lower concentrations?
The second challenge is in identification of safe com-
pounds for prolonged therapy. The most serious side effects
of prolonged therapy are drug-drug interactions, since a
compound-specific toxicity profile is usually addressed in
the safety studies. This aspect can be studied very early in
the drug discovery cascade, thus the focus of the unmet chal-
lenge shifts to identification of chemical entities with rapid
kill kinetics, since this is fundamental for the quick reduction
of the bacterial load and, eventually, sterilization. Hence, an
anti-TB compound should be able to act on MTB in different
metabolic states. It is worth noting that the success of
target-based drug discovery is mainly dependent on the qual-
ity of the target and the level to which it has undergone vali-
dation [70]. Hence, from this perspective and while excluding
the ribosome, the currently available repertoire of anti-TB
drugs reveals only a small number of comprehensively vali-
dated targets, namely RNA polymerase, DNA gyrase, NADH-
dependent enoyl-(acyl-carrier-protein) reductase, and ATP
synthase [71,72]. Given the need to increase the chances of
success at a time when attrition rates are quite high in the
early phase of anti-TB drug discovery, lack of information
on several validated targets poses severe limitations on the
diversity of efforts, raising fundamental questions as to
whether alternative ‘lead generation’ approaches are more
suitable for anti-TB drug discovery. Fortunately, advances in
genome sequencing technology can perhaps augment the ‘
whole-cell-screening’ approaches since it can enable the
identification of more targets [29,73]. It is worth noting that,
whole-cell screening (phenotypic screening) clearly enables
the early identification of compounds with killing ability
(cidal activity) and their progression along the drug discoverypathway. In addition, it also enables testing of cidality of com-
pounds on different metabolic states of MTB and, thus, over-
comes a key challenge early in the drug discovery path [74].Combination of whole cell and target-based
screening approaches
The inability to convert target inhibition into growth inhibi-
tion and eventually to bacterial cell death, which bedevils
the target-driven approach, is circumvented by identifying
compounds with potent anti-TB activity by whole-cell screen-
ing, which is clearly a feasible starting point. The whole-cell
screening approach does not provide information in regard
to mechanism of action and possible toxicity, which is espe-
cially relevant in anti-TB therapy because of the prolonged
duration of treatment. This challenge can be partially miti-
gated by merging the whole-cell screening with the target-
based approach by carrying out studies on mechanism of
action and toxicity studies on the potent whole-cell active
compound. This will facilitate identification of the pathway
and/or target before extensive studies on medicinal chemistry
are started. Even though further medicinal chemistry can,
indeed, be driven by MIC-based structure activity relationship
patterns, knowledge of the target and or mechanism of action
would enable studies on possible mechanisms of toxicity [74].
Alternatively, another promising method is to use high-
content screening systems [75], where a confocal microplate
imaging reader is used to monitor inhibition of intracellular
mycobacterial growth and possible cytotoxic effects, using
infected macrophages simultaneously [74].Anti-TB in vitro bioassays
Agar diffusion
The common disc or well-diffusion assays employed in many
antimicrobial assays of natural products only indicate that
there is growth inhibition at some unknown concentration
along the concentration gradient, but are not quantitative
when used to evaluate extracts or new compounds. The sizes
of inhibition zones can only be interpreted as indicative of
microbial susceptibility or resistance in a clinical setting with
well-characterized antibiotics, since the size of the zone of
inhibition depends upon both the rate of diffusion of the
active agent and the rate of growth of the target organism.
Diffusion assays with mycobacteria need to be avoided, since
these organisms with a very lipid-rich, hydrophobic cell wall
are often more susceptible to less-polar compounds [76].
Hence, non-polar compounds will diffuse more slowly than
polar compounds of similar molecular weight in the aqueous
agar medium resulting in relatively small inhibition zones,
giving the erroneous impression of weak bioactivity. In addi-
tion, active low molecular weight, polar compounds may dif-
fuse to equilibrium before colony growth of slow-growing
mycobacteria is apparent, and if the concentration at equilib-
rium is below the MIC, then there will be no zone of inhibi-
tion; hence, the compound bioactivity will not be reflected
[46].
I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3 173Macro- and micro-agar dilution
Testing known concentrations of extracts, fractions or com-
pounds in an agar medium allows for MIC value determina-
tion and the quantitation of bioactivity. The majority of
mycobacterial strains, including MTB, will grow well on
Middlebrook 7H11 agar supplemented with oleic acid, albu-
min, dextrose and catalase (OADC supplement, Difco), with
the exception of a few fastidious species. Test samples can
be added to the molten media (held at 50 C) at 1% v/v final
concentration and then either 100–200 ll medium to 96-well
microplates, 1.5 ml to 24-well microplates, 4 ml to 6-well
microplates or 20 ml added to standard 150 mm diameter
Petri-dishes. Following the hardening of the medium, the
inoculum can be spotted on the surface with a micro pip-
ette. Suggested volumes of inoculum are: 1–5 ll for 96-well
plates, 10 ll for 6- or 24-well plates and 100 ll (spread
evenly) for standard Petri dishes. The plates are then incu-
bated at 37 C overnight, after which they can then be
inverted for the remainder of the incubation period. The
major disadvantage with such a bioassay is that it requires
at least 18 days to visibly detect growth of the mycobacte-
rial colonies [46].
Radiorespirometry
The inhibition or growth of MTB growth can be determined
in a period of 1 week by evaluating the extent of oxidation
of [1–14C] palmitic acid in a liquid mycobacterial
Middlebrook 7H12 medium (BACTECTM 460TB 12B) to 14CO2,
which is measured in the BACTEC 460 instrument [77].
Radiorespirometry was the method of choice in the devel-
oped world for the greater part of the 1980s and 1990s for
clinical mycobacterial drug susceptibility testing since
results were obtained more rapidly compared with conven-
tional agar dilution methods. The relative activity of various
samples can either be compared by testing at only one or
two concentrations and determining the percentage inhibi-
tion of 14CO2 production relative to drug-free controls [78],
or multiple concentrations can be tested and an MIC calcu-
lated [79,80]. Readings can be taken at various time inter-
vals, usually after every 24 h and, thus, this technique can
provide a kinetic picture of mycobacterial growth or inhibi-
tion. The main drawback of this bioassay are the costs
involved, including isotope disposal costs in some countries
and the large volumes of medium required, which in turn
requires a large amount of sample to be tested, usually in
the range of 50–100 ll. More recent non-radiometric auto-
mated systems for clinical use utilize indicators of oxygen
consumption [81], carbon dioxide production [82], or head
space pressure [83] to determine mycobacterial growth or
inhibition, but otherwise, they do have the same disadvan-
tages as the BACTEC 460. These systems include the
BACTEC TB-460 radiometric system (Becton Dickinson,
Sparks, MD, USA) and, more recently, the mycobacterial
growth indicator tube (MGIT) (Becton Dickinson) in both its
automated and manual versions [84–87]. The radiorespiro-
metric technique detects the metabolic activity ofmycobacteria, as opposed to mycobacterial growth as colo-
nies on a solid medium [46].
Micro-broth dilution
Evaluation of activity (susceptibility) of natural products in a
96-well microplate format offers the advantages of small
sample requirements, low cost, and high-throughput, includ-
ing the potential for automation. Mycobacteria are usually
cultivated in Middlebrook 7H9 broth supplemented with
0.5% glycerol, 0.1% casitone, 0.05% Tween-80 and 10% OADC
(oleic acid, albumin, dextrose and catalase), 7H9GC-Tween
80. The growth of many strains of mycobacteria can be quan-
titatively evaluated by turbidity in a liquid medium, but the
tendency of mycobacteria to clump together makes this a dif-
ficult test. Crude extracts from natural products may in addi-
tion impart some turbidity to the culture medium, making
interpretation of results difficult. The use of an oxidation–re-
duction indicator dye such as Alamar Blue (Trek Diagnostics,
Westlake, Ohio) makes micro-broth dilution a more rapid and
a sensitive bioassay. This method was first proposed by Yajko
et al. [88] in a study that evaluated the activity of the first-line
anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol
and streptomycin) against clinical isolates of MTB. Alamar
blue, a proprietary reagent, had been used previously to study
both metabolism and viability in other microorganisms
[89,90]. In addition, it has also been used to measure toxicity
in both prokaryotic and eukaryotic cells [91]. The reagent
(Alamar blue) is blue in color in the oxidized state, but it turns
pink when reduced due to bacterial metabolism. The two col-
ors (blue and pink) can easily be differentiated with the naked
eye. The study of Yajko et al. [88] was important, since it
showed for the first time that MICs of essential anti-TB drugs
(isoniazid, rifampicin, streptomycin and ethambutol) could be
determined following incubation of MTB isolates for only one
to 2 weeks in the presence of the test drugs. This colorimetric
method (Alamar blue) was also proposed by Collins and
Franzblau [92] for use in a microplate format, microplate
Alamar blue assay (MABA), for high-throughput screening of
compounds against MTB and Mycobacterium avium, and by
Shawar et al. [93], for rapid screening of natural products for
activity against MTB. The results of the MABA can be read
visually [94] and do not require any instruments. The reduced
form of Alamar blue can also be quantitated colorimetrically
by measuring absorbance at 570 nm (and subtracting absor-
bance at 600 nm; the peak for the oxidized form), or fluoro-
metrically [77] by exciting at 530 nm and detecting emission
at 590 nm; the latter mode has been shown to be more sensi-
tive. For non-fluorometric readouts, micro-broth dilution
tests can also be performed by using the non-proprietary
resazurin [95,96] or tetrazolium dyes [97–100]. Hence, it is pos-
sible to conduct high-throughput, anti-TB assays in micro-
plate format using a microplate spectrophotometer or
microplate fluorometer, which are more quantitative bioas-
says capable of detecting partial inhibition, thus making it
ideal for determination of the relative activity of fractions
from natural product crude extracts using one or two
concentrations.
174 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3Nitrate reductase assay/Greiss method
Nitrate reductase assay (NRA), a new approach for the rapid
colorimetric detection of drug resistance in TB or suscepti-
bility of natural products against MTB is based on the
capacity of MTB to reduce nitrate to nitrite. The NRA, also
known as the Griess method, is an old method that has
also been used to differentiate MTB from other species of
mycobacterium [101]. This technique has been introduced
for the rapid detection of drug resistance in TB [102], as
well as in the evaluation of anti-TB activity of natural prod-
ucts. Following the incorporation of potassium nitrate in
the culture medium, reduction of nitrate to nitrite can be
detected using specific reagents, which produce a colored
reaction. The sensitivity and specificity of NRA, compared
with the BACTEC 460-TB system, were 100% and 100% for
rifampicin, 97% and 96% for isoniazid, 95% and 83% for
streptomycin, and 75% and 98% for ethambutol, respec-
tively, when a panel of MTB strains with various resistance
patterns was tested. The majority of results were available
following incubation for 7 days, and NRA was able to iden-
tify most resistant and susceptible strains positively.
Recently, two studies have described the use of the NRA
directly with sputum samples. Musa et al. [103] evaluated
the NRA for drug susceptibility testing (DST) of MTB directly
on smear-positive sputum samples containing more than
ten acid-fast bacilli (AFB) per microscopy field, while Solis
et al. [104] compared the sensitivity and specificity of the
direct NRA with the proportion method in Lowenstein
Jensen (LJ) medium for determination of resistance to isoni-
azid (INH) and rifampicin (RIF) in clinical isolates of MTB.
These two studies have shown the feasibility of implement-
ing the NRA as a direct method for detecting drug-resistant
clinical isolates of MTB in sputum samples [87]. Nitrate
reductase assay (NRA) can also be used in susceptibility
testing of MTB against natural products.
Flow cytometry
Flow cytometry was first used at the beginning of the 1980s
to study the effects of antimicrobial agents in prokaryotes
[105,106]. The number of scientific articles addressing the
antimicrobial responses of bacteria (including mycobacte-
ria), fungi, and parasites to antimicrobial agents consider-
ably increased in the 1990s, due to interesting advances in
the field of flow cytometry from microbiology laboratories
[107]. Norden et al., has reported the use of both
Fluorescein diacetate (FDA) (a non-fluorescent diacetyl fluo-
rescein ester that becomes fluorescent upon hydrolysis by
cytoplasmic esterases) staining and flow cytometry for sus-
ceptibility testing of MTB [108]. In addition, Pina-Vaz et al.
stained MTB with SYTO 16 (a nucleic acid fluorescent stain
that only penetrates into cells with severe lesions of the
membrane) in the absence or presence of antimycobacterial
drugs [109]. Flow cytometry is a promising technique which
needs to be considered in the setting of a clinical mycobac-
terial laboratory, since it gives fast results, compared with
the time needed to obtain susceptibility results of MTB
using classical methodologies, which are currently too long.The main disadvantage of this method is the high cost of
equipment [105].
Reporter gene assays
Several species of firefly, beetle, crustacean, bacteria and the
sea pansy have been used to clone genes which encode luci-
ferase enzymes [110]. In addition, fluorescent proteins, such
as the red fluorescent protein (RFP) and green fluorescent
protein (GFP), have also been introduced into mycobacterial
plasmids. These proteins permit rapid determination of
bacterial viability by measuring the expression of an
introduced fluorescent or luminescent protein [105]. These
fluorescent proteins do not require an exogenous substrate,
thus simplifying quantitation and enabling easy determina-
tion of growth and/or inhibition kinetics [111,112]. This
method can be applied in a multi-well format with more
convenient high throughput detection. Luciferase proteins
from the firefly [113–115] and from Vibrio harveyi [116] utilize
luciferin and n-decylaldehyde substrates, respectively, with
n-decylaldehyde yielding a higher signal in mycobacteria.
Luciferase enzymes are not ideal for kinetic measurements
since they require the addition of a substrate, but they are
potentially useful for susceptibility testing in MTB-infected
macrophages [113] and in mice [116,117] since the lumines-
cence measurements, performed in a luminometer, have a
much higher signal-to-background ratio than is obtained
from fluorescence assays. The major disadvantage of reporter
gene assays is that their use for commercial applications is
often limited by patent restrictions; hence, the number of
mycobacteriology laboratories using this method for suscep-
tibility testing of MTB against natural products is fairly small
[105].
Dormant tubercle bacilli bioassay/low oxygen bioassay
The therapeutic challenges encountered in eradicating dor-
mant tubercle bacilli in MTB infection is responsible for pro-
longed treatment of active disease [118], making TB control
difficult [119]. Dormancy of tubercle bacilli has been identified
as the principle cause for the majority of the problems asso-
ciated with TB therapy [120]. Current anti-TB drugs cannot
effectively kill the dormant forms of MTB [121], and the lack
of a screening bioassay for chemical molecules with activity
against dormant tubercle bacilli has been an obstacle towards
the development of novel drugs against latent TB [121].
Currently, researchers are using non replicating mycobacteria
[122] and hypoxic adapted (low oxygen adapted mycobacte-
ria) mycobacteria that are subsequently exposed to test sam-
ples [123]. Under the test conditions, mycobacteria are in a
state of dormancy. L.G. Wayne has devised Wayne’s hypoxic
model which is currently used for in vitro evaluation of
new compounds; however, this method possesses a low
throughput capability [121,124]. In addition, Cho et al. [125]
has implemented a high-throughput, luminescence-based,
low-oxygen-recovery assay for screening of compounds
against non-replicating MTB using an MTB pFCAluxAB strain
(this is the MTB H37Rv strain containing a plasmid with an
acetamidase promoter driving a bacterial luciferase gene).
I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3 175Recently, Khan and Sarkar [121], while using Wayne’s hypoxic
model and nitrate reductase activity in M. bovis BCG (Bacillus
Calmette-Guérin) culture, have developed a dormant stage
specific antitubercular screening protocol in microplate for-
mat [121].High-performance liquid chromatography mycolic acid
analysis
Identification of mycobacterial strains isolated from clinical
specimens by mycolic acid analysis using HPLC and p-
bromophenacyl bromide derivatizing reagent for UV detec-
tion is a well-established method [126,127]. The total area
under the mycolic acid (TAMA) can be used as a good estima-
tor of mycobacterial growth and also as a means of suscepti-
bility testing of MTB, since a linear relationship between the
TAMA chromatographic peaks of a culture of MTB and
logCFU/mL has been found to exist [128,127]. Even though
the reagents and supplies for HPLC are cheaper compared
with those needed for the BACTEC radiometric method, the
main drawback of this method is the initial cost of the
equipment.Numerical evaluation
It is important to design experimental evaluation protocols
that are independent of the typical 2-fold dilution scheme
that is commonly used in susceptibility testing of MTB for
the purpose of establishing structure–activity relationships.
A numerical evaluation scheme has been established that
allows the determination of more precise MIC values and
extrapolation of the precise endpoint (99% inhibition of
mycobacterial metabolic activity in BACTEC and 90% growth
inhibition in MABA) [129]. This scheme also allows for the
quantitation of the bioassay-guided isolation protocol. The
importance of quantification of bioactivity in search for novel
anti-TB agents has also been emphasized by Eloff [130], point-
ing out that quantitation is a requirement for the detection of
synergistic effects [46].Macrophage bioassays
The intracellular activity of anti-mycobacterials has been
evaluated using in vitro models of macrophage infection by
Mycobacterium species. Macrophages can be sourced from
humans, mice and rabbits. Different mycobacterial species
(MTB H37Ra, H37Rv, Erdman, and clinical isolates, M. bovis
BCG and M. avium) have been used, and hence a source of
variability of results [131,105]. The monocytic cell line THP-1
(ATCC/TIB-202) obtained from American Type Culture
Collection (Rockville, MD) can be used to examine the inhibi-
tory activity of crude extracts, fractions and isolated com-
pounds against intracellular bacilli. THP-1 cells (5 · 104
cells/ml) are treated with 100 nM of phorbol myristate acetate
in a culture flask for 24 h to convert them into macrophages.
These macrophages are incubated for 12 h with MTB H37Ra at
a multiplicity of infection of 1:100 for infection. Extracellular
mycobacteria are removed by washing twice with
phosphate-buffered saline and then adding fresh mediumto adhered cells. Crude extracts, fractions and isolated com-
pounds can then be added to these infected macrophages at
different concentrations. 2-Nitroimidazole is used as the pos-
itive control. The effect of the test samples is monitored by
determining the bacterial load within macrophages by lysing
them with hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2 and
10 mM KCl) and spreading the samples on Dubos agar plates
at different time intervals to enumerate colonies after 21 days
[121].Anti-TB ex vivo bioassay
Patient Peripheral Blood Mononuclear Cell (PBMC) bioassay
Before novel formulations (hits) can proceed for proof of con-
cept evaluation in animal models, efficacy against MTB can be
tested further in an ex vivo model. In countries where TB is
widely prevalent, Peripheral Blood Mononuclear Cell (PBMC)
isolated from tubercular patients can be considered as a very
good ex vivo model. The clinical efficacy of a compound will be
better revealed following its evaluation on collections of a
wide variety of patient samples in different stages of the
infection with different strains of mycobacterium (MDR or
XDR) in a diverse spectrum of disease situation. A series of
immune pathological events happen following MTB infection
as reported by several studies using animal models. For
example, infected cells from active MTB patients have been
shown to produce significant amounts of nitric oxide com-
pared with non-infected cells [132–134]. In addition, IFN-
gamma elevation is also observed in human PBMC infected
with MTB. Animal models infected with MTB exhibit a gross
down-regulation of gene expression associated with innate
and adaptive immunity. In particular, a lower relative expres-
sion of key innate immunity related genes, including the Toll-
like receptor genes (TLR genes 2 and 4), lack of differential
expression of indicator adaptive immune gene transcripts
(IFNG, IL2, IL4) and lower major histocompatibility complex
class I (BOLA) and class II (BOLA-DRA) gene expression, has
been shown to be consistent with innate immune gene
expression in M. bovis (BTB)-infected animals [133]. This
diversity in differential gene expression will affect the effect
of drugs in PBMC isolated from patients in comparison with
the non-tubercular counterparts. Hence, novel compounds
tested on patient PBMC ex vivo bioassay before exploring the
in vivo animal model will be more informative and cost-
effective.Anti-TB in vivo bioassay
Drug candidates (hits) for clinical evaluation must be active in
an in vivo animal model of MTB infection at a dosage that can
be well-tolerated in human subjects. Mice are usually infected
via aerosol exposure to virulent strains of MTB, resulting in
the deposition of low numbers of tubercle bacilli in the lungs
[46]. Following multiplication of tubercle bacilli and host
immune response, therapy is commenced either during the
phase of rapid multiplication (up to 1 month) [135,136], or dur-
ing the non-replicating/dormancy phase [119], which can last
176 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3for months. Recently, long-term models evaluating the steril-
izing ability of novel compounds have been described [137]. In
vivo models (non-human primates) that can be used to assess
the activity of novel compounds in latent infection have
recently been described [138].Toxicity evaluation
Toxicity is a leading cause of attrition of novel compounds at
all stages of the drug development process [139,140]. In vitro
toxicology studies are usually conducted before the first
in vivo toxicity studies, usually to predict those compound-
related toxicities that can limit the progression of a novel
chemical molecule. Following the evaluation of novel com-
pounds for activity against MTB, cytotoxicity to mammalian
cell lines should be evaluated to determine if the compound
is only toxic to mycobacterial cells (selective toxicity). There
are several bioassays, including colorimetric methods based
on the formation of formazan-like products [79,141], as well
as the Alamar Blue dye and bioluminescent analysis of ade-
nosine triphosphate (ATP), which appears to offer the answer
to the demands of speed and simplicity, providing the
required sensitivity to screen out for cytotoxicity [142].
Hence, the determination of general cytotoxicity is important
in the course of drug discovery against MTB.Selectivity and criteria for antimycobacterial
activity
Studies on the selectivity index (SI) should be performed dur-
ing the early phases of drug discovery against MTB. Based on
the simultaneous determination of the general median cyto-
toxic concentration of a novel compound to a mammalian
cell line (IC50) and the lowest concentration inhibiting
mycobacterial growth, the mycobacterial MIC selectivity
index (SI) [135,143] can be determined as the ratio of both
(i.e., IC50/MIC), and taken into account throughout the
bioactivity-guided fractionation of active principles in the
drug discovery process. Relevant mycobacterial activity as
defined by the Clinical and Laboratory Standards Institute
(CLSI) relates to MIC values below 128 lg/ml (<128 lg/ml) for
plant extracts, below 0.25% v/v for essential oils, and below
25 lM (<25 lM) for pure compounds [144]. A selectivity index
of greater than 10 (>10) is considered to be of interest during
the drug discovery process, especially to the pharmaceutical
industry [145].Natural products chemistry
Minor considerations
The majority of natural product collections usually start as
crude extracts of fresh or dried material processed by differ-
ent methods using various chemical solvents. Hydrophilic
compounds are extracted using polar solvents, such as
methanol, ethanol or ethyl-acetate, while lipophilic com-
pounds are extracted using non-polar solvents, such as
dichloromethane (DCM) or a mixture of DCM and methanol(1:1). Crude natural product extracts are complex mixtures
of perhaps hundreds of different compounds working
together in synergy when the extract is administered as a
whole. Discovery of natural product hits and their progression
towards development includes extraction of the crude extract
from the source, concentration, lyophilization (in cases where
polar solvents have been used), fractionation and purification
to yield a single bioactive compound. Chromatography is one
of the most useful means for separation of complex com-
pound mixtures, and also as a technique for both compound
purification and identification. Chromatographic methods
that are primarily used in isolation and identification of nat-
ural products include thin-layer chromatography (TLC), liquid
column chromatography (LC), gas chromatography (GC), high-
performance liquid chromatography (HPLC), fast protein liq-
uid chromatography (FPLC), immobilized metal-ion affinity
chromatography and antibody affinity chromatography [105].
Traditional bioassay-guided fractionation techniques may
only be run for a few months in an intensive screening cam-
paign, and the purification of active compounds may not be
possible in that time frame; hence, they are generally
regarded as being too slow to fit into the pace of high-
throughput screening [146]. The chances for success in isolat-
ing a potent antimycobacterial compound from a semi-
purified extract may or may not depend on the generated
MIC values. The possibility exists that an extract with a rela-
tively low MIC (high activity) may contain large amounts of
only very few moderately active major compounds, while
moderately active crude natural product extracts could con-
tain minor constituents with high activity. An example for
the former is the DCM extract of Alpinia galanga with an
MIC of 1 lg/ml (H37Ra, MABA), which contains 5% of acetoxy-
chavicole acetate as the main active phytoconstituent with an
MIC of 1 lg/ml. An example for the latter is the methanolic
crude extract of Ajuga remota with an MIC of 100 lg/ml (98%
inhibition in BACTEC/H37Rv at that concentration), which
has been shown to contain 0.1% of ergosterol-5, 8-
endoperoxide with an MIC of 1 lg/ml [147]. The combination
of moderately active natural product crude extracts with syn-
thetic analogs has been shown to bear great potential of
increasing antimicrobial activity by two orders of magnitude,
hence increasing the motivation for rigorous studies on
extracts with moderate activity. In addition, any structural
class of natural products that is consistently found to have
activity against MTB shall be considered to be more attractive
for further development as an anti-TB agent than a single
compound with high potency, but no reported anti-TB activity
of related natural analogs [46]. Hence, a rational drug discov-
ery program against MTB should employ a bioassay-guided
fractionation protocol that is capable of isolating minor con-
stituents from a crude extract with interesting activity against
the target organism, MTB. The major advantage of such a pro-
tocol is that isolation of large quantities of an entire series of
structurally related anti-TB compounds is greatly enhanced,
and hence basic structure–activity relationships can be estab-
lished following isolation of primary hits using bioactivity-
guided fractionation. In addition, such a protocol can help
to prioritize classes of natural products for further evaluation
towards lead compound identification for anti-TB activity. For
example, carbazole alkaloids are a class of natural products
I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 3 177with moderate but consistent activity with a potential for
development as lead compounds against MTB [148,149].
However, in the course of bioactivity-guided fractionation of
Micromelum hirsutum, it is worth noting that the ‘‘best hit’’
approach focused further development on micromolide, the
fatty acid lactone with an MIC of 1 lg/ml, versus its carbazole
counterparts with lesser bioactivity (MICs of 16 to >128 lg/ml)
[147].
Structure elucidation of natural products
Isolation, purification and structure elucidation of target com-
pounds from complex crude extract mixtures are the major
bottlenecks in natural products chemistry. Currently, the
main spectroscopic tools for structure elucidation of natural
products are nuclear magnetic resonance (NMR) and mass
spectroscopic (MS) techniques, in addition to infrared (IR)
and ultraviolet–visible spectrophotometric (UV–Vis) methods,
which are also equally important [150]. Structure elucidation
of natural products in small, sub-milligram quantities of
material have currently been made possible by recent
advances in NMR spectroscopy and MS techniques. The time
line for dereplication, isolation and structure elucidation of
natural individual compounds present in the crude extracts
has been significantly reduced by the recent development in
the hyphenated techniques, which combine separation tech-
nologies such as HPLC and solid-phase extraction (SPE) with
NMR and MS techniques [151]. The sensitivity of modern
hyphenated MS methods is in the nano or pictogram range;
hence, it is well below the detection limit of a bioactive com-
pound. Excellent reviews on these topics are available
[46,152–154]. The major contributing factors in NMR are the
superconducting magnet technology [155], micro- and cryo-
probe technology [156–158], and the establishment of a myr-
iad of multi-pulse experiments that cover all routine aspects
of organic structure determination [159]. The need to analyze
natural product quantities that are sufficient for the anti-TB
bioassay (100 lg for a MABA anti-TB test) make sensitive 1H
NMR methods most useful for structural characterization of
active compounds in a bioassay-guided drug discovery pro-
gram. All proton-detecting experiments, such as 2D COSY,
HSQC/HMBC and NOESY, are powerful tools in both the
dereplication and structure elucidation of bioactive natural
products besides the routine 1D proton NMR [160–162]. It is
important to note that 1D NMR experiments that apply selec-
tive excitation pulses as part of classical COSY, TOCSY and
NOESY sequences are particularly valuable sources of struc-
tural information [163–165]. For example, the sesquiterpene
2,10-bisaboladiene- 1,4-endoperoxide from Rudbeckia laciniata,
an anti-TB natural product, was characterized using
Gaussian-shaped pulses in a selective COSY experiment [46].
Dereplication and NMR fingerprinting of natural products
The driving force behind much phytochemical research is the
discovery of new biologically active compounds with antimy-
cobacterial activity. Bioassays, then, must be carried out in
order to identify promising plant extracts, to guide the sepa-
ration and isolation and finally to evaluate hit compounds.
Dereplication (positive identification of known naturalproducts) is not a trivial task [166–170] since comprehensive
sets of standards are rarely available. Improved efficiency in
dereplication of active principles is of dual importance.
First, it lowers the overall efforts during bioassay-guided frac-
tionation, since a relatively small number of (ubiquitous) con-
stituents can blur the view of the natural products chemists
for new compounds with desired activity. Secondly, it allows
the concentration of resources on the elucidation of novel
compounds. Dereplication, however, must be definitive,
which in light of the structural complexity of natural products
(e.g., multiple [stereo] isomers) places strong demands on the
quality and comprehensiveness of the analytical data [46].
One simplified yet highly significant approach to this problem
is to dereplicate compounds by 1H NMR fingerprint analysis of
their hyper complex proton signals [171–173]. This methodol-
ogy makes use of the distinct fingerprint patterns of proton
signals arising from the complex proton spin system con-
tained in most natural products. An additional benefit of the
aforementioned selective 1D NMR experiments is that they
facilitate compound dereplication by providing coupling
and/or shift-edited sub-spectra of the often crowded 1H
NMR spectra and are suitable to generate high-resolution data
for 1H NMR fingerprint analysis [46].
Countercurrent separation of natural products
Currently, the most common chromatographic methods
applied in natural product separation include: adsorption
chromatographic methods, such as TLC; LC; GC; HPLC;
FPLC; immobilized metal-ion affinity chromatography; and
antibody affinity chromatography [174,175]. In addition, par-
tition chromatography is another separation technique that
has so far been applied by rather few scientists [46].
Nevertheless, countercurrent chromatography (CCC) and
centrifugal partition chromatography (CPC), collectively
known as countercurrent separation, are powerful tools in
both the early and advanced stages of the fractionation pro-
cess of crude natural product extracts [176]. There are recent
reviews on this subject [46,177]. Modern CCC methods, such
as high-speed countercurrent chromatography (HSCCC) and
[fast] centrifugal partition chromatography ([F] CPC), reduce
the difficulties involved in natural product drug discovery,
i.e., expensive and time-consuming steps to isolate active
constituents, and have the ability to attain high resolution
[46]. Current instrumental developments have been summa-
rized [178–180], and continuously updated information is
available online [181]. The major advantages of countercur-
rent/partition chromatography evolve from the complete
lack of a solid stationary phase, translating into the lack of
any irreversible absorption [46], which is essential for a
bioassay-directed search for anti-TB lead compounds. The
chances of ‘‘losing’’ the anti-TB activity of a natural product
during fractionation are eliminated [182], or at least reduced
to the unavoidable possibility of degradation in solution at
room temperature (c.f. with ubiquitous rotary evaporation
at elevated temperatures), since CCC/CPC provides a means
of loss-free fractionation, which in the course of bioassay-
guided fractionation is particularly valuable. Dr. Yoichiro
Ito (NHLBI/NIH), the inventor of modern CCC has recently
created a timely and invaluable documentation of more than
178 I n t e r n a t i o n a l J o u r n a l o f M y c o b a c t e r i o l o g y 4 ( 2 0 1 5 ) 1 6 5 –1 8 330 years of experience in the field by formulating his 18
Golden Rules and Pitfalls in selecting optimum conditions
for CCC [183]. Significant loss of activity between subsequent
countercurrent/partition chromatographic steps can be con-
fidently explained as several natural product constituents
acting in synergy, which (synergy) has recently been identi-
fied as a major factor in explaining the overall antimicrobial
activity of plant-derived agents [46]. For example, the signif-
icant antimicrobial effect of berberine alkaloids contained in
the Berberis extracts has been associated with the flavono-
lignan 5V-methoxyhydnocarpin, which inhibits multidrug-
resistance pumps [184]. While using the Oplopanax horridus
(Devil’s Club), it has been demonstrated that the polarity
window can be chosen such that the activity could be
enriched into a sub-fraction of less than 90% (w/w) [185].
This enabled the in vivo evaluation of a crude natural pro-
duct with only one literature report on the anti-TB activity
of hops constituents [186]. Recently, using CCC, the anti-TB
polarity window of O. horridus has been shown to contain
polyynes (polyacetylenes) by GC–MS analysis, in addition to
other constituents. Moreover, it exhibited no cytotoxicity
on Vero cells, which is not only a prerequisite for in vivo
testing, but also came as a surprise since numerous litera-
ture reports have associated polyynes with cytotoxicity
[46,187–194].New perspectives
New and drug-resistant strains of MTB continue to emerge
because of the remarkable genetic and adaptable plasticity
of the microbiota. Natural products have been and will con-
tinue to be a rich source of novel drugs. New natural products
chemistry tools and new mycobacteriological bioassays with
relevance to MTB virulence are available and await eager
employment by interdisciplinary research teams. The
extended mycobacteriology toolbox allows for the detection
of relevant biological activities at various levels correspond-
ing to the disease, including mycobacterial growth (MABA),
pathogen self-defense (LORA), host defense (macrophage),
and in vivo (animal) regardless of the source of the natural
product (plant, marine, animal or microorganisms).
Researchers in all institutions will be able to contribute to
the development of understanding and utilization of natural
product resources, due to the continuous development of
sensitive, rapid and inexpensive bioassays. Innovative proce-
dures for preparative analysis can be tailored by taking
advantage of new bioassays, new separation (CCC/CPC) and
detection and identification (NMR) methods outlined in this
review in order to improve access to and benefit from the
favorable chemo diversity of nature. These methods will open
new perspectives in the discovery of useful anti-TB agents
when seamlessly combined. The increasing frequency of
MDR-TB, XDR-TB and currently, totally TDR-TB, and limited
therapeutic options emphasize the urgent need for novel
drugs against TB.
Finally, it shall be noted that, from both the biological/my
cobacteriological and the natural products chemistry per-
spective, the various aspects of the collaborative challenges
faced during drug discovery against TB from natural productsare applicable to other infectious diseases. It is believed that
the interplay of co-developed innovative mycobacteriological
and natural product chemistry methods on both ends will
greatly impact the early phases of anti-TB drug discovery
and increase the chances of success.Conflict of interest
We have no conflict of interest to declare.Acknowledgments
We wish to acknowledge the Bill and Melinda Gates
Foundation, through Noguchi Memorial Institute for Medical
Research postdoctoral training fellowship in infectious dis-
eases for support and the shared vision of making a contribu-
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