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ORIGINAL PAPER
Exploring anti-TB leads from natural products libraryoriginated from marine microbes and medicinal plants
Xueting Liu • Caixia Chen • Wenni He • Pei Huang •
Miaomiao Liu • Qian Wang • Hui Guo • Krishna Bolla •
Yan Lu • Fuhang Song • Huanqin Dai • Mei Liu • Lixin Zhang
Received: 18 April 2012 / Accepted: 12 July 2012 / Published online: 20 July 2012
� Springer Science+Business Media B.V. 2012
Abstract Multidrug-resistant tuberculosis (MDR-
TB) and TB–HIV co-infection have become a great
threat to global health. However, the last truly novel
drug that was approved for the treatment of TB was
discovered 40 years ago. The search for new effective
drugs against TB has never been more intensive.
Natural products derived from microbes and medic-
inal plants have been an important source of TB
therapeutics. Recent advances have been made to
accelerate the discovery rate of novel TB drugs
including diversifying strategies for environmental
strains, high-throughput screening (HTS) assays, and
chemical diversity. This review will discuss the
challenges of finding novel natural products with
anti-TB activity from marine microbes and plant
medicines, including biodiversity- and taxonomy-
guided microbial natural products library construction,
target- and cell-based HTS, and bioassay-directed
isolation of anti-TB substances from traditional
medicines.
Keywords Mycobacterium tuberculosis �MDR-TB � Natural products library �Marine microbes � Traditional medicines �Tanshinones
Abbreviations
TB Tuberculosis
Mtb Mycobacterium tuberculosis
MDR-TB Multidrug-resistant Mycobacterium
tuberculosis
XDR-TB Extensively drug-resistant TB
HIV Human immunodeficiency virus
NPL Natural products library
HTS High-throughput screening
PKS Polyketide synthases
NRPS Non-ribosomal peptide synthetases
ICL Isocitrate lyase
MS Malate synthase
BPL Biotin protein ligase
Fab Fatty acids biosynthase
MmpL Mycobacterial membrane protein, large
GyrB DNA gyrase subunit B
DHFR Dihydrofolate reductase
TMPK Thymidine monophosphate kinase
HRS High-resolution screening
Xueting Liu and Caixia Chen have contributed equally to this
paper.
X. Liu � C. Chen � W. He � P. Huang � M. Liu �Q. Wang � H. Guo � K. Bolla � Y. Lu � F. Song �H. Dai � M. Liu � L. Zhang (&)
Key Laboratory of Pathogenic Microbiology and
Immunology, Institute of Microbiology, Chinese
Academy of Sciences, Beijing 100190, China
e-mail: [email protected]; [email protected]
P. Huang � M. Liu � Q. Wang � H. Guo
Graduate University of Chinese Academy of Sciences,
Beijing 100049, China
Y. Lu
Anhui University, Hefei 230601, Anhui, China
123
Antonie van Leeuwenhoek (2012) 102:447–461
DOI 10.1007/s10482-012-9777-0
ALIS Automated ligand identification system
rpsD Ribosomal protein small-subunit D
PtpA Protein tyrosine phosphatase A
Introduction
Tuberculosis (TB) is a contagious airborne disease
caused by the pathogen Mycobacterium tuberculosis
(Mtb) and accompanies human for a long history
(WHO 2011). TB is second only to human immuno-
deficiency virus (HIV) as the leading infectious killer
of adults worldwide and is the leading infectious cause
of deaths among people with HIV/AIDS (http://new.
tballiance.org/why/tb-threat.php). One third of the
world’s population is currently infected with Mtb,
which kills someone approximately for every 20 s—
nearly 4,700 people everyday. In 2010 there were 8.8
million (range, 8.5–9.2 million) incident cases of TB,
1.1 million (range, 0.9–1.2 million) deaths from TB
among HIV-negative people and an additional 0.35
million (range, 0.32–0.39 million) deaths from
HIV-associated TB according to the report from the
World Health Organization (WHO) (World Health
Organization 2011). Multidrug-resistant Mtb (MDR-
TB), defined as resistance to at least isoniazid and
rifampin, was documented in nearly every country sur-
veyed from 1994 to 2000 by the World Health Orga-
nization—International Union against Tuberculosis and
Lung Disease as part of the Global Drug Resistance
Surveillance Project (Gupta and Espinal 2003).
Furthermore, the emergence of extensively drug-resis-
tant TB (XDR-TB) has been noted on a global scale
(Centers for Disease Control and Prevention 2006a, b).
The ability for Mtb to remain dormant or persistent
within host cells for many years with the potential to
be activated allows the bacterium to escape the
activated immune system of the host (Meena and
Rajni 2010). A person infected with Mtb incurs a
5–10 % risk of developing active TB. The latent or
persistent TB infection is a major obstacle in the cure
and prevention of TB. Reactivation of latent TB is a
high factor for disease development particularly in
immunocompromised individuals such as those co-
infected with HIV, on an anti-tumor necrosis factor
therapy or with diabetes (Koul et al. 2011).
Streptomycin was the first drug introduced in 1944
for the treatment of TB but almost immediately after
its introduction many patients started showing
resistance to this antibiotic (Youmans et al. 1946;
Pyle et al. 1947; Medical Research Council 1948).
Para-aminosalicylate (PAS) was introduced in 1946,
which largely overcame the emergence of resistant
strains (Medical Research Council 1950). A few years
later, isonicotinylhydrazine (isoniazide, INH) was
developed and initial treatment with both INH and
streptomycin was even more effective. To date, many
drugs are available, which are classified into two
categories. First line therapy includes five medica-
tions: isoniazide, pyrazinamide (analog of nicotin-
amide), ethambutol [(S,S0)-2,20(ethylenediimino)
di-1-butanol], rifampicin (lipophilic ansamycine) and
streptomycin (aminocyclitol glycoside) (Gilman
1990; Goldberger 1988). Second line therapy, which
is used exceptionally in the cases of drug resistance,
includes cycloserine, capreomycin, fluoroquinolones,
ethionamide, PAS, thioacetazone, rifabutin, clofazi-
mine and some macrolides (Iseman 1993).
However, the last truly novel drug that was
approved for the treatment of TB was discovered
40 years ago. Search for new effective drugs against
TB has never been more intensive.
For thousands of years, natural products have
played an important role throughout the world in
treating and preventing human diseases. Natural
product medicines have come from various source
materials including plants, microorganisms, verte-
brates, and invertebrates. The value of natural prod-
ucts could be assessed using 3 criteria: (1) the rate of
introduction of new chemical entities of wide struc-
tural diversity, including serving as templates for
semisynthetic and total synthetic modification, (2) the
number of diseases treated or prevented by these
substances, and (3) their frequency of use in the
treatment of disease (Chin et al. 2006).
Our laboratory focuses on bioactivity or mecha-
nism of action-directed isolation and characterization
of active compounds from marine microbes and
Traditional Chinese Medicines (TCMs) (Zhang et al.
2007; Song et al. 2010; Ashforth et al. 2010; Zhuo
et al. 2010; Liu et al. 2012). This review will discuss
the challenges of finding novel natural products with
anti-TB activity from marine microbes and medicinal
plants, which focuses on three aspects: (1) biodiver-
sity- and taxonomy-guided microbial natural products
library construction: the sourcing and de-replication of
microbial metabolites from novel species isolated
from a variety of environments, including extreme and
448 Antonie van Leeuwenhoek (2012) 102:447–461
123
marine habitats, and those species in symbiosis with
other organisms, (2) target- and cell-based high-
throughput screening (HTS), and (3) anti-TB sub-
stances from traditional medicines. As shown in
Fig. 1, the subsequent preclinical optimization of a
lead compound is a cyclic process of obtaining
bioassay screening results, analyzing activity data,
designing new target compounds, and synthesizing
new analogues.
Construction of diversified natural products
library from marine microbes and medicinal plants
Natural products from marine microbes
The construction of a high-quality microbial natural
products library is assessed on two criteria: (1)
diversity of the microbial sources and microbial
gene resources: biodiversity- and taxonomy-guided
isolation of the samples collected from undiscovered
environments is normally used for diversifying the
microbial sources, and metagenomics is promising in
efforts to gain access to uncultured microorganisms,
and (2) diversity of crude extracts, fractions, and pure
compounds.
Diversified microbial strains and gene resources
Undiscovered species inhabiting unique environments
with differing environmental constraints have been
thought to be resources of novel compounds (Bull
et al. 1992; Jensen and Fenical 1996). In order to get
high hit rates of novel leads from HTS, more extensive
collections of microorganisms and further exploration
in the ability to culture diverse species are performed.
The untapped sources from marine and other extrem-
ophilic environment (such as hyper-arid, high tem-
perature, etc.) could provide many novel chemicals for
Fig. 1 The flow chart of
leads identification from
NPL
Antonie van Leeuwenhoek (2012) 102:447–461 449
123
use in drug discovery assays (Freundlich et al. 2010;
Rateb et al. 2011).
Functional (meta-) genomics can provide an insight
into the genes and gene clusters involved in the
production of metabolites. Genomic de-replication and
genome prediction will provide the information of the
physico-chemical properties and the structures of the
metabolites and thus enable taxonomically unique struc-
tures to be produced. Identification of silent (orphan)
biosynthetic gene clusters combined with nonstandard
fermentation or genetic manipulation, which would
activate the silent biosynthetic gene clusters, would
greatly enhance the ability to isolate novel products.
Ecopia BioSciences Company succeeded in revealing an
antibacterial compound (ECO-0501) of a new structural
class from the vancomycin-producer strain Amycolatop-
sis orientalis (Banskota et al. 2006).
Design and use of primers targeting special genes
involved in the compound synthesis afford a direc-
tional method for discovery of specific structures
(Ouyang et al. 2011). Molecular techniques such as
PCR, electrophoresis and amplification of 16S rRNA
genes enabled the identification of novel and known
type-I PKS genes and NRPS genes.
PKS as well as NRPS are of great academic and
industrial interest because many of the compounds
produced by them have potent medicinal utility and
display unusual chemistry. Shotgun sequencing of
environmental DNA and subsequent data analysis also
have the potential to identify genes encoding new
structures. Two products of NRPS with predicated
biological activity have been recently identified via
heterologous expression (Penesyan et al. 2010).
Only the strains that have the genetically potential to
produce the compounds of interest and are likely to
produce chemical novelty are selected for fermentation.
Different fermentation conditions were designed to
stimulate the expression of the genes of interest. In
general, about 50 types of media and different culture
conditions (temperature, pH, etc.) were designed for
small-scale fermentation. The composition of the metab-
olites was significantly influenced by growth conditions.
De-replication of crude extracts and evaluation
of natural product library
One of the most challenges for natural products
discovery is to avoid rediscovery of known com-
pounds. In order to identify the active compounds with
new scaffold and/or new activities rapidly and
efficiently, different technologies have been applied
for de-replication during the drug discovery from
natural sources. There are two primary approaches to
the discovery of novel natural products from extracts:
bioassay-guided fractionation and the singling out of
agents possessing unique structural features and/or
novelty as important representatives of different
chemical classes.
The combined use of LC, solid-phase extraction
(SPE) and NMR spectroscopy (LC-SPE-NMR) has
been developed and continues to undergo refinement
via expansion of the types of NMR analysis one can
perform on samples once they’ve been separated,
removed from LC mobile phase and placed into
deuterated solvents (Larsen et al. 2005; Bobzin et al.
2000; Gu et al. 2006; Konishi et al. 2007; Lambert
et al. 2005; Hu and Xiao 1989; Mukherjee et al. 2010;
Velho-Pereira et al. 2011). However, cost and effort
requirements, for the foreseeable future, limit the
availability of such techniques to many academic
natural product laboratories. In such settings, de-
replication efforts will likely continue to focus on the
use of MS, UV–Vis and possibly, ‘‘second round’’
bioassays to narrow down those fractions of natural
product extracts that warrant more focused structure
elucidation efforts.
To screen a large numbers of compounds for
growth/inhibition kinetics, simple HTS techniques are
needed. Alamar Blue, validated against slow-growing
tuberculosis (TB H37Rv) in 1997, is a rapid and
inexpensive way to measure cell growth by the extent
of pink fluorescence resulting from an oxidation/
reduction reaction (Shawar et al. 1997). Reporter
genes expressed in Mtb can also be used as a surrogate
for growth in HTS. The green fluorescence protein
(GFP) assay measures bacterial growth by direct
readout of fluorescence. Recent improvements in GFP
using an acetamidase promoter have increased the
signal-to-background ratios, making it preferable over
alternative reporters, including luciferase (Changsen
et al. 2003; Pauli et al. 2005). Several other advantages
of GFP include its intrinsic fluorescent nature, pre-
cluding the need for a substrate, and better biosafety (if
the minimal bactericidal concentration is not being
determined), as the microplate can remain closed after
inoculation. Also, GFP measurements in intracellular
environments preclude the need for host cell lysis,
since a substrate is not required, allowing direct and
450 Antonie van Leeuwenhoek (2012) 102:447–461
123
repeated measurements of cell viability, thus offering
easy kinetic monitoring and low cost.
Utilizing of these new technologies on one hand
provide insight into the role and function of orphan
gene clusters which encode novel metabolites, on the
other hand, make the activity screening process more
facile and therefore accelerate the process of new
drugs discovery.
Natural products library from medicinal plants
TCM, which encompasses many different practices, is
based on a clear rationale and a well-established
theoretical approach. The Chinese materia medica
contains hundreds of medicinal substances, primarily
plants, but also some minerals and animal products,
classified by their perceived action in the body.
Different parts of plants such as the leaves, roots,
stems, flowers, and seeds are used.
As an example, we selected 500 traditional plant
medicines, which have been used for the treatment of
TB and/or other infectious diseases, and constructed
the crude extracts library and fractions library for
further anti-TB discovery. The chemical diversity of
these libraries has been evaluated by HPLC–UV,
LC–MS, and NMR analysis.
HTS to evaluate the natural product library
HTS has now evolved into a mature discipline that is a
crucial source of chemical starting points for drug
discovery. Using automation, miniaturized assays and
large-scale data analysis, HTS aims to accelerate the
pace of discovery of new drugs by screening hundreds
of thousands of compounds (Ashforth et al. 2010).
The release of the complete genome sequence of
Mtb has facilitated a more rational and directional
approach to search for new drug targets (Chopra et al.
2003). Recent developments in mycobacterial molec-
ular genetic tools such as transposon mutagenesis,
signature-tagged mutagenesis, gene knock-out and
gene transfer will facilitate the identification and
validation of new drug targets essential for the survival
and persistence of tuberculosis bacilli not only in vitro
but also in vivo (Zhang 2005). In general, gene
products involved in persistence or latency, energy
metabolism, signal transduction [serine/threonine pro-
tein kinases, tyrosine phosphatase and two-component
systems (Chopra et al. 2003)], transcription factors,
cell wall synthesis, virulence factors and unique
physiology of Mtb would be potential targets for the
development of new drugs (Chopra et al. 2003; Zhang
2005). Much effort should be made to evaluate and
validate these novel targets with inhibitors that can be
tested in infection models (Mdluli and Ma. 2007).
The assays described in this review are used to
screen the natural product library derived from marine
microbes and medicinal plants, and include those that
focus on: the targeted screening of enzymes and
pathways; the effect of a compound on the whole cell
of the microbe; and the validation of compounds in
intracellular tests.
Target-based assays
The current TB drugs come into effect mostly by
inhibiting cell wall synthesis (isoniazid, ethambutol,
cycloserine), inhibiting nucleic acid synthesis (rifam-
pin, quinolones), inhibiting protein synthesis (strepto-
mycin) and inhibiting or depleting membrane energy
(pyrazinamide) (Zhang 2005). Due to the drug-resis-
tant TB problem, it is important to develop new drugs
inhibiting novel targets, which are different from those
of currently used drugs. To avoid significant toxicity,
it would be better that the targets of inhibition should
be present in bacteria but not in the human host.
Isocitrate lyase (ICL) and malate synthase (MS) are
two key enzymes in the glyoxylate shunt, which has
been implicated in the persistence (McKinney et al.
2000; Smith et al. 2004) of Mtb and pathogenesis of
several bacteria and fungi (Dunn et al. 2009). The
glyoxylate shunt is not present in mammals and
therefore represents an attractive drug target (Lorenz
and Fink 2002).
In Mtb, the cell wall comprises a complex lipid bi-
layer composed primarily of mycolic acid. It is estimated
that fatty acids account for 40 % of the mycobacterial dry
cell weight and this feature greatly enhances the bacte-
ria’s ability to resist chemical damage, survives in the
hostile environment created by macrophages and limits
its susceptibility to most commercially available antibi-
otics (Loerger and Sacchettini 2009). Biotin protein
ligase (BPL) which play key roles in the synthesis of fatty
acids required for the biogenesis and maintenance of cell
membranes (Soares et al. 2012), were suggested prom-
ising drugable targets for further antitubercular therapeu-
tic development (Purushothaman et al. 2008; Duckworth
Antonie van Leeuwenhoek (2012) 102:447–461 451
123
et al. 2011). Inner membrane transporter MmpL3, a
member of the MmpL (mycobacterial membrane protein,
large) family, was recently suggested to be a target of
pyrrole derivatives (Grzegorzewicz et al. 2012; La Rosa
et al. 2012) and SQ109 (Tahlan et al. 2012).
GlgE is an essential maltosyltransferase that elon-
gates linear a-glucans as part of a synthetic lethal
biosynthetic pathway. Inactivation of GlgE causes
accumulation of a toxic phosphosugar intermediate,
maltose 1-phosphate, which drives the bacilli into a
suicidal self-poisoning cycle that elicits a complex
stress profile, eventually resulting in DNA damage and
death of Mtb. The apparent lack of similar enzymes in
human to GlgE and its many favorable properties
make it a highly attractive novel drug target for
chemotherapy of TB (Jacobs et al. 2010; Kalscheuer
et al. 2010).
Increasing resistance to fluoroquinolones target the
gyrase subunit A has driven interest in targeting DNA
gyrase subunit B (GyrB). GyrB was demonstrated a
new target for TB, and has been validated by two
small-molecules (Chopra et al. 2012).
Dihydrofolate reductase (DHFR), which catalyzes
the NADPH-dependent reduction of dihydrofolate to
tetrahydrofolate that is essential for DNA synthesis, is
an attractive novel drug target for developing anti-TB
drugs. Structure of Mtb DHFR and human DHFR
reveals key differences in the active sites and these
differences could be exploited for the design and
screening of novel anti-TB drugs (Kumar et al. 2010).
Thymidine monophosphate kinase (TMPK), which
is the last specific enzyme in the pyrimidine biosyn-
thetic pathway and catalyses the ATP-dependent
reversible phosphorylation of deoxythymidine 50
monophosphate into deoxythymidine 50 diphosphate
and lies at the point where the salvage and de novo
synthetic pathways meet in nucleotide synthesis, has
emerged as an attractive drug target in Mtb since
blocking it will affect both the pathways involved in
the thymidine triphosphate synthesis. Morerover, the
unique differences at the active site of TMPK enzyme
in Mtb and humans led to more expectation for novel
effective drugs with anti-TB activity (Kumar et al.
2011).
Mtb protein tyrosine phosphatase A (PtpA) and
PtpB are two enzymes secreted by mycobacterial cells
and related to the survival of mycobacteria in their
host macrophages (Koul et al. 2004). Since Mtb strains
with disrupted ptpB genes were impaired in their
ability to survive in guinea pigs, these two enzymes
are suggested as new drug targets (Singh et al. 2005).
Cyclic depsipeptides of the stevastelin family were
reported to be inhibitors for Mtb PtpA (Manger et al.
2005).
The striking degree of microbial resistance to
traditional antibiotics provides us with an overwhelm-
ing impetus to investigate new targets and approaches
to identify novel means to combat pathogenic bacteria.
These obvious drug targets have now been extensively
investigated. Significant differences in organization,
structure of enzymes and the specific roles between the
related systems of human and bacteria make these
systems attractive targets for antibacterial drug dis-
covery and enhance the opportunity for developing
new effective drugs.
Whole-cell-based assays
Whole-cell assays and target-based assays are nor-
mally used in high throughput screening. Target-based
assays are more directional but some natural products
with good inhibition of pure enzymes may also show a
limited inhibitory effect on Mtb cells due to their
limited membrane permeability (Dhiman et al. 2005;
Henriksson et al. 2007). In such case the proper
modification of the hit compounds will be proposed to
overcome the problem. In order to combine the
advantages of the traditional whole-cell assays and
the target-inhibiting assay, target-based whole-cell
assays were developed in recent years to make the
HTS process more facile and efficient.
Arnoldo identified the first known inhibitor of the
Pseudomonas aeruginosa virulence protein, Exos,
using a yeast cell-based phenotypic assay combined
with chemical genomics and it is the first report to
employ compound screening against S. cerevisiae to
identify small molecule inhibitors of human patho-
genic bacteria (Arnoldo et al. 2008).
Of the most significant contributions are the natural
product isolates from two different strains of Strepto-
myces platensis, platensimycin and platencin, which
have been reported as inhibitors against fatty acids
biosynthase (Fab) F and FabF/FabH, respectively
(Singh et al. 2006; Wang et al. 2007a, b). FabH, the
initiation condensing enzyme, and FabF/B, the elon-
gation condensing enzymes are essential components
of Type-II Fatty acid synthesis pathway, which is
significantly different from the human fatty acid
452 Antonie van Leeuwenhoek (2012) 102:447–461
123
synthesis system. Platensimycin and platencin was
discovered by target-based whole-cell screening strat-
egy using antisense differential sensitivity assay. An
essential enzyme, FabI, is responsible for reduction of
the double bond in the enoy-acyl carrier protein
derivative, and is a proven drug target with two
marketed antibacterial agents, triclosan and isoniazid
(an first-line anti-TB agent) (Wang et al. 2007a, b).
Several novel FabI-directed antibacterial agents were
discovered using antisense-based screening strategy
(Payne et al. 2002; Yum et al. 2007). Antisense-based
screening strategy can also be designed in different
pathogens direct to different essential targets, such as
rpsD (ribosomal protein small-subunit D), the ribo-
somal protein S4 of the 30s ribosomal subunit (John
et al. 2007), and SecA, a central member of the protein
secretion machinery (Parish et al. 2009).
HTS programs targeting BPL, MS, ICL, PtpA and
PtpB and Fab series essential enzymes of type-II fatty
acid synthase pathway have led to the discovery of
attractive molecules. The potential inhibitor hits
identified from the HTS will be further validated by
testing in vitro against the target protein to determine
its potency and followed by assays for growth
inhibition against whole cells.
Anti-TB compounds from natural products library
Anti-TB microbial compounds
A series of anti-TB microbial metabolites have
recently been identified from a variety of aquatic and
terrestrial sources (Fig. 2). A marine Pseudomonas
species isolated from a marine alga and tube worm
produced the cyclic depsipeptides massetolide A (1)
and viscosin (2) which showed anti-TB activity with
MIC values of 5.0–10.0 and 2.5–5.0 lg/mL, respec-
tively (Sayed et al. 2000). A new nucleosidyl-peptide
antibiotic, sansanmycin (3), was isolated from an
Streptomyces sp. and exhibited its antibacterial activ-
ity against Mtb and P. aeruginosa with MIC values of
10.0 and 12.5 lg/mL, respectively, but possesses very
poor activities against the other gram-positive and
gram-negative microorganisms (Xie et al. 2007). Two
analogs of Sansanmycins were also discovered with
inhibitory activity against Mtb H37Rv and MDR-TB
strain, with MIC values ranging from 8.0 to 20.0 lg/mL
(Xie et al. 2008).
Halicyclamine A (4), isolated from Insonesian
marine sponge of Haliclona sp. was rediscovered as
anti-TB agent, which is effective to Mtb in both active
and dormant states with MIC values of 1.0–5.0 lg/mL,
by a screening system in hypoxic condition inducing
Mtb dormant state (Arai et al. 2008). Caprazamycin B
(5), as a novel anti-TB antibiotic from the culture broth
of the strain Streptomyces sp. MK730-62F2, showed
excellent anti-mycobacterial activity in vitro against
drug-susceptible and MDR-TB strains with MIC values
3.13–12.5 lg/mL and was considered to be the prom-
ising candidate as an anti-TB drug (Igarashi et al.
2003). The cyanobacterium Tychonema sp. produces
the new cyclic hexapeptides brunsvicamides B (6) and
C (7), which selectively inhibit the Mtb protein PtpB
with IC50 values of 7.3 and 8.0 lM, respectively
(Muller et al. 2006). Three new aminolipopeptides,
designated trichoderins A (8), A1 (9), and B (10) were
isolated from a culture of marine sponge-derived
fungus of Trichoderma sp. as anti-mycobacterial
substances. Trichoderins showed potent anti-mycobac-
terial activity against Mtb H37Rv under standard
aerobic growth conditions as well as dormancy-induc-
ing hypoxic conditions, with MIC values in the range
of 0.02–2.0 lg/mL (Pruksakorn et al. 2010). Pseudop-
teroxazole (11) from the hexane extracts of the West
Indian gorgonian Coral Pseudopterogorgia elisabethae
(Bayer) was a diterpenoid exhibiting potent inhibitory
activity (97 %) of 12.5 lg/mL (Rodriguez 1999).
The natural products (-)-abyssomicin C (12) and its
atropisomer (-)-atrop-abyssomicin C (13), isolated
from Verrucosispora strain Ab-18-032 with activity
against the para-aminobenzoate biosynthetic pathway
initially, were found to exhibit antibacterial activity
against Mtb H37Rv with MIC values of 3.6 and
7.2 lM, respectively. More specifically, (-)-abyssomi-
cin C was bactericidal. This complex natural product
and its analogs, thus, hold promise as chemical tools in
the study of Mtb metabolism (Freundlich et al. 2010).
The indication of quality control in any natural
product library is in the novelty of the active natural
products identified. We find that around 30 % of our
purified compounds have novel structures, and have
recently identified 20 natural product scaffolds of
chemotherapeutic interest. During a pilot screen of
5,000 crude extracts from the library against the
exponentially growing TB vaccine strain BCG, the
known compounds isonitrile, nucleosidyl peptide and
ansamycin were identified as active chemicals present
Antonie van Leeuwenhoek (2012) 102:447–461 453
123
in the library (Fu et al. 2009). Some identified anti-TB
compounds were shown in Fig. 3 from our NPL.
Beauvericin (14), avermectin B1a (15), valinomycin
(16), nanomycin bA (17), and nanomycin aA (18)
showed inhibitory activity against Mtb H37Rv with
MIC values 4.0, 16.0, 1.0, 8.0 and 8.0 lg/mL,
respectively. A series of active polyketides possessing
a unique scaffold were investigated from marine
Verrucosispora (unpublished).
Anti-TB Tanshinones and other plant-derived
natural products
TCM, which encompasses many different practices, is
based on a clear rationale and a well-established
theoretical approach. The Chinese materia medica
contains hundreds of medicinal substances, primarily
plants, but also some minerals and animal products,
classified by their perceived action in the body.
Fig. 2 Anti-TB microbial compounds reported in literature
454 Antonie van Leeuwenhoek (2012) 102:447–461
123
Different parts of plants such as the leaves, roots,
stems, flowers, and seeds are used.
This section will outline the anti-TB natural
products research from our NPL derived from TCM.
Anti-TB tanshinones
Tanshinones are a series of abietane-type norditerpe-
noid quinones isolated from the roots of Salvia
miltiorrhiza (‘tanshen’), a well-known TCM by Nak-
ano and Fukushima for the first time in 1930. Tanshen
is an annual sage plant and has been used in TCM for
the treatment of coronary heart diseases, particularly
angina pectoris and myocardial infarction (Wang et al.
2007a). It has been used for hemorrhage, dysmenor-
rhea, miscarriage, swelling, insomnia, and inflamma-
tory diseases such as edema, arthritis, and endangitis
(Wu et al. 1991; Ryu et al. 1997; Jang et al. 2003). Fu
Fang Dan Shen (a mixture of S. miltiorrhiza, Panax
notoginseng, and Cinnamomum camphora) is regis-
tered as a drug in several countries outside China,
including Vietnam, Russia, Cuba, Korea, and Saudi
Arabia. In 2010, Tanshen was the first TCM to pass US
phase II clinical trials for cardiovascular indications.
Diterpenoid tanshinones were found exclusively in
the genus Salvia and attracted particular attention from
many researchers for their wide-ranging pharmaco-
logical activities such as antibacterial, antioxidant,
anti-inflammatory and antineoplastic (Wang et al.
2007a). Tanshinone I (19), tanshinone IIA (20), and
cryptotanshinone (21) are the major constituents of S.
miltiorrhiza. A related plant S. columbariae, which
has been used by California Indians in the treatment of
strokes, also contains tanshinones, especially crypto-
tanshinone (Adams et al. 2005, 2006). Anti-tumor
activities of tanshinones, especially tan-I, tan-IIA and
neo-tanshilactone, have been fully studied both in
vitro and in vivo against a variety of different human
cancer cells such as glioma cancer, leukemia tumor,
breast cancer, promyelocytic cancer, erythroleukemia
cancer, gastric carcinoma, human colon carcinoma,
and hepatocellular cancer. Their molecular mecha-
nisms have been investigated in inducing apoptosis,
inhibiting invasion, metastasis, inhibiting angiogene-
sis and down-regulating epidermal growth factor
receptors. In addition, tanshinones were reported as a
group of compounds isolated from S. miltiorrhiza
responsible in the treatment of coronary artery disease
and hypertension. However, the anti-TB activity of
tanshinones was rarely studied. In 1980’s, Luo et al.
isolated a series of tanshinones, which showed anti-TB
activity with an MIC range of 0.31–5.0 lg/mL (Luo
et al. 1982). Further modification on these tanshinones
led to several new compounds and their anti-TB were
evaluated as well. Luo discussed the structure–activity
relationship and indicated that the quinone group was
the necessary group responsible for the activity (Luo
et al. 1988).
In order to evaluate the anti-TB potentials of
tanshinones, a small tanshinones library, containing 78
tanshinone-type compounds, has been constructed in
our NPL. A high throughput screening on these
tanshinones against Mtb H37Rv was applied and ten
tanshinones showed promising activity (Table 1). Inter-
estingly, the major constituents of Tanshen, tanshinone I
(19), tanshinone IIA (20), and cryptotanshinone (21),
showed a similar activity against TB with MIC value in
the range of 1.17–26.57 lM. Different substitutions
were introduced to the a-position of ring D of Tan I by
Mannich reaction to obtained two tanshinone analogues
27 and 28 (Scheme 1) and dramatically decreased MIC
values were observed, 1.17 and 2.09 lM, respectively.
Fig. 3 Anti-TB natural products identified by the NPL derived from marine microbes
Antonie van Leeuwenhoek (2012) 102:447–461 455
123
Table 1 Anti-TB
tanshinones identified by
the NPL
Cmpd Name Structure Anti-TB MIC (lM)
19 Tanshinone I 11.32
20 Tanshinone IIA 26.57
21 Cryptotanshinone 10.56
22 Diacetoxytanshinone IIA 8.22
23 Tanshinone IIB 20.16
24 Przewaquinone A 10.08
25 1,2-Dihydrotanshinone I 11.24
26 Dihydrotanshinone I 5.62
27 2-(N,N-dimethyl)-tanshinone I 1.17
28 2-(N-pyrrolidine-alkyl) tanshinone I 2.09
456 Antonie van Leeuwenhoek (2012) 102:447–461
123
Anti-TB sesquiterpenoids and plant-derived organic
acids
The genus Ferula (Apiaceae) comprises about 130
species distributed throughout the Mediterranean area
and Central Asia and its chemical substances have
been studied in many research groups. The widespread
sesquiterpene compounds in this genus are character-
istic daucanes, humulanes, himachalanes, germacr-
anes, eudesmanes, and guainanes (Gonzalez and
Barrera 1995). Several species of the genus Ferula
have been used in traditional medicine for a variety of
therapeutic purposes such as tranquilizers, and for the
treatment of digestive disorders, rheumatism, head-
ache, arthritis, dizziness, toothache, etc. (Gonzalez
and Barrera 1995). Ferula hermonis Boiss. commonly
known as ‘Shilsh-el-zallouh’ or ‘Hashishat-al-kattira’,
is a small shrub that grows abundantly on the Hermon
Mountain between Syria and Lebanon (Said et al.
2002; Lev and Amar 2002). This plant has long been
used in the Middle East as an aphrodisiac, and for the
treatment of frigidity and impotence (El-Taher et al.
2001; Hadidi et al. 2003).
Our anti-TB bioassay-guided investigation on the
crude extracts of the root of Ferula hermonis Boiss.
identified seventeen daucane sesquiterpenoid esters,
(a)
(b)
Scheme 1 Reagents and conditions: a 37 % HCHO, HN(Me)2,
CH3COOH, 125 �C, 12 h; b 37 % HCHO, pyrrolidine,
CH3COOH, 125 �C, 12 h (Yang and Luo 1998)
Table 2 Anti-TB sesquiterpenoids and organic acids identified by the NPL
Cmpd Name Structure Anti-TB MIC (lM)
29 Ferutinin 5.62
30 Teferin 20.72
31 Anthelminthicin A 5.54
32 Anthelminthicin B 16.70
33 Anthelminthicin C 4.38
34 Chaulmoogric acid 9.82
35 Ethyl chaulmoograte 16.80
Antonie van Leeuwenhoek (2012) 102:447–461 457
123
including two anti-TB compounds, ferutinin (29) and
teferin (30), which showed strong activity with MIC
values of 5.62 and 20.72 lM, respectively (Ibraheim
et al. 2011).
Hydnocarpus anthelminthica Pierre ex Laness
(Flacourtiaceae) is a tall evergreen tree, which mainly
grows in Southeast Asia. Its seeds have been used as a
well known folk medicine against leprosy, tinea,
gonorrhea, and TB (Corporation of Zhonghua Bencao
1998). Anti-TB activity directed isolation on the
extracts of Hydnocarpus anthelminthica seeds led to
the isolation of three new compounds, anthelminthic-
ins A–C (31–33), and two known ones, namely
chaulmoogric acid (34) and ethyl chaulmoograte
(35) (Table 2). These compounds (31–35) signifi-
cantly inhibited Mtb H37Rv growth with MIC values
of 5.54, 16.70, 4.38, 9.82, and 16.80 lM, respectively
(Wang et al. 2010).
Conclusion and future prospects
MDR-TBs and TB–HIV co-infection have become a
great threat to global health. However, the last truly
novel drug that was approved for the treatment of TB
was discovered 40 years ago. Search for new effective
drugs against TB has never been more intensive. For
discovery of new molecules that meet the lead criteria,
natural products which possess high chemical diver-
sity, the effects of evolutionary pressure to create
biologically active molecules, the structural similarity
of protein targets across many species and so on, have
been a rich source of lead molecules in drug discovery.
Around 80 % of medicinal products up to 1996 were
either directly derived from naturally occurring com-
pounds or were inspired by a natural product (Harvey
2007). Natural products derived from microbes and
medicinal plants have played a critical role in TB drug
discovery. Recent advances have been made to
accelerate the discovery rate of novel TB drugs
including diversifying strategies for environmental
strains, HTS assays, and chemical diversity.
This review outlined the strategy of finding novel
natural products with anti-TB activity from marine
microbes and plant medicines, including biodiversity-
and taxonomy-guided microbial natural products
library construction, target- and cell-based HTS, and
bioassay-directed isolation of anti-TB substances from
traditional medicines. Anti-TB tanshinones identified
from our NPL were highlighted and further lead
optimization on these anti-TB leads.
Acknowledgments Part of this work was performed under
research collaboration between the Global Alliance for TB Drug
Development (TB Alliance) and the Institute of Microbiology of
the Chinese Academy of Sciences (IMCAS). We acknowledge
Drs. Zhenkun Ma, Anna Upton, and Christopher B. Cooper from
the TB Alliance for their scientific input during the performance
of this work. One of the authors, Krishna Bolla is thankful to
TWAS & CAS for the financial support. This work was
supported in part by grants from National Natural Science
Foundation of China (81102369, 30911120483, 81102356,
30901849, 30973665, 30911120484), the CAS Pillar Program
(XDA04074000) and the Ministry of Science and Technology of
China (2011ZX11102-011-11, 2007DFB31620). LZ is an
Awardee for National Distinguished Young Scholar Program
in China.
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