Perspective

pubs.acs.org/jmc

Development of Mycobacterium tuberculosis Whole Cell Screening

Hits as Potential Antituberculosis Agents
Miniperspectives Series on Phenotypic Screening for Antiinfective Targets
Christopher B. Cooper*
Global Alliance for TB Drug Development (TB Alliance), 40 Wall Street, 24th Floor, New York, New York 10005, United States

ABSTRACT: The global pandemic of drug sensitive tuber-

culosis (TB) as well as the increasing threat from various
multidrug resistant forms of TB drives the quest for newer,
safer, more effective TB treatment options. The general lack of success in progressing novel chemical matter from high
throughput screens of Mycobacterium tuberculosis (M.tb) biochemical targets has prompted resurgence in interest and efforts in
prosecuting mycobacterial phenotypic screens. Whole cell active compounds identified from such screens offer significant
intrinsic advantages over biochemical screening hits, and derivatives of many of these have proven invaluable in helping to fill the
current TB drug development pipeline. Modern techniques for “de-orphaning” such screening hits (i.e., determining their specific
biological mechanism of action) offer the possibility of ultimately identifying improved next-generation chemical series by
screening these essential, pharmacologically validated biochemical targets as well.

■ INTRODUCTION
O Death! The poor man’s dearest friend The kindest and
the best! Welcome the hour, my aged limbs Are laid with
thee at rest! The great, the wealthy, fear thy blow, From
pomp and pleasure torn! But, oh! A blessed relief to those
That weary-laden mourn.
From: “Man Was Made To Mourn. A Dirge,” by Scottish poet and
tuberculosis victim, Robert Burns
Tuberculosis (TB) represents an enduring, deadly infectious
disease for all of mankind. Nearly two billion people are
currently infected with TB with a staggering 13.7 million active
cases worldwide (see Figure 11 ). The World Health
Organization (WHO) estimates that nearly 1.5 million people
die from TB each year, with the overwhelming majority of these
from developing portions of the world.2 In recent years, the
significance of the disease has increased dramatically as TB is
also the major cause of death among patients coinfected with
HIV. In addition, new drug-resistant strains of TB have
emerged which are exceedingly difficult to treat successfully.
Every year, nearly half a million new cases of multidrug resistant
tuberculosis (MDR-TB) are estimated to occur.3 Extremely
drug resistant TB (XDR-TB) is fatal in a large proportion of
cases.4
For those suffering from drug sensitive (DS) tuberculosis, the
standard treatment involves an oral, four-drug combination
(isoniazid, rifampin, pyrazinamide, and ethambutol) given daily
over a period of 6−9 months. Under optimal conditions, this
treatment will cure ∼85−90% of drug-sensitive TB patients if
the treatment regimen is strictly adhered to.1 Each of these
drugs, however, exhibits a range of deleterious side-effects,
including gastrointestinal inflammation/pain, liver toxicity, and
neurological/behavioral manifestations.5 In many circumstan-
ces, the requirement to remain on this regimen for a prolonged
period of time is wholly unacceptable and patients abandon
their treatments before sterilization is fully complete. This then
leads to the emergence of various forms of drug resistant (DR)
TB, including multidrug resistant, extremely drug resistant, and
the recently diagnosed totally drug resistant (TDR) TB.6 Not
surprisingly, treatment options for patients with these forms of
TB are even more limited, with much longer treatment periods
(12−18 months or longer) requiring second-line, injectable
agents which can only be administered in hospital/clinic
settings. The incidence rate of TDR TB, while numerically still
a very small percentage, underscores the genuine urgency to
address this significant unmet medical need.7
■ SOURCES OF NEW ANTITUBERCULAR AGENTS
Target-Based Screens. With the transcription of the
Mycobacterium tuberculosis genome in 1998,8 efforts have
continued unabated to identify and interrogate specific,
essential TB biochemical targets. The construction of rapid
and robust high throughput assays against such targets could
lead to the identification of novel chemical hits for elaboration
ultimately into “drug-like” chemical series of interest. The
underlying problem with this target-based approach remains
the translation of potent, selective in vitro biochemical activity
into whole cell, antimycobacterial activity. Efforts to enhance
binding affinity and specificity can lead to molecules with
unsatisfactory physicochemical properties for uptake by M.tb
and/or exclusion by innate xenobiotic efflux mechanisms.
A range of target-based screens has been employed in recent
years for early TB drug discovery. One example is the
identification of inhibitors of the protein tyrosine phosphatases
Special Issue: Miniperspectives Series on Phenotypic Screening for
Antiinfective Targets
Received: March 14, 2013
Published: August 8, 2013

© 2013 American Chemical Society 7755 dx.doi.org/10.1021/jm400381v | J. Med. Chem. 2013, 56, 7755−7760
Journal of Medicinal Chemistry Perspective

Figure 1. Estimated global TB incidence rates.1

Figure 2. Lead compounds identified using fragment based techniques against protein tyrosine phosphates PtpA and PtpB. Compound 4 was
identified against PtpB using structure based “lead hopping” approaches based on the structure of 3.

Figure 3. Examples of compounds identified from phenotypic screening methods. TMC207 (Bedaquiline) 5 has recently been approved by the
FDA. PA-824 6, OPC- 67683 (Delamanid) 7, and SQ-109 8 are in phase II clinical trials. In early phase drug discovery (MIC values showing activity
against M. tuberculosis H37Rv), BTZ043 9, 10a, and 10b are leading 2-aminothiazole-4-carboxylates (ATC) and 11 is a macrolide derivative.

PtpA and PtpB. For these two targets, a fragment-based
approach was adopted in order to find low molecular weight
inhibitors as chemical “starting points” for further elaboration
and development. From this screen, compounds 1 and 2 were
found to inhibit PtpA with Ki values of 1.4 and 1.6 μM,
respectively9,10 (see Figure 2). Compound 3 was found to bind
to PtpB with a Ki of 1.3 μM with subsequent structure-based
“lead hopping,” resulting in the identification of analogue 4
which bound to this enzyme with a Ki value of 0.69 μM.11,12
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Phenotypic Screens. Despite numerous biochemical target
screening campaigns (by multiple large pharmaceutical
organizations), success in identifying attractive, advanceable
chemical series for infectious disease indications has been
limited.13 As a result, there is a pronounced trend toward
returning to the phenotypic screening strategies which have
proven successful in the past.14 It is important to recognize that
all current TB drugs were either identified by or derived from
M.tb (or surrogate) whole cell screening “hits”. A recent
dx.doi.org/10.1021/jm400381v | J. Med. Chem. 2013, 56, 7755−7760
Journal of Medicinal Chemistry
Figure 4. GSK phenotypic screening progression paradigm for the identification of novel antitubercular whole cell actives.26
increase in commitment, resources, and access to large
pharmaceutical company infrastructures for TB drug discovery
has provided expanded opportunities for high throughput
whole cell screens of pharma collections against M.tb (or
surrogate mycobacteria). In addition, genomic/proteomic
mechanisms now exist to help “de-orphan” whole cell actives
and thus elucidate new, viable, pharmacologically validated M.tb
targets. Work in this field can be further supported by
computational/structure-based drug design (SBDD) efforts to
help identify improved, next-generation agents.
The success of this whole cell screening approach may be
best illustrated with the discovery of the diarylquinoline
TMC207 (bedaquiline 5, see Figure 3).15 Discovered by
researchers at Tibotec (Janssen), this compound was prepared
by medicinal chemistry optimization of a lead originally
identified from a corporate collection screening campaign
against Mycobacterium smegmatis and was subsequently
developed as an antituberculosis agent. It is highly active
against both drug sensitive and drug resistant strains of M.
tuberculosis and was recently approved by the Food and Drug
Administration (FDA) for MDR TB.16 Similar success has been
seen with the development of the nitroimidazoles PA-824 617
and OPC-67683 (Delamanid) 718 and the diamine SQ-109 8,19
which are also in phase II clinical trials. Other recent examples
of novel active series in the early discovery phase include the
benzothiazinones (BTZ) 9,20 identified from the NM4TB
program, and the 2-aminothiazole-4-carboxylates (ATC’s) 10a
and 10b from the TBD-UK consortium.21 Additionally, a
number of recent screening campaigns carried out with M.
tuberculosis H37Rv have led to the identification of a large
number of potential new lead compounds with indications of
both whole cell antituberculosis activity and selectivity against
representative eukaryotic cell lines.22,23 One such example was
the identification of macrolide derivatives 11 as reported by
Falzari et al.24 Through the support of the Bill and Melinda
Gates Foundation (BMGF), efforts are also underway to
conduct M.tb phenotypic screens under “persistent” conditions
(i.e., low oxygen, low nutrient, high reactive oxygen species
(ROS), NO exposure, etc.). In such a manner, it is envisioned
that novel chemotypes may be identified which could prove
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effective against nonreplicating, persistent M.tb subpopula-
tions.25
GSK Antimycobacterial Phenotypic Screen: Hit Iden-
tification. Recently, researchers at GlaxoSmithKline (GSK)
conducted a large (∼2 M compound) phenotypic screening
campaign in hopes of identifying novel antimycobacterial
chemical series for further TB pursuit.26 Like several other large
pharmaceutical companies, GSK opted to use Mycobacterium
bovis BCG as an M. tuberculosis surrogate for primary screening
purposes. They initially selected and tested a representative
compound collection subset of ∼20,000 compounds to confirm
the use of M. bovis BCG as a predictor for M.tb activity overall.
Compounds belonging to this subset were characterized by
good membrane permeability, GSK available stocks, and
reasonable overall “drug-like” properties. Compounds were
assayed against both M. bovis BCG (“BCG”) and M. tuberculosis
H37Rv (“H37Rv”) in two independent experiments at a single
10 μM concentration. Similar hit rates were found in both
experiments: 127 primary hits were identified as inhibitors of
BCG growth and 112 hits as inhibitors of H37Rv. Retesting
both sets of hits confirmed 88 of the compounds detected in
the BCG screen (69%) and 73 of the primary hits from H37Rv
(65%) as active.
The GSK team then evaluated the full 2 M compound
collection against BCG with an initial cutoff of >50% growth
inhibition at a single, 10 μM concentration. This resulted in the
identification of ∼62,000 primary hits. A series of in silico
filtering (removing known antibacterials) and physicochemical
property clustering operations reduced this original number to
∼15,000 hits. As depicted in Figure 4 (courtesy of L. Ballell,
GSK), these hits were further interrogated to determine their
minimum inhibitory concentration (MIC) values vs M. bovis
BCG as well as their HepG2 cytotoxicity potential (IC50s).
Only those compounds which demonstrated M. bovis BCG
MIC’s <10 μM as well as therapeutic indices [(IC50HepG2)/
(MIC50BCG)] > 50 were taken forward for M.tb MIC
determinations. In this manner, this set of 850 “BCG” hits
was reduced to 177 “M.tb” hits which had M.tb MIC95s <10 μM
while retaining therapeutic indices [(IC 50 HepG2)/
(MIC95H37Rv)] > 50.
dx.doi.org/10.1021/jm400381v | J. Med. Chem. 2013, 56, 7755−7760
Journal of Medicinal Chemistry
Figure 5. GSK antitubercular whole cell active chemotypes of interest.
GSK Antimycobacterial Phenotypic Screen: Hit Char-
acterization and Expansion. To further elucidate the lead
progression potential of the antitubercular hit list, the GSK
team selected a range of chemical clusters based on their
intrinsic, in vitro antimycobacterial activities (M.tb H37Rv
MIC’s < 1 μM) and the number of structural analogues
represented within the full hit list. This provided seven
chemical clusters (see Figure 5), representatives of which
were selected for further characterization. Biological and
physicochemical profiling consisted of MIC determination
against various M. tuberculosis strains, characterization against
intracellular growth, activity profiling against an internal panel
of eight different bacteria for early assessment of the potential
for wide-spectrum activity, aqueous solubility measurement,
evaluation of cytotoxicity, artificial membrane permeability
studies, cytochrome P450 (CYP) inhibition profiling against a
representative panel of isoforms,27 and early in vitro metabolic
studies to establish the stability of the hit structures in mouse
and human microsomal fractions (CLint and t1/2 values).
Working with TB Alliance, GSK remains actively engaged in
advancing two of these series (THPP’s and Spiros) through
lead optimization medicinal chemistry.
■ FUTURE PROSPECTS
“De-orphaning” Whole Cell Screening Hits. Despite the
genuine success of identifying active chemical matter through
antimycobacterial phenotypic screens, the conversion of such
hits into progressible chemical series and ultimately into clinical
candidates is hampered by the lack of knowledge regarding the
specific biochemical target(s) which such species inhibit and/or
upregulate. Recent advances in M.tb genomics/proteomics can
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provide opportunities to “de-orphan” such screening hits (or
advanced analogues derived from initial hits) and thus: (a) aid
in further SAR development for a given chemical series or (b)
help identify new, essential, druggable M.tb targets of interest.
These new targets can then be exploited further through
subsequent dedicated high throughput screens to identify
entirely different chemical starting points. Furthermore,
coupling target identification work with structure-based drug
design (SBDD) efforts may facilitate the synthesis of novel,
improved compounds/chemical series against such pharmaco-
logically validated targets of interest.
The determination of the precise mechanism(s) of action
(MOA) of whole cell screening actives remains challenging.
The inherent slow cycle time of M.tb and the diminished
viability of mutants lacking essential targets create significant
difficulties in the generation of M.tb compound-specific
resistant mutants. Additionally, the surfeit of “non-MOA”
targets which have recently been identified (e.g., efflux pump
substrates, growth media-specific targets, etc.) further compli-
cates this process.28 Experience indicates that target identi-
fication efforts for pleiotropic TB compounds, while exceed-
ingly effective as therapeutic agents, are increasingly complex
and difficult. Finally, the use of M.tb surrogates, while
technically more convenient, can lead to the identification of
false M.tb targets (i.e., wide range of target binding affinity
differences observed between mycobacteria, and even among
different M.tb strains29). Despite these challenges, efforts
continue on a global level at multiple universities and institutes
to: (a) generate and sequence M.tb mutants following increased
exposures to novel phenotypic screening hit materials, (b)
establish their precise biochemical target(s), and (c) confirm
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Journal of Medicinal Chemistry
such on-target activities through the generation of knockout
(KO), knockdown (KD), and overexpresser (OE) M.tb strains.
Coupling SBDD with M.tb Target Validation/New
Lead Discovery Efforts. Despite world-class diligent efforts
by many hundreds of researchers over the past four decades,
there are still very few pharmacologically validated TB drug
targets to work on. The BMGF TB Drug Accelerator program
is attempting to improve the current “barren landscape” by
expanding the list of validated M.tb drug targets.25 In concert
with this genomic/proteomic work, the University of Toronto-
based Structural Genomics Consortium (SGC) has proposed
to use structure-guided methods to design and generate novel
antitubercular drug leads de novo.30 Their strategy is to express
and purify milligram quantities of such validated protein targets
and characterize the interactions with their corresponding
phenotypic screening hits (or advanced analogues derived from
the original hits). In cases where binding is confirmed, they
hope to cocrystallize the target with its bound inhibitor.
Furthermore, the SGC plans to expand attractive hit molecules
into progressible compound series through medicinal chem-
istry. These new compounds would then be assessed in protein-
and cell-based assays to generate data for structure−activity
relationship (SAR) analysis. Additionally, those analogues with
promising SAR and physicochemical properties would be
cocrystallized with their targets and, with costructures available,
an iterative lead optimization cycle would ensue comprising
structure-guided design of new molecules, target assays to
confirm and characterize target inhibition, cell-based assays to
characterize potency of anti-M.tb activity, in vitro ADMET
assessment, and finally rodent PK/efficacy testing. The goal of
this exercise will be the generation of antitubercular lead
molecules which meet the criteria for early drug discovery leads
and which demonstrate proof of concept efficacy in vivo.
Further lead optimization of such series may ultimately produce
“next generation” antitubercular preclinical candidates.
■ CONCLUSIONS
The search for new TB drugs remains a challenging albeit
vitally important task. With target-based screening approaches
offering few tangible successes, the global TB community has
predominantly adopted whole cell screening methods for the
primary source of novel, tractable sources of lead molecules/
series. New whole cell screening campaigns conducted under
various simulated physiological conditions should reveal novel
chemotypes which are active under chronic, persistent
conditions. Improved technologies for deorphaning phenotypic
screening hits hold promise for rigorous evaluations of M.tb-
specific biochemical targets (or pathways) through future
target-based screens. Finally, the large-scale preparation of
M.tb-specific protein targets may permit the inclusion of
structure-guided drug design approaches to create entirely
novel chemical matter for future TB drug development.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: 646-616-8634. E-mail: christopher.cooper@tballiance.
org.
Notes
The authors declare no competing financial interest.
Biography
Christopher B. Cooper received his Ph.D. in organic chemistry with
Professor Paul A. Wender in 1988. From 1988 through 1998, he was
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engaged in medicinal chemistry research at Pfizer, Inc., at both their
Groton, Connecticut, and Sandwich, England, sites. In 1998, Chris
joined Bristol-Myers Squibb, Inc., in Princeton, New Jersey, to launch
the Lead Synthesis Group spearheading the design, development, and
synthesis of novel, drug-like medicinal chemistry arrays. In 2009, he
came to the Global Alliance for TB Drug Development (TB Alliance),
where he is currently Senior Director, Chemistry, overseeing the
Alliance’s global discovery and preclinical development chemistry
activities. Chris has published over 60 peer-reviewed papers and is
coinventor on 23 U.S. patents.
■
ACKNOWLEDGMENTS
I thank the members of the TB Alliance research staff for their
helpful comments and advice in reviewing this paper. In
addition, I would like to acknowledge Lluis Ballell, Modesto
Remuinan, and David Barros from GSK (Tres Cantos, Spain)
for their collective assistance in preparing this manuscript.
■
ABBREVIATIONS USED
ADMET, absorption, distribution, metabolism, excretion, and
toxicity; ATC, amino-4-thiazole carboxylic acid; BMGF, Bill
and Melinda Gates Foundation; BTZ, benzothiazones; CLint,
intrinsic clearance; CYP, cytochrome P450; DR, drug resistant;
DS, drug sensitive; FDA, Food and Drug Administration; GSK,
GlaxoSmithKline; HepG2, HepG2 human liver carcinoma cell
line; HIV, human immunodeficiency virus; KD, knockdown;
KO, knockout; M. bovis BCG, Mycobacterium bovis Bacille de
Calmette et Guérin; MDR-TB, multidrug resistant TB; MIC,
minimum inhibitory concentration; MOA, mechanism of
action; M.tb, Mycobacterium tuberculosis; NM4TB, New
Medicines for TB; PK, pharmacokinetics; PtpA, protein
tyrosine phosphatase A; PtpB B, protein tyrosine phosphatase
B; ROS, reactive oxygen species; SBDD, structure-based drug
design; SGC, Structural Genomics Consortium; TB, tuber-
culosis; TDR TB, totally drug resistant TB; THPP,
tetrahydropyrrolopyrimidine; WHO, World Health Organiza-
tion; XDR-TB, extremely drug resistant TB
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